University of Veterinary Medicine Hannover
Institute of Virology
Analysis of viral and host factors
influencing Alphacoronavirus life
cycle in chiropteran and porcine
cell lines
THESIS
Submitted in partial fulfillment of the requirements for the degree
Doctor rerum naturalium
(Dr. rer. nat.)
awarded by the University of Veterinary Medicine Hannover
by
Anna-Theresa Rüdiger
Sömmerda, Germany
Hannover, Germany 2016
Supervisor:
PD Dr. Christel Schwegmann-Weßels
Supervision Group:
PD Dr. Christel Schwegmann-Weßels
Prof. Dr. Andreas Beinecke
PD Dr. Eike Steinmann
1th Evaluation:
PD Dr. Christel Schwegmann-Weßels
Institut für Virologie
Tierärztliche Hochschule Hannover
Prof. Dr. Andreas Beinecke
Institut für Pathologie
Tierärztliche Hochschule Hannover
PD Dr. Eike Steinmann
Institut für Experimentelle Virologie
TWINCORE, Hannover
2nd Evaluation:
Prof. Dr. Volker Thiel
Institut für Virologie
Universität Bern
Date of final exam:
04.04.2016
This work was financed by the Emmy Noether Programme of the DFG and the
Georg-Christoph-Lichtenberg scholarship.
Parts of the thesis have been submitted:
Rüdiger A-T, Müller MA, Schwegmann-Weßels C. Release of infectious virus
particles as well as spike and membrane protein expression pattern differ within
chiropteran cell lines infected by the Alphacoronavirus TGEV.
Rüdiger A-T, Mayrhofer P, Ma-Lauer Y, Pohlentz P, Müthing J, v. Brunn A,
Schwegmann-Weßels C. Tubulins interact with porcine & human S proteins of the
genus Alphacoronavirus and support successful assembly and release of infectious
viral particles.
Results of this thesis were presented in the following events:
Poster presentations
Rüdiger A-T, Schwegmann-Weßels C. (2013) Production of infectious virus particles
of Alphacoronaviruses differs in a variety of bat species. 5th European Congress of
Virology, Lyon (France).
Rüdiger A-T, Schwegmann-Weßels C. (2014) The released amount of infectious
virus particles of Alphacoronaviruses is different in a variety of bat species. 24th
Annual Meeting of the Society for Virology, Alpach (Austria).
Rüdiger A-T, Schwegmann-Weßels C. (2014) Depending on the last 16 amino acids
of the cytoplasmatic tail coronavirus spike proteins show different transport behaviors
in not infected cells. 7th European Meeting on Viral Zoonoses, Saint-Raphaël
(France).
Rüdiger A-T, Schwegmann-Weßels C. (2014) Analysis of the charge-rich region of
the cytoplasmic domain of zoonotic relevant coronavirus spike proteins. 7 th Graduate
School Day HGNI, Hannover.
Rüdiger A-T, Mayrhofer P, Ma-Lauer Y, Pohlentz P, Muething J, v. Brunn A,
Schwegmann-Weßels C. (2015) Analysis of host cell protein interaction partners of
porcine and human coronavirus spike protein cytoplasmic domains. 25th Annual
Meeting of the Society for Virology, Bochum.
Oral presentations
Rüdiger A-T, Schwegmann-Weßels C. (2013) Production of infectious virus particles
of Alphacoronaviruses differs in a variety of bat species. 6th Graduate School Day
HGNI, Hannover.
Rüdiger A-T, Mayrhofer P, Ma-Lauer Y, Pohlentz P, Muething J, v. Brunn A,
Schwegmann-Weßels C. (2015) Tubulins interact with porcine & human S proteins of
the genus α-Coronavirus and influence the viral replication cycle of the porcine
coronavirus TGEV. Xth International Congress of Veterinary Virology, Montpellier
(France).
To my mom & sister,
to Sue & Larry
No matter what people tell you, words and ideas can change the world.
(Robin Williams)
Table of contents
Table of contents
Table of contents .............................................................................................. I
List of abbreviations....................................................................................... VI
List of figures and tables ................................................................................ X
Summary ........................................................................................................ xii
Zusammenfassung ....................................................................................... xiv
1 Introduction ................................................................................................ 1
1.1 Coronaviruses ........................................................................................ 1
1.1.1 Taxonomy ............................................................................................ 1
1.1.2 Coronavirus particle ............................................................................. 2
1.1.3 Genome organization ........................................................................... 3
1.1.4 Transmissible gastroenteritis coronavirus (TGEV) ............................... 4
1.1.5 Coronavirus life cycle ........................................................................... 6
1.1.5.1 Entry ........................................................................................ 7
1.1.5.2 Replication and translation ...................................................... 7
1.1.5.3 Assembly and release ............................................................. 8
1.1.6 Coronavirus structural proteins of interest ........................................... 9
1.1.6.1 Spike (S) protein...................................................................... 9
1.1.6.2 Membrane (M) protein ........................................................... 11
1.1.7 Coronavirus-host interaction .............................................................. 12
1.1.7.1 Coronavirus interaction with host cytoskeleton ..................... 14
1.2 Chiroptera ............................................................................................. 17
1.2.1 Zoonosis and virus transmission from chiropterans to humans ......... 19
1.2.2 Coronaviruses detected in chiropterans ............................................. 20
1.3 Aims of the study .................................................................................. 23
1.4 References ........................................................................................... 24
I
Table of contents
2 Manuscript I
Release of infectious virus particles as well as spike and membrane
protein expression pattern differ within chiropteran cell lines
infected by the Alphacoronavirus TGEV ............................................... 45
2.1 Introduction ........................................................................................... 46
2.2 Material & Methods ............................................................................... 47
2.2.1 Cell lines and virus strains ................................................................. 47
2.2.2 Plasmids ............................................................................................ 48
2.2.3 Immunofluorescence analysis ............................................................ 48
2.2.3.1 Viral infection ......................................................................... 48
2.2.3.2 Viral protein localization after TGEV infection ....................... 49
2.2.4 Growth curve...................................................................................... 50
2.2.5 Determination of receptor expression levels by flow cytometry.......... 50
2.2.5.1 Determination of pAPN expression ....................................... 50
2.2.5.2 Determination of alpha-2,6-linked sialic acids ....................... 50
2.2.5.3 Determination of low density lipoprotein receptor (LDL)
receptor expression ............................................................... 51
2.2.5.4 Determination of alpha 2,3-linked sialic acids ....................... 51
2.3 Results .................................................................................................. 51
2.3.1 TGEV infection is restricted to pAPN expressing cells although the
receptor expression level does not fully correlate with the amount
of released infectious viral particles ................................................... 51
2.3.2 The infection rate of SIV-H3N2 was similar in all tested chiropteran
cell lines despite different expression levels of the receptor
determinant ........................................................................................ 56
2.3.3 Highest VSV infection rate in TB1Lu cells while receptor
expression levels were similar on bat cell surfaces............................ 59
2.3.4 The Gammacoronavirus IBV was able to enter chiropteran cell
lines ................................................................................................... 63
2.3.5 TGEV S and M protein expression pattern differed in the tested
chiropteran cell lines .......................................................................... 67
II
Table of contents
2.4 Discussion ............................................................................................ 70
2.4.1 Infection of different chiropteran cells by TGEV, SIV, VSV, and IBV . 70
2.4.2 Localization of TGEV proteins within chiropteran cell lines ................ 72
2.5 Conclusion ............................................................................................ 73
2.6 References ........................................................................................... 74
3 Manuscript II
Tubulins interact with porcine & human S proteins of the genus
Alphacoronavirus and support successful assembly and release of
infectious viral particles .......................................................................... 78
3.1 Introduction ........................................................................................... 79
3.2 Material & Methods ............................................................................... 81
3.2.1 Cell lines and virus strains ................................................................. 81
3.2.2 Plasmids ............................................................................................ 81
3.2.3 GFP Trap® pull down assay & SDS-PAGE ........................................ 82
3.2.4 In gel digestion and mass spectrometry ............................................. 83
3.2.5 Immunofluorescence analysis ............................................................ 83
3.2.6 Plaque assay ..................................................................................... 84
3.2.7 Virus particle assay ............................................................................ 85
3.3 Results .................................................................................................. 85
3.3.1 Co-immunoprecipitation of tubulins with the TGEV S protein
cytoplasmic domain ........................................................................... 85
3.3.2 Co-immunoprecipitation of tubulins with corresponding parts of the
human CoV 229E and CoV NL63 S protein cytoplasmic domains ..... 86
3.3.3 The TGEV Swt full length protein partly co-localizes with authentic
cellular β-tubulins ............................................................................... 87
3.3.4 S proteins are differentially distributed after treatment with
Nocodazole ........................................................................................ 88
3.3.5 In infected cells, TGEV S protein distribution differs in DMSO and
NOC treated cells, and is expressed near the ERGIC and Golgi
compartment whereby both compartments are scattered
throughout the cell after NOC treatment ............................................ 92
III
Table of contents
3.3.6 Release of infectious virus particles is reduced in NOC treated ST
cells .................................................................................................... 94
3.3.7 Less S protein is incorporated into virions after NOC treatment of
infected ST cells................................................................................. 95
3.4 Discussion ............................................................................................ 96
3.5 Conclusion .......................................................................................... 100
3.6 References ......................................................................................... 101
4 Manuscript III
Analysis of tyrosine motifs within the cytoplasmic domain of the
coronavirus spike (S) and membrane (M) proteins and their
functions during S-M interaction .......................................................... 109
4.1 Introduction ......................................................................................... 110
4.2 Material and Methods ......................................................................... 112
4.2.1 Cell lines and virus strains ............................................................... 112
4.2.2 Plasmids .......................................................................................... 112
4.2.3 Transfection ..................................................................................... 113
4.2.4 Immunoflurescence analysis of single S or M protein expression .... 113
4.2.5 Immunofluorescence analysis of S and M-HA co-expression .......... 114
4.2.6 Immunofluorescence analysis of S and Mwt co-expression ............. 114
4.2.7 Immunofluorescence analysis of S Y/A and M Y/A mutant coexpression........................................................................................ 115
4.2.8 Co-immunoprecipitation, surface biotinylation, and cell lysis ........... 115
4.3 Results ................................................................................................ 117
4.3.1 S expression pattern alters when co-expressed with M proteins
resulting in intracellular S accumulation ........................................... 117
4.3.2 TGEV M Y/A protein mutants still lead to S protein retention ........... 122
4.4 Discussion .......................................................................................... 125
4.5 Conclusion .......................................................................................... 129
4.6 References ......................................................................................... 130
IV
Table of contents
5 Discussion .............................................................................................. 135
5.1 Replication of the Alphacoronavirus TGEV within chiropteran cell
lines .................................................................................................... 135
5.2 Alphacoronavirus interaction with host cell microtubules ................... 137
5.3 Interaction of coronaviral S and M proteins ........................................ 140
5.4 Conclusion .......................................................................................... 142
5.5 References ......................................................................................... 143
Appendix ...................................................................................................... 149
Affidavit
Acknowledgement
V
List of abbreviations
List of abbreviations
A
Adenine
aa
Amino acid
AB
Antibody
ACE2
Angiotensin-converting-enzyme-2
ACN
Acetonitrile
APN
Aminopeptidase N
BCoV
Bovine coronavirus
BHK
Baby hamster kidney
bp
Base pair
BSA
Bovine serum albumin
CD
Cytoplasmic domain
cDNA
Complementary DNA
CEACAM1
Carcinoembryonic antigen cell adhesion molecule 1
COP
Coat protein complex
CoV(s)
Coronavirus(es)
CRM
Cysteine-rich motif
CT/ C-terminus
Carboxy-terminus
Cy3
Carbocyanine-3
DAPI
4',6-diamidino-2-phenylindole
DMEM
Dulbecco's Modified Eagle's Medium
DMSO
Dimethyl sulfoxide
DMV
Double-membrane vesicle
DNA
Deoxyribonucleic acid
DPP4
Dipeptidyl peptidase-4
E protein
Envelope protein
Env
Envelope protein of HTLV-1
EpoNi
Epomops franqueti kidney
ER
Endoplasmatic reticulum
ERGIC
Endoplasmatic reticulum-Golgi intermediate compartment
F
Phenylalanine
FIPV
Feline infectious peritonitis virus
FITC
Fluorescein isothiocyanate
VI
List of abbreviations
g
Gravitational force
GFP
Green fluorescence protein
h
Hour(s)
HA
Hemagglutinin
hAPN
Human aminopeptidase N
HCoV 229E
Human coronavirus 229E
HCoV NL63
Human coronavirus NL63
HCoV OC43
Human coronavirus OC43
HE protein
Hemagglutinin esterase protein
HEK
Human embryonic kidney
HeLa
Henrietta Lacks (cervical cancer cells)
HKU
University of Hong Kong
hnRNP
Heterogeneous nuclear ribonucleoprotein
hpi
Hours post infection
hpt
Hours post transfection
HTLV-1
Human T-lymphotropic virus type 1
HypNi
Hypsignathus monstrosus kidney
I
Isoleucine
IBV
Infectious bronchitis virus
ICTV
International Committee for the Taxonomy of Viruses
IFA
Immunofluorescence analysis
Ig
Immunoglobulin
K
Lysine
kb
Kilobases
kDa
Kilodalton
L
Leucine
LANUV
Landesamt für Naturschutz und Umwelt
LDL
Low density lipoprotein receptor
LLC-PK1
Lilly Laboratories cell porcine kidney
M
Methionine
M protein
Membrane protein
MAA
Maackia Amurensis
mAb
Monoclonal antibody
MADP1
Zinc finger CCHC-type and RNA binding motif 1
VII
List of abbreviations
MAP
Microtubule-associated proteins
MDCKII
Madin-Darby canine kidney
MERS
Middle-East respiratory syndrome
MHV
Murine hepatitis virus
min
Minute(s)
ml
Milliliter
mM
Millimolar
MOI
Multiplicity of infection
mRNA
Messenger RNA
MT
Microtubule
MTOC
Microtubule organizing center
MyDauDa
Myotis daubentonii intestine
N protein
Nucleocapsid protein
NaCl
Natrium chloride
NOC
Nocodazole
nsp
Non-structural protein
NT/ N-terminus
Amino-terminus
ORF
Open reading frame
pAB
Polyclonal antibody
pAPN
Porcine aminopeptidase N
PBS
Phosphate buffered saline
PCR
Polymerase chain reaction
PEDV
Porcine epidemic diarrhea virus
PFA
Paraformaldehyde
PipNi
Pipistrellus pipistrellus kidney
pp
Polypeptide
PRCoV
Porcine respiratory coronavirus
RBD
Receptor binding domain
RBS
Receptor binding site
RNA
Ribonucleic acid
S protein
Spike protein
SARS
Severe acute respiratory syndrome
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
sg
Subgenomic
VIII
List of abbreviations
SIV
Swine influenza virus
SNA
Sambucus Nigra
ST
Swine testis
T
Tween
Tb1Lu
Tadarida brasiliensis lung
TGEV
Transmissible gastroenteritis virus
TGN
Trans-Golgi network
TM
Transmembrane domain
TUBA4A
Tubulin alpha-4A chain
TUBB2A
Tubulin beta-2A chain
TUBB4A
Tubulin beta-4A chain
TUBB6
Tubulin beta-6 chain
V
Valine
Vero
African green monkey kidney epithelial
VLP
Virus-like particle
VSV
Vesicular stomatitis virus
wt
Wildtype
X
Any amino acid
Y
Tyrosine
°C
Degree Celsius
µg
Microgram
µl
Microliter
µM
Micromolar
Φ
Hydrophobic amino acid
IX
List of figures and tables
List of figures and tables
Fig. 1-1 Taxonomy of the family Coronaviridae according to the International Committee for the
Taxonomy of Viruses (ICTV), modified (CHAN et al. 2015). .................................................... 1
Fig. 1-2 Phylogenetic tree of several members of coronaviruses, modified (CHAN et al. 2015). .......... 2
Fig. 1-3 Coronavirus particle. ................................................................................................................. 3
Fig. 1-4 Coronavirus genome organization, modified (UJIKE & TAGUCHI 2015). ................................ 4
Fig. 1-5 Severe acute respiratory syndrome coronavirus (SARS-CoV) life cycle, modified (DU et al.
2009). ........................................................................................................................................ 6
Fig. 1-6 Schematic representation of coronavirus spike proteins, modified (MOU et al. 2013). .......... 10
Fig. 1-7 C-terminal end of transmissible gastroenteritis coronavirus (TGEV) spike protein. ............... 11
Fig. 1-8 Schematic drawing of microtubules and actin filaments and their corresponding molecular
motors, modified (GAUDIN et al. 2013). ................................................................................. 16
Fig. 1-9 Phylogenetic relations between bat hosts and coronaviruses, modified (DREXLER et al.
2014). ...................................................................................................................................... 22
Fig. 2-1 TGEV infection study: Immunofluorescence analysis of pAPN and TGEV S protein
expression. .............................................................................................................................. 53
Fig. 2-2 TGEV infection study: Growth curve. ...................................................................................... 54
Fig. 2-3 pAPN-GFP expression level: Flow cytometry ......................................................................... 55
Fig. 2-4 SIV-H3N2 infection study: Immunofluorescence of SIV-H3N2 nucleocapsid protein. ............ 57
Fig. 2-5 SIV-H3N2 infection study: Growth curve. ............................................................................... 58
Fig. 2-6 Alpha-2,6-linked sialic acid expression level: Flow cytometry. ............................................... 59
Fig. 2-7 VSV infection study: Immunofluorescence of VSV glycoprotein. ............................................ 61
Fig. 2-8 VSV infection study: Growth curve.......................................................................................... 62
Fig. 2-9 LDL expression level: Flow cytometry..................................................................................... 63
Fig. 2-10 IBV infection study: Immunofluorescence of IBV. .................................................................. 64
Fig. 2-11 IBV infection study: Growth curve. ......................................................................................... 65
Fig. 2-12 Alpha-2,3-sialic acid expression level: Flow cytometry. ......................................................... 66
Fig. 2-13 Localization of TGEV spike (S) protein 1 dpi. ........................................................................ 67
Fig. 2-14 Localization of TGEV membrane (M) protein 1 dpi. ............................................................... 68
Fig. 2-15 Localization of TGEV nucleocapsid (N) protein 1 dpi. ........................................................... 69
Fig. 2-16 Localization of TGEV envelope (E) protein 1 dpi. .................................................................. 70
Fig. 3-1 Co-immunoprecipitation of TGEV S-GFP-NT fusion protein and different tubulins via GFP®
Trap . ...................................................................................................................................... 86
Fig. 3-2 Co-immunoprecipitation of alphacoronavirus S fusion proteins and different tubulins via GFP®
Trap . ...................................................................................................................................... 87
Fig. 3-3 Co-localization study of TGEV Swt proteins and authentic β-tubulin. .................................... 88
Fig. 3-4 S protein expression in untreated and NOC treated ST cells 7 hpt. ....................................... 90
Fig. 3-5 S protein expression in untreated and NOC treated chiropteran cells 7 hpt. ......................... 91
Fig. 3-6 TGEV-S expression in untreated and NOC treated cells 7hpi. ............................................... 93
X
List of figures and tables
Fig. 3-7 TGEV-S expression in untreated and NOC treated ST cells 7hpi. ......................................... 94
Fig. 3-8 Quantification of released infectious virus particles in mock-treated and NOC treated cells via
plaque assay. .......................................................................................................................... 95
Fig. 3-9 TGEV particle assay 24 hpi. .................................................................................................... 96
Fig. 4-1 Surface and total protein expression of S constructs in single expressing or M-HA coexpressing cells (part 1). ....................................................................................................... 118
Fig. 4-2 Surface and total protein expression of S constructs in single expressing or M-HA coexpressing cells (part 2). ....................................................................................................... 119
Fig. 4-3 TGEV SSARS-S-Tail and TGEV M wildtype protein expression in co-transfected BHK-21 cells.
.............................................................................................................................................. 120
Fig. 4-4 TGEV SMERS-S-Tail and TGEV M wildtype protein expression in co-transfected BHK-21 cells.
.............................................................................................................................................. 121
Fig. 4-5 TGEV SMERS-S-Tail single expression ....................................................................................... 121
Fig. 4-6 Surface expression of parental TGEV S or TGEV S mutant proteins and total protein
expression of S and TGEV Mwt proteins. ............................................................................. 122
Fig. 4-7 Immunofluorescence analysis of TGEV M Y/A protein mutants single-expressed or coexpressed with TGEV S Y/A protein in BHK-21 cells. .......................................................... 124
Fig. 4-8 Surface biotinylation and cell lysates of BHK-21 cells single transfected with TGEV M Y/A
mutant constructs or co-transfected with S Y/A cDNA. ........................................................ 125
Fig. 5-1 Intracellular protein transport, modified (KREIS et al. 1997; GLICK & NAKANO 2009). ...... 140
Tab. 1 TGEV titer measured by plaque assay 24 hpi. .......................................................................... 54
Tab. 2 SIV-H3N2 titer measured by plaque assay 24 hpi. .................................................................... 58
Tab. 3 VSV titer measured by plaque assay 24 hpi. ............................................................................. 62
Tab. 4 IBV titer measured by plaque assay 24 hpi. .............................................................................. 65
Tab. 5 Last 39 amino acid stretches of coronavirus cytoplasmic domains linked to GFP. ................... 81
Tab. 6 Amino acid sequences of coronavirus (CoV) spike (S) charge-rich regions............................ 113
Tab. 7 Amino acid sequence of TGEV M Y/A mutants. ...................................................................... 113
XI
Summary
Summary
Analysis of viral and host factors influencing Alphacoronavirus life cycle in
chiropteran and porcine cell lines
Anna-Theresa Rüdiger
Coronaviruses (CoVs) are enveloped, single-stranded RNA viruses with positive
orientation. Their envelope consists of three to four structural proteins, the spike (S)
protein, membrane (M) protein, envelope (E) protein, and some members of
Betacoronaviruses contain the hemagglutinin esterase protein. Within the virion the
nucleocapsid protein is associated with the viral RNA. CoVs infect mammals,
including humans, and birds. Several animal-to-human as well as animal-to-animal
transmissions are known for CoVs. Since the occurrence of the severe acute
respiratory syndrome coronavirus (SARS-CoV), research has focused on bats which
are considered the reservoir species of most CoVs. The entry, replication, and
release of transmissible gastroenteritis virus (TGEV), a representative of the genus
Alphacoronavirus, in different chiropteran cell lines was examined. Surface
expression of the specific receptor, porcine aminopeptidase N, was necessary for
TGEV to enter the chiropteran cells. Differences in receptor expression levels as well
as differences in viral titers were measured in the analyzed chiropteran cells.
Furthermore, a diverse expression pattern of the TGEV S and M protein was
observed. We demonstrated that the lack of a specific receptor is responsible for
species restriction and is the first hurdle which the virus has to overcome for
successful entry and replication. However, infectious titers of released viral particles
indicate that additional factors may influence viral replication in chiropteran cells. The
differences in distribution of the TGEV S and M protein during infection underline the
assumption that host factors depending on bat species and organ source play an
important role for a successful CoV infection.
Therefore, cellular components associated with CoV S proteins were investigated.
For several viruses microtubules are utilized for the transport of viral components
within the host cell. The S protein mediates host-cell-attachment and virus entry. In
this study we demonstrated that the last 39 amino acid stretch of the S cytoplasmic
domains of TGEV and human CoVs 229E and NL63 interact with tubulin alpha and
beta chains. Additionally, a partly co-localization of TGEV S proteins with authentic
xii
Summary
host
cell
β-tubulin
was
observed.
Furthermore,
drug-induced
microtubule
depolymerization led to changes in S protein distribution. Here, TGEV S proteins
partly co-localized with the ER-Golgi intermediate compartment (ERGIC) and Golgi
complex, while these compartments were scattered throughout the cell after
nocodazole (NOC) treatment. Moreover, a reduction in the release of infectious virus
particles and lower amounts of TGEV S protein incorporated into virions were
detected in NOC treated cells. In CoV S transfected or TGEV infected chiropteran
cells similar results were obtained. These data demonstrate that interactions of
Alphacoronavirus S proteins with tubulin support the virus during assembly and
release and that this strategy seems to be a conserved mechanism.
Beside virus-host interactions also the interaction between viral proteins is
influencing the CoV life cycle. CoV replication takes place in the host cytosol
whereas its assembly and budding occurs at the ERGIC. Protein-protein as well as
protein-RNA interactions are important during these steps. The S, M, and E proteins
contain signals targeting them to the site of budding where they interact with each
other for efficient incorporation. Especially, the incorporation of S proteins is
necessary for emergence of infectious progeny, accomplished by S-M interaction. S
proteins contain an ectodomain, transmembrane domain as well as a cytoplasmic
tail. The cytoplasmic domain consists of a cysteine- and charge-rich region. Some
members have a tyrosine-based motif within their S charge-rich region leading to
intracellular accumulation. Regarding mouse hepatitis virus and SARS-CoV the S
cytoplasmic tail, particularly the charge-rich region, plays a major role during
association with M. The corresponding region of different coronavirus S proteins was
examined for their impact on S-M interaction. Hereby, the charge-rich region of the
TGEV S protein cytoplasmic domain was replaced by human- and bat-derived CoV S
charge-rich regions from the genus Alpha- and Betacoronavirus. Different S protein
expression pattern due to the charge-rich regions were identified, when expressed
alone. Nevertheless, S-M interaction was neither completely abolished nor inhibited.
In S and M co-expressing cells S was retained near the nucleus and close to the M
protein. Neither amino acid changes within the tyrosine motif of CoV S proteins nor in
their charge-rich region influenced S-M interaction. Additionally, three TGEV M Y/A
mutants were tested for their ability to retain the S protein. All of them seem to
associate with the TGEV S protein visible by S retention, although less S was
retained compared to S and Mwt protein co-expressing cells.
xiii
Zusammenfassung
Zusammenfassung
Analyse viraler und zellulärer Faktoren sowie deren Einfluss auf den
Lebenszyklus von Alphacoronaviren in Fledertier- und Schweinezelllinien
Anna-Theresa Rüdiger
Coronaviren (CoV) sind behüllte, einzelsträngige RNA Viren mit positiver
Orientierung. Sie besitzen drei bis vier Hüllproteine, das Spike-Protein (S), MembranProtein (M), Envelope-Protein (E) und einige Vertreter der Betacoronaviren weisen
das Hämagglutinin-Esterase-Protein auf. Innerhalb des Viruspartikels befindet sich
das Nukleokapsid-Protein, welches mit der RNA assoziiert ist. CoV infizieren
Säugetiere, einschließlich des Menschen, sowie Vögel. Es ist bekannt, dass
Coronaviren vom Tier auf den Menschen, wie auch zwischen verschiedenen
Tierarten übertragen werden können. Seit dem Ausbruch des schweren akuten
Atemwegssyndroms (SARS), ausgelöst durch SARS-CoV, stehen insbesondere
Fledermäuse im Fokus der Forschung, da sie als Reservoir für SARS-CoV, wie auch
für weitere CoV gesehen werden. Das Virus der übertragbaren Gastroenteritis
(TGEV), ein Vertreter der Alphacoronaviren, diente in dieser Studie als ModellCoronavirus. Der Eintritt, die Replikation, sowie das Freisetzen von TGEV wurden in
verschiedenen Fledertier-Zelllinien untersucht. Die Oberflächenexpression des
spezies-spezifischen Rezeptors, der porzinen Aminopeptidase N, war dabei
notwendig um den Eintritt von TGEV in Fledertierzellen zu ermöglichen. Abhängig
von
der
getesteten
Fledertierspezies
wurden
unterschiedliche
Rezeptor-
Expressionslevel, sowie Virustiter gemessen. Des Weiteren wurden verschiedene
Expressionsmuster für das TGEV S- und M-Protein beobachtet. Es konnte gezeigt
werden,
dass
die
Expression
des
spezifischen
Rezeptors
eine
wichtige
Voraussetzung für den viralen Eintritt darstellt. Die kalkulierten Virustiter aus den
Zellkulturüberständen deuten darauf hin, dass weitere Faktoren die Replikation
innerhalb der Fledertierzellen beeinflussen. Betrachtet man die unterschiedlichen
Verteilungen der TGEV S- und M-Proteine während der Infektion, scheinen
Wirtsfaktoren, sowohl abhängig von der Fledermausart als auch vom betroffenen
Organ, eine wichtige Rolle für eine erfolgreiche Coronavirusinfektion zu spielen.
Aus diesem Grund wurden zelluläre Faktoren, welche mit dem CoV S-Protein
interagieren, näher untersucht. Das S-Protein vermittelt das Anheften an den
xiv
Zusammenfassung
Wirtszellrezeptor, sowie den Viruseintritt. Für einige Viren konnte bereits gezeigt
werden, dass Mikrotubuli für den Transport viraler Komponenten innerhalb der
Wirtszelle genutzt werden. In dieser Studie konnte demonstriert werden, dass die
letzten 39 Aminosäuren der zytoplasmatischen Domäne des S-Proteins von TGEV
und den humanen CoV 229E und NL63 mit Tubulin-alpha- und -beta-Ketten
interagieren. Zudem co-lokalisierte das TGEV S-Protein mit β-Tubulinen der
Wirtszelle. Eine
induzierte Depolymerisierung der Mikrotubuli führte zu einer
Änderung in der TGEV S-Protein Verteilung in infizierten Zellen. Hierbei colokalisierten TGEV S-Proteine mit dem ER-Golgi intermediären Kompartment
(ERGIC) und mit dem Golgi-Apparat, wobei diese Kompartimente, nach NocodazoleBehandlung (NOC) verstreut in der Zelle vorlagen. Zusätzlich wurden ein reduzierter
Austritt von infektiösen Viruspartikeln und ein verminderter Einbau der S-Proteine in
die Virushülle detektiert. Diese Ergebnisse demonstrieren, dass eine Interaktion des
CoV S-Proteins mit zellulärem Tubulin den Zusammenbau und die Freisetzung von
Viruspartikeln begünstigt.
Neben der Virus-Wirts-Interaktion beeinflusst auch das Zusammenspiel der viralen
Proteine den Vermehrungszyklus von CoV. Deren Replikation findet im Zytosol der
Wirtszelle statt, wobei der Zusammenbau und die Knospung der Viruspartikel am
ERGIC erfolgen. Protein-Protein, sowie Protein-RNA Interaktionen sind in diesen
Schritten von großer Bedeutung. Das virale S-, M und E-Protein enthalten
Signalsequenzen, welche zur Lokalisation im Bereich der Knospung führen. Dadurch
erfolgt eine Inkorporation dieser Strukturproteine in neugebildete Viruspartikel.
Insbesondere für den Einbau der S-Proteine ist die Interaktion mit dem M-Protein
essentiell.
S-Proteine
bestehen
aus
einer
Ektodomäne,
einer
Transmembrandomäne, sowie einer zytoplasmatischen Domäne, wobei letztere eine
Cystein-reiche und eine ladungsreiche Region besitzt. Zudem weisen einige
Vertreter ein Tyrosin-basiertes Motiv innerhalb der ladungsreichen Region auf,
welches zur intrazellulären Retention oder Akkumulation führen kann. Es ist bekannt,
dass der zytoplasmatische Abschnitt, besonders die ladungsreiche Region des SProteins des Maus Hepatitis Virus, sowie des SARS-CoV bei der Interaktion mit MProteinen eine essentielle Rolle spielt. In dieser Arbeit wurde die zytoplasmatische
Domäne diverser CoV S-Proteine bezüglich des Zusammenspiels mit M-Proteinen
untersucht. Die ladungsreiche Region des TGEV S-Proteins wurde dabei durch
analoge Sequenzen, von humanen CoV und Fledermaus-CoV S-Proteinen
xv
Zusammenfassung
stammend, ersetzt. Hierbei wurden Sequenzen von Vertretern der Gattungen Alphaund Betacoronavirus gewählt. Abhängig von der Aminosäurensequenz der
ladungsreichen
Region
Sortierungssignalen,
wie
beziehungsweise
z.B.
eines
dem
Tyrosin-basierten
Vorhandensein
Signals,
wurden
von
in
Einzelexpression unterschiedliche Expressionsverhalten für die validierten SProteine detektiert. Dennoch war die Interaktion mit dem TGEV M-Protein weder
komplett zerstört noch beeinträchtigt. In den Zellen, die S- und M-Proteine coexprimierten, wurde das S in der Nähe des Zellkerns und der M-Proteine beobachtet.
Die Ergebnisse zeigen, dass ein Aminosäurenaustausch weder im Tyrosin-basierten
Motiv, noch innerhalb der ladungsreichen Region von CoV S-Proteinen die
Interaktion mit M-Proteinen beeinflusst. Zusätzlich wurden drei TGEV M Y/A
Konstrukte, mit mutierten Tyrosinen innerhalb der zytoplasmatischen Domäne,
kloniert und für S-M Interaktionsstudien verwendet. Alle getesteten TGEV M Y/A
Proteine führten zum Rückhalt des S-Proteins in co-exprimierenden Zellen.
Allerdings erschien die S-M Interaktion leicht beeinträchtigt, da im Gegensatz zur CoExpression von TGEV S und TGEV Mwt weniger S-Protein in der Zelle
zurückgehalten wurde.
xvi
Introduction
1 Introduction
1.1 Coronaviruses
1.1.1 Taxonomy
Coronaviruses (CoV) are single-stranded RNA viruses with positive orientation
belonging to the order Nidovirales and the family Coronaviridae (Fig. 1-1). The
subfamily Coronavirinae is divided into four genera: Alpha-, Beta-, Gamma- and
Deltacoronaviruses. Alpha- and Betacoronaviruses infect only mammalian species
ranging from humans to bats, primarily their respiratory and gastrointestinal systems.
Famous members of Alphacoronaviruses are human coronavirus 229E (HCoV 229E)
and human coronavirus NL63 (HCoV NL63), porcine epidemic diarrhea virus (PEDV)
and transmissible gastroenteritis virus (TGEV), as well as feline infectious peritonitis
virus (FIPV), (Fig. 1-2). The severe acute respiratory syndrome coronavirus (SARSCoV),
Middle-East
respiratory
syndrome
coronavirus
(MERS-CoV),
human
coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV) and murine hepatitis
virus (MHV) are well known members of the genus Betacoronavirus (Fig. 1-2).
Gammacoronaviruses like infectious bronchitis virus (IBV) are mainly present in
avian species (Fig. 1-2). Bat coronaviruses seem to be the gene source for the
Alpha- and Beta- genera, whereas Gamma- and Deltacoronaviruses are derived from
avian gene pools (WOO et al. 2012).
Fig. 1-1 Taxonomy of the family Coronaviridae according to the International Committee for
the Taxonomy of Viruses (ICTV), modified (CHAN et al. 2015).
1
Introduction
Fig. 1-2 Phylogenetic tree of several members of coronaviruses, modified (CHAN et al. 2015).
Partial nucleotide sequences of the RNA-dependent RNA polymerase and neighbor-joining method
(MEGA 5.0) were used for constructing this tree. Scale bar displays assessed number of substitutions
per 20 nucleotides.
1.1.2 Coronavirus particle
The virions are about 100-140 nm in diameter (Fig. 1-3). Coronaviruses are
enveloped viruses while the large, petal-shaped spike proteins form a crown (Latin:
corona), which give the family its name (ALMEIDA 1968). Embedded in its lipid
bilayer one can find: the spike protein (S) which forms trimers and is necessary for
receptor binding as well as membrane fusion; the membrane protein (M) with the
highest abundance which is essential for virus assembly; the nucleocapsid protein
2
Introduction
(N) which is associated with the viral RNA to arrange a helical nucleocapsid; the
envelope protein (E) with the lowest abundance is involved in virus assembly and
shows ion channel activity; and the hemagglutinin esterase protein (HE; only found in
some members of Betacoronaviruses) which facilitates virus entry (WENTWORTH &
HOLMES 2007).
Fig. 1-3 Coronavirus particle.
(a) Schematic representation, modified (VABRET et al. 2009); (b) Electron microscopy of severe acute
respiratory syndrome coronavirus, bar: 100 nm, modified (UJIKE & TAGUCHI 2015).
1.1.3 Genome organization
The CoV genome is the largest known for RNA viruses with a size of about 26-32 kb
(Fig. 1-4). The RNA owns a 5’ cap structure and a poly-adenosin (A)-tail at its 3’ end
(YOGO et al. 1977; LAI & STOHLMAN 1981). It contains six to ten open reading
frames (ORFs) depending on the genus. Two-thirds of the CoV genome consists of
ORF1a and ORF1b which encodes for non-structural proteins (nsps). The last third
encodes for structural proteins in a highly conserved 5’ to 3’ order (HE)-S-E-M-N as
well as for accessory proteins (BRIAN & BARIC 2005). The accessory proteins are
strain-specific but assumed to be not essential for coronavirus replication (DE HAAN
et al. 2002; CASAIS et al. 2005; GORBALENYA et al. 2006; HODGSON et al. 2006).
Nevertheless, accessory proteins are involved in virus-host interactions. For
3
Introduction
example, accessory protein 4a of MERS-CoV is identified as type I interferon
antagonist which blocks interferon induction (NIEMEYER et al. 2013). In the case of
FIPV its accessory protein 7a is shown to be a type I interferon antagonist as well,
while ORF 3-encoded proteins have to be present (DEDEURWAERDER et al. 2014).
Furthermore, accessory proteins determine coronavirus virulence like it is known for
ORF 3a of TGEV and porcine respiratory coronavirus (PRCoV) (PAUL et al. 1997). In
the case of IBV, a mutant with a truncated 3b gene is shown to be more virulent in
chicken embryos than wildtype IBV (SHEN et al. 2003).
Fig. 1-4 Coronavirus genome organization, modified (UJIKE & TAGUCHI 2015).
Open reading frame (ORF) 1a & 1b: green; genes encoding for structutal proteins hemagglutininesterase (HE, only for some members of Betacoronaviruses), spike (S), envelope (E), membrane (M),
and nucleocapsid (N) protein: yellow; genes for accessory proteins: red; Transmissible gastroenteritis
virus (TGEV), bovine coronavirus (BCoV), infectious bronchitis virus (IBV), bulbul coronavirus HKU11
(Bul CoV).
1.1.4 Transmissible gastroenteritis coronavirus (TGEV)
The main focus of this work is laying on TGEV, which was used as model
coronavirus. TGEV belongs to the genus Alphacoronavirus. TGE was first described
in 1935 and the pathogen was identified in 1946 (DOYLE & HUTCHINGS 1946;
SMITH 1956). It is an enteropathogenic CoV infecting pigs. Virus uptake starts via
oral route of infectious material such as feces or sow’s milk. Next, villous epithelial
cells of the small intestine become infected, although replication within the respiratory
tract or mammary gland is possible as well. The infection leads to cell necrosis
4
Introduction
followed by villous atrophy, diarrhea and vomiting (SAIF et al. 2012). In two week old
or younger piglets, TGEV infection shows a mortality rate of nearly 100 %, whereas
in adult pigs a rate of only 5 % is found. Due to industrialization, economic losses
have been recorded for Europe and the United States, but since the 1980’s,
outbreaks have declined (ENJUANES & VAN DER ZEIJST 1995). An explanation for
the regression may be the propagation of a natural variant of TGEV that was first
isolated in 1986 from the respiratory tract of infected pigs in Belgium (PENSAERT et
al. 1986). This variant was called porcine respiratory coronavirus (PRCoV) and
shared a 96 % sequence homology with TGEV. In contrast to the TGEV genome,
PRCoV has one deletion in the 5’ end of its S gene and deletions in ORF 3a and/or
ORF 3b (resulting in lack of or truncated protein expression) leading to strong
alterations in virus-host interactions (RASSCHAERT et al. 1990; WESLEY et al.
1991; SANCHEZ et al. 1992; LAUDE et al. 1993; KIM et al. 2000). The PRCoV
deletion in its S gene may be the reason for the altered tissue tropism (WESLEY et
al. 1990a; WESLEY et al. 1990b; WESLEY et al. 1991). In contrast to TGEV, PRCoV
infects mainly the respiratory tract resulting in mild symptoms (COX et al. 1990). Due
to close serological relatedness of both viruses PRCoV infected pigs are naturally
immune to TGEV (CALLEBAUT et al. 1988).
5
Introduction
1.1.5 Coronavirus life cycle
The replication cycle can be divided into the following functional steps: entry,
replication and translation, assembly, and release (Fig. 1-5).
Fig. 1-5 Severe acute respiratory syndrome coronavirus (SARS-CoV) life cycle, modified (DU et
al. 2009).
Angiotensin-converting enzyme 2 (ACE2), viral replicase polyprotein 1a and 1ab (pp1a, pp1ab),
structural proteins (S, E, M, N), accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b, 9b), endoplasmatic
reticulum (ER), ER-Golgi intermediate compartment (ERGIC), Golgi apparatus (Golgi).
6
Introduction
1.1.5.1
Entry
Initially, the viral S protein interacts with host cells by binding to its specific receptor.
Thus, S proteins seem to be a critical determinant for cell tropisms, species
specificity, and virulence (RAO & GALLAGHER 1998; NAVAS et al. 2001; NAVAS &
WEISS 2003). Several receptors have been identified so far. MHV binds to
carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), SARS-CoV and
HCoV NL63 to angiotensin-converting-enzyme-2 (ACE2), whereas MERS-CoV
interacts with dipeptidyl peptidase-4 (DPP4) to enter the host cell (DVEKSLER et al.
1991; BEAUCHEMIN et al. 1999; W. LI et al. 2003; PYRC et al. 2007; RAJ et al.
2013). Most members of the genus Alphacoronavirus like HCoV 229E, FIPV, PEDV,
and TGEV use aminopeptidase N (APN), which is expressed at the apical site of host
epithelial cells in respiratory and intestinal tracts (DELMAS et al. 1992; YEAGER et
al. 1992; DELMAS et al. 1994; TRESNAN et al. 1996; LIU et al. 2015). By receptormediated endocytosis and subsequent fusion of the endosomal membrane with the
viral envelope, the viral nucleocapsid is released into the cytoplasm where replication
takes place (HANSEN et al. 1998; CANN 2005).
1.1.5.2
Replication and translation
The life cycle of CoVs lacks a nuclear stage. Therefore, the virus has to synthesize
and/or recruit all factors which are required for its mRNA synthesis. Additionally, the
virus has to ensure recognition by the host cell translation machinery (ZIEBUHR &
SNIJDER 2007). Initiation of RNA synthesis depends on replicase gene expression.
Translation of 5’-located ORF1a occurs. This strategy allows the expression of all
downstream genes except from replicase ORF1b. The ORF1b expression relies on a
ribosomal frameshift. Resulting polypeptides (pp) 1a and 1ab encode for enzymatic
functions (Papain-like and Poliovirus 3C-like proteases) and polyprotein cleavage
into 15 or 16 nsps occurs (ZIEBUHR et al. 2000; BRIAN & BARIC 2005; ZIEBUHR &
SNIJDER 2007; PERLMAN & NETLAND 2009). Nsps assemble with the viral RNAdependent RNA polymerase and form the replication complex, which synthesizes
full-length negative orientated single-stranded RNA. The viral replication complex
associates with double-membrane vesicles (DMVs) to create a microenvironment
where the genome replication and transcription take place (GOSERT et al. 2002;
SNIJDER et al. 2006). DMVs are hijacked from the endoplasmatic reticulum (ER)
7
Introduction
and protect viral RNA from the host defense mechanisms (GOSERT et al. 2002;
KNOOPS et al. 2008). Regarding TGEV, replication mostly occurs in nucleaseresistant DMVs (SETHNA & BRIAN 1997). Furthermore, a discontinuous
transcription leads to the synthesis of a set of sg RNAs with negative orientation,
which are used as template to synthesize positive-sense mRNA (SAWICKI &
SAWICKI 1998). A second hypothesis implies the generation of sg RNAs during the
synthesis of positive-orientated mRNAs (SAWICKI & SAWICKI 1998). Dominantly,
the 5’ ORF is translated by using the cellular transcription machinery. Hereby, the
translation of host proteins is inhibited during coronaviral infection whereas the
translation rate of viral proteins is increased (KYUWA et al. 1994; TAHARA et al.
1994; BANERJEE et al. 2000; BANERJEE et al. 2002).
1.1.5.3
Assembly and release
Contrary to most other enveloped RNA viruses, coronavirus assembly and budding
take place at the endoplasmatic reticulum-Golgi intermediate compartment (ERGIC),
whereas rhabdoviruses, orthomyxoviruses or retroviruses use the plasma membrane
for this step (TOOZE et al. 1984; KRIJNSE-LOCKER et al. 1994; VENNEMA et al.
1996). Similar to other enveloped viruses, protein localization and protein-protein
interactions are required for successful assembly. Therefore, M, E, and S proteins
contain trafficking signals which result in their targeting or accumulation near the site
of assembly and budding. For IBV, MHV and TGEV M proteins localization at the
Golgi compartment is described (KLUMPERMAN et al. 1994). The IBV E protein is
expressed in high levels near the Golgi complex in infected cells as well (CORSE &
MACHAMER 2000). The TGEV S protein comes with a tyrosine-based retention
signal within its cytoplasmic domain which leads to intracellular retention at the
ERGIC (SCHWEGMANN-WESSELS et al. 2004). The IBV S cytoplasmic tail
contains a canonical dilysine endoplasmic reticulum retrieval signal as well as a
tyrosine motif (LONTOK et al. 2004; WINTER et al. 2008). For an incorporation of
ribonucleoproteins as well as the E and S proteins into virus particles, a heterotypic
interaction with the M protein at the budding site is necessary (OPSTELTEN et al.
1995; NGUYEN & HOGUE 1997; NARAYANAN et al. 2000; RAAMSMAN et al. 2000;
DE HAAN & ROTTIER 2005; MASTERS et al. 2006). N proteins bind to genomic
RNA at multiple regions within the cytoplasm and the helical nucleocapsid is formed
8
Introduction
(HILSER & THOMPSON 2007; TAKEDA et al. 2008; CHANG et al. 2009). At the
budding site, the nucleocapsid may interact with the M protein to trigger the assembly
of the nucleocapsid with the viral envelope and budding (CHANG et al. 2014). By
using the cellular secretory pathway, immature virus particles get transported from
the ERGIC over the Golgi compartment to the plasma membrane. On this exocytotic
way, several maturation processes of the S protein occur such as glycosylation,
proteolysis, and modification of oligosaccharides (STERN & SEFTON 1982;
VENNEMA et al. 1996; SALANUEVA et al. 1999). At the plasma membrane, mature
virions are released in the extracellular space via exocytosis (VENNEMA et al. 1996).
1.1.6 Coronavirus structural proteins of interest
1.1.6.1
Spike (S) protein
The S protein is a membrane glycoprotein type I which forms trimers at the virion’s
surface. With its 21-35 potential N-glycosylation sites, it is highly glycosylated, but
also acetylated (ROTTIER et al. 1981; STERN & SEFTON 1982; DAVID
CAVANAGH 1995). Some coronavirus S proteins like the MHV S or IBV S proteins
are post-translationally cleaved into S1 and S2 subunits at the Golgi compartment
(DE HAAN et al. 2004; YAMADA & LIU 2009). The S1 subunit contains the receptorbinding domain and is essential for binding, while the S2 domain contains a fusion
peptide and two heptad repeat sequences to mediate viral entry and membrane
fusion (LUYTJES et al. 1987; YOO et al. 1991; SUZUKI & TAGUCHI 1996), (Fig.
1-6). The TGEV S protein shows a functional S1 and S2 domain as well.
Nevertheless, a cleavage by host or exogenous proteases is not known yet.
9
Introduction
Fig. 1-6 Schematic representation of coronavirus spike proteins, modified (MOU et al. 2013).
Spike (S) proteins aligned at their S1-S2 junctions, numbers represent amount/position of amino
acids. Ectodomain: blue; transmembrane domain (TM): green; cytoplasmic domain (CD): red;
receptor-binding domain: grey box. Transmissible gastroenteritis virus (TGEV), human coronavirus
NL63 (HCoV NL63), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East
respiratory syndrome coronavirus (MERS-CoV), mouse hepatitis virus (MHV), aminopeptidase N
(APN), angiotensin-converting-enzyme-2 (ACE2), dipeptidyl peptidase-4 (DPP4), N-terminal located
carcinoembryonic antigen cellular adhesion molecule (N-CEACAM).
The TGEV S protein consists of 1,449 amino acids (aa) and has a molecular weight
of about 220 kDa (SPAAN et al. 1988). It can be divided into a large N-terminal
ectodomain (1,387 aa), a single hydrophobic transmembrane domain (20 aa) and a
hydrophilic cytoplasmic domain (40 aa) (GALLAGHER & BUCHMEIER 2001), (Fig.
1-6). TGEV S’s cytoplasmic tail contains a cysteine-rich motif (CRM) and a chargerich region (Fig. 1-7). In the case of the MHV S protein both are partly overlapping.
The CRM is present in all coronaviral S proteins and shows a cysteine content of
about 35 %. For TGEV, MHV and SARS-CoV S, cysteine residues are modified by
palmitic acids, an essential characteristic for S incorporation into virus-like particles
(VLPs) or virions (THORP et al. 2006; C. M. PETIT et al. 2007; GELHAUS et al.
2014). Concerning the TGEV and IBV S charge-rich region, a tyrosine-based
retention signal is located resulting in S intracellular retention (SCHWEGMANNWESSELS et al. 2004; WINTER et al. 2008). In contrast, PEDV contains an ER
retrieval signal involved in intracellular retention (SHIRATO et al. 2011).
Betacoronavirus S proteins lack tyrosine-based retention signals and are transported
to the plasma membrane where they promote cell-to-cell fusion, but when co-
10
Introduction
expressed with the M protein, S is retained near the budding site (OPSTELTEN et al.
1995; MCBRIDE et al. 2007; UJIKE & TAGUCHI 2015).
Furthermore, S proteins are dispensable for virion and VLP formation. If S is present
during virion assembly, then it is incorporated and infectious progeny emerges
(VENNEMA et al. 1996; BAUDOUX et al. 1998; CORSE & MACHAMER 2000;
HUANG et al. 2004). Another premise for S incorporation is the interaction with M
proteins (OPSTELTEN et al. 1995; NGUYEN & HOGUE 1997). Here, the
endodomain of the S protein is recruited by the M protein for virus assembly as
shown for MHV (GODEKE et al. 2000; BOSCH et al. 2005).
Fig. 1-7 C-terminal end of transmissible gastroenteritis coronavirus (TGEV) spike protein.
Amino acid sequence of TGEV spike transmembrane and cytoplasmic domain. Cysteine residues of
the cysteine-rich motif: red, positive charged amino acids (+); negative charged amino acids (-);
tyrosine-based retention signal: green; dibasic ER retrieval signal: purple.
1.1.6.2
Membrane (M) protein
The M protein is a transmembrane protein type III. In the case of TGEV, M consists
of 262 aa and has a molecular weight of approximately 28 kDa. M proteins contain a
short glycosylated ectodomain, three transmembrane domains and a long C-terminal
cytoplasmic tail (HOGUE & MACHAMER 2008). The last one is divided into an
amphipathic and a hydrophobic domain. On its N-terminus, it has a trafficking signal
leading to intracellular retention at different Golgi regions depending on the virus
species (ROTTIER & ROSE 1987; KLUMPERMAN et al. 1994; JACOMINE KRIJNSE
LOCKER et al. 1995). Here, M proteins may interact with each other and are able to
form homomultimeric protein complexes (J. K. LOCKER et al. 1992). The TGEV M
protein exists in two topologies: Nexo-Cendo and Nexo-Cexo. Nexo-Cendo
orientation means that the N-terminus is on the surface of the virion, whereas the Cterminus is inside the virus particle. If both termini are located at the virus particle
surface, it is called Nexo-Cexo orientation (RISCO et al. 1995; ESCORS et al. 2001).
11
Introduction
Regarding its N-terminal ectodomain, all Alpha- and Gammacoronavirus M proteins
are N-glycosylated while all Betacoronavirus M proteins undergo O-glycosylation
(NIEMANN et al. 1984; DE HAAN et al. 1998; OOSTRA et al. 2006). CoV M proteins
play a crucial role in virus assembly and are able to interact with viral S, E and N
proteins (LANSER & HOWARD 1980; STURMAN et al. 1980; OPSTELTEN et al.
1995; NARAYANAN et al. 2000; LIM & LIU 2001). If only M and E proteins are
expressed, nucleocapsid independent assembly takes place and VLPs are formed.
This was shown for MHV, IBV, TGEV, and bovine CoVs (VENNEMA et al. 1996;
BAUDOUX et al. 1998; CORSE & MACHAMER 2000; GELHAUS et al. 2014).
1.1.7 Coronavirus-host interaction
Viruses depend on the host cell machinery to drive their life cycle. For successful
replication viral proteins or their RNA have to interact with cellular and viral
components (SAKAGUCHI et al. 1996; OP DE BEECK & CAILLET-FAUQUET 1997;
KONAN et al. 2003; CHOE et al. 2005; BELOV et al. 2007; BESKE et al. 2007;
MOFFAT et al. 2007; OOSTRA et al. 2007). CoV infection leads to modifications in
transcription and translation pattern, cell cycles, innate immune as well as stress
responses, cytoskeleton, in the machinery of autophagy, and in cell death pathways
of the host cell (L. C. MILLER & FOX 2004; ENJUANES et al. 2006; D. CAVANAGH
2007; DE HAAN & REGGIORI 2008; COTTAM et al. 2011). Therefore, infected host
cells underlay changes in their gene expression pattern – either gene up- or
downregulation – resulting in differently expressed proteins. In ST (swine testis) cells
infected by TGEV 146 proteins show significant alterations in their expression pattern
at 48 hours post-infection (hpi) and even 219 proteins display changes in the
expression at later stages of infection (ZHANG et al. 2013). In MHV infected Hela
cells 116 proteins just within the Golgi-enriched fractions alter their abundance
compared to mock infected cells (VOGELS et al. 2011). Furthermore, alterations in
host gene expression may explain CoV pathologies. For example, in SARS-CoV
infected cells more genes affecting inflammation, stress and coagulation are
upregulated compared to HCoV 229E infected cells (B. S. TANG et al. 2005). SARSCoV infection leads to respiratory failures, whereas HCoV 229E causes only mild
diseases in the upper respiratory tract (ENJUANES et al. 2006). However, CoVs
have evolved several strategies to minimize recognition by the host cell and to
12
Introduction
suppress type I interferon responses. One way is that viral nsp16 creates a 5’-cap for
new synthesized mRNAs which is analogue to the cellular mRNA (LAI et al. 1982;
DECROLY et al. 2008; Y. CHEN et al. 2011; PICARD-JEAN et al. 2013).
Additionally, incoming CoVs may induce stress in host cells such as ER stress.
Infected cells respond to this via translation attenuation or mRNA degradation as well
as increased protein folding and ER membrane expansion (FUNG et al. 2014).
Attenuation of translation and degradation of selected mRNAs serve as a defense
mechanism against CoVs, although some viruses are resistant (BECHILL et al.
2008). Nevertheless, increased protein folding and enhanced appearance of
chaperons as a side effect may support the excessive production of viral proteins
(FUNG et al. 2014). Because CoVs use microenvironments for shielded replication
and transcription, ER membrane expansion supplies additional sources to form
DMVs (GOSERT et al. 2002; SNIJDER et al. 2006; KNOOPS et al. 2008; FUNG et
al. 2014).
Nonetheless, CoVs can affect host cell cycle on several more steps. For example,
after MHV infection, inhibition of the host translation machinery results in an
increased synthesis of viral proteins (KYUWA et al. 1994; TAHARA et al. 1994;
BANERJEE et al. 2000; BANERJEE et al. 2002). Furthermore, after SARS-CoV
infection, a downregulation of host genes involved in translation of cellular proteins
occurs (LEONG et al. 2005). In contrast, IBV is able to boost its replication and
protein production by arresting the host cell cycle at phase S and G2/M (F. Q. LI et
al. 2007). Another mechanism of CoVs to control host cells and to enhance viral
mRNA synthesis is by locating viral proteins within the nucleus. Here, TGEV, MHV
and IBV N proteins have been found in the host nucleus, whereas in case of SARSCoV, nsp 3b is localized there (HISCOX et al. 2001; WURM et al. 2001; H. CHEN et
al. 2002; X. YUAN et al. 2005). However, this strategy seems to be cell dependent.
Regarding TGEV, N proteins have been detected in the nucleus of Vero and LLCPK1 cells, but are not found in ST cells (WURM et al. 2001; CALVO et al. 2005).
Controversially, several host factors positively affect virus life cycle particularly during
the replication and transcription steps (ZHONG et al. 2012). The heterogeneous
nuclear ribonucleoprotein (hnRNP) A1, which is involved in alternative splicing of
cellular RNAs, was found associated with MHV negative-sense leader sequence as
well as with its N protein (H. P. LI et al. 1997; Y. WANG & ZHANG 1999; LUO et al.
2005; HE & SMITH 2009; OKUNOLA & KRAINER 2009). During TGEV RNA
13
Introduction
synthesis, several cellular proteins such as poly(A)-binding protein, hnRNP Q, and
glutamyl-prolyl-tRNA synthetase interact with TGEV 3’end and facilitate virus
infection (GALAN et al. 2009). A zinc finger CCHC-type and RNA-binding motif 1
(MADP1) interacts with 5’ untranslated regions of SARS-CoV and IBV. In the case of
IBV, an increased replication and transcription occurs (TAN et al. 2012). Further
cellular proteins like DEAD box helicase 1 or β-actin interact with IBV nsp14 or M
protein to enhance virus replication or may be involved during assembly and budding
processes (J. WANG et al. 2009; XU et al. 2010). The release of coronavirus
particles is influenced by the association of nsp15 and the host retinoblastoma tumor
suppressor protein (BHARDWAJ et al. 2012).
1.1.7.1
Coronavirus interaction with host cytoskeleton
The cytoskeleton is a complex, three-dimensional network built of filaments and
tubes to maintain cell shape, organelle organization, cell division and movement. It
consists of three major elements: (i) the small microfilaments with 6 nm in diameter
composed of actin; (ii) the medium-sized intermediate filaments with a diameter of 10
nm; and (iii) the large microtubules with 25 nm diameter made of tubulin. These
filaments, as well as accessory proteins, are linked to each other and to cellular
components. Intra- and intercellular communication and signal transduction is also
possible. It is known that animal as well as plant virus particles or virus proteins
interact with cytoskeletal filaments or cytoskeletal components to reach their site of
replication (LUFTIG 1982; RADTKE et al. 2006).
Within eukaryotic cells, actin is the most abundant protein, which is highly conserved
(WINDER & AYSCOUGH 2005; J. WANG et al. 2009). Actin filaments or
microfilaments form a flexible network underneath the plasma membrane and
interact with the membrane via the help of several actin-binding proteins (SCHLIWA
1981; WEATHERBEE 1981). They provide structure and motility in animal cells, play
a role during endocytosis, cytokinesis, transport of organelles, and locomotion
(RIDLEY et al. 2003; DOHERTY & MCMAHON 2009; POLLARD & COOPER 2009).
For vaccinia virus, an association of virus particles with microfilaments was observed
as well as a reorganization of actin filaments when infected by poliovirus (STOKES
1976; HILLER et al. 1979; LENK & PENMAN 1979). Japanese encephalitis virus and
West Nile virus entry require actin filaments (J. J. CHU & NG 2004; HENRY SUM
14
Introduction
2015). Within the coronavirus life cycle, actin filaments and actin associated proteins
play a crucial role as well. In FIPV infected monocytes, myosin light chain kinase and
myosin 1 are decisive during virus internalization. For the IBV M protein, an
interaction with β-actin is suggested to promote virus assembly and budding (J.
WANG et al. 2009; DEWERCHIN et al. 2014). Regarding TGEV and PEDV virus
attachment, internalization, nuclear targeting, transport of virus progeny as well as
their release is supported by actin filaments (ZHAO et al. 2014).
The wavy intermediate filaments form cage-like structures (LUFTIG 1982). Their
main function is to maintain separate compartments within the cells as well as to
integrate cytoplasmic organelles (LAZARIDES 1980; LUFTIG 1982). In the case of
intermediate filament components, 5 different protein classes are known: vimentin,
prekeratin and desmin found in epithelial or muscle cells as well as neuro- and glia
filaments found in neurons and glia cells (LUFTIG 1982). Reovirus infection causes
disruption as well as reorganization of vimentin filaments (SHARPE et al. 1982). In
HeLa cells infected by poliovirus, a rearrangement of intermediate filaments is
observed (LENK & PENMAN 1979). Vimentin plays a critical role in virus entry of
human cytomegalovirus (M. S. MILLER & HERTEL 2009), Japanese encephalitis
virus (LIANG et al. 2011), and cowpea mosaic virus (KOUDELKA et al. 2009).
Interaction between vimentin and virus outer capsid protein VP2 of bluetongue virus
or vimentin and nonstructural protein 1 of dengue virus is also essential for virus
replication and/or egress (BHATTACHARYA et al. 2007; KANLAYA et al. 2010).
Nevertheless, during TGEV replication, vimentin is required as well. Thus, an
interaction of TGEV N protein with vimentin can be detected (ZHANG et al. 2015).
Microtubules are dynamic, polarized, cylindrical tubes with microtubule-associated
proteins (MAPs) as sidearms (LUFTIG 1982). Their protofilaments consist of globular
α- and β-tubulins, which form stable heterodimers with extensive noncovalent
bindings to adjacent dimers. Approximately 13 connected protofilaments form a
helical microtubule (Fig. 1-8). Binding, hydrolysis or exchange of GFP on β-tubulin
monomers result in polymerization (assembly) or depolymerization (disassembly) of
microtubules (DRÁBER & DRÁBEROVÁ 2012). The main functions of microtubules
are external cell movement as well as intracellular transport of vesicles and
organelles (LUFTIG 1982).
15
Introduction
Fig. 1-8 Schematic drawing of microtubules and actin filaments and their corresponding
molecular motors, modified (GAUDIN et al. 2013).
Arrows show direction of cargo transport. Kinesin moves towards microtubule plus-end and dynein
towards microtubule minus-end.
Several viruses like adenovirus type 2 and 5, reovirus type 1 and 3, murine leukemia
virus, and herpes simplex virus are known to associate with microtubules among
others for nuclear targeting during viral entry or successful virus assembly (DALES &
CHARDONNET 1973; BABISS et al. 1979; MILES et al. 1980; SATAKE et al. 1981;
MABIT et al. 2002). However, the cytoplasm is extremely crowded and free
movement of virus-sized particles is strongly restricted. Thus, viruses can utilize the
microtubules as trails to get to their site of replication (LEOPOLD & PFISTER 2006).
Microtubule associated motor proteins like dynein transport cargos towards the
minus end, generally from the cell periphery to the cell center (SHARP et al. 2000;
VALLEE et al. 2004). In contrast, kinesin moves cargos towards the plus end,
generally from the cell center to the cell periphery (HIROKAWA & TAKEMURA
2005). Dynein interacts with several herpes simplex virus 1 proteins, such as its
nuclear or capsid protein and its helicase protein (YE et al. 2000; MARTINEZMORENO et al. 2003). African swine fever protein 54, human immunodeficiency
virus integrase, rabies virus phosphoprotein or foamy virus Gag protein directly
interact with dynein (RAUX et al. 2000; ALONSO et al. 2001; POISSON et al. 2001;
RODRIGUEZ-CRESPO et al. 2001; DE SOULTRAIT et al. 2002; C. PETIT et al.
2003). A direct interaction of kinesin with viral proteins of herpes simplex virus 1 and
vaccinia
virus has been described as well
(DIEFENBACH et al. 2002;
BENBOUDJEMA et al. 2003; WARD & MOSS 2004; KOSHIZUKA et al. 2005). In
addition, some representative members of CoVs also utilize microtubules for their
16
Introduction
own advantage. During the FIPV replication cycle, its internalized vesicles switch
tracks from actin and move via microtubules towards the microtubule organizing
center (DEWERCHIN et al. 2014). MHV replication as well as neuronal transport of
viral proteins depend on microtubules. Furthermore, protein sequence mimicry of the
MHV nucleocapsid protein and the microtubule-associated protein tau has also been
noticed (PASICK et al. 1994; BISWAS & DAS SARMA 2014). Proteome profile
analysis of TGEV infected ST cells identified a tubulin beta-2B chain as one of the
differentially expressed proteins versus mock infected cells (ZHANG et al. 2013).
1.2 Chiroptera
The order Chiroptera belongs to the class Mammalia and is subdivided into
Microchiroptera (echolocating bats) and Megachiroptera (Old World fruit bats and
flying foxes). These probably originated 52-50 million years ago in the early Eocene
era. This possibly was the result of increasing temperatures, plant diversity and
abundance, and high insect diversity (SIMMONS 2005; TEELING et al. 2005). About
20 % of all mammalian species are represented by bats (over 930) and they are the
second largest order beside rodents (order Rodentia) (VAN DER POEL et al. 2006;
WONG et al. 2007). Chiropterans are the only mammals that have the ability to
actively fly. They exist worldwide apart from Arctic, Antarctic, and some oceanic
islands. Chiropterans show a high diversity in biology and ecology. Their diet varies
from plants, insects, small mammals, fish, and blood, although the latter is unique
among mammals (CALISHER et al. 2006; WONG et al. 2007). While searching for
food, they are able to cover distances of up to 10-18 km and Megachiroptera reach
up to 50 km (NEUWEILER 2000). Hibernation (not in case of megachiropterans) as
well as migration was determined during winter. Here, a migration distance ranging
from 200 km to nearly 2000 km has been recorded (FLEMING & EBY 2003).
Although chiropterans are quiet small mammals, they develop slowly and may reach
a relatively long life span of more than 30 years (CALISHER et al. 2006). Most of
these animals are nocturnal and stay in roosts during the day such as caves, rock
crevices, trees or man-made habitats including abandoned mines, bridges or
buildings. Bats live in colonies from less than 10 to more than 200,000 individuals
(KUNZ & LUMSDEN 2003). Several bat species may share their roost, which
facilitates
virus
transmission
between
17
different
bat
species.
Regarding
Introduction
Alphacoronaviruses, an interspecies transmission between two different bat
suborders is known (LAU et al. 2012).
Thus, due to their great species diversity, worldwide distribution, ability to fly as well
as their population size and density, chiropterans represent a high potential reservoir
for animal and human pathogens as well as a high risk factor for zoonotic
transmission (CALISHER et al. 2006). Presently, more than 80 different viruses from
the family Rhabdoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae,
Flaviviridae, Arenaviridae, Herpesviridae, Picornaviridea, etc. have been isolated
from bats (CALISHER et al. 2006). Bats rarely show clinical symptoms, although they
may carry a huge amount of pathogens (whether naturally or experimentally infected)
(SULKIN et al. 1966; SWANEPOEL et al. 1996; WILLIAMSON et al. 1998;
WILLIAMSON et al. 2000; LEROY et al. 2005; MIDDLETON et al. 2007; LEROY et
al. 2009; TOWNER et al. 2009). Only the rabies virus and Australien bat lyssavirus
display clinical signs in bats (FIELD et al. 1999; MCCOLL et al. 2002). These findings
suggest that a coexistence of bats and viruses lead to more efficient control
mechanisms of viral replication compared to most other mammalian species.
Knowledge about the immune system of chiropterans is incomplete. However, they
are known to show components as reported for the mammalian immune complex.
Macrophages, B cells as well as T cells with similar characteristics in mice and
humans have been identified in lymph nodes and spleen of Pteropus giganteus
(SARKAR & CHAKRAVARTY 1991). Lymphocytes, neutrophils, basophils, and
eosinophils have been detected in Tadarida brasiliensis (TURMELLE et al. 2010). In
the case of Pteropus alecto and Rousettus leschenaultii, toll-like receptors have been
described (IHA et al. 2009; COWLED et al. 2011). Furthermore, different
immunoglobulins like IgM, IgG, and IgA are transcribed in bats, which are
homologues to the corresponding human ones (MCMURRAY et al. 1982;
CHAKRAVARTY & SARKAR 1994; BUTLER et al. 2011). In wild bats, neutralizing
antibodies to ebola virus, Hendra virus, and SARS-like CoV have been observed
(HALPIN et al. 2000; LAU et al. 2005; LEROY et al. 2005). In Rousettus leschenaultii
cytokine genes have been identified similar to interleukin and tumor necrosis factor
(IHA et al. 2010).
18
Introduction
1.2.1 Zoonosis and virus transmission from chiropterans to humans
Zoonosis means any infection or disease that is naturally transmitted from animals to
humans or/and vice versa (WHO 2015). This can be the transmission of bacteria,
parasites or viruses. About 75 % of emerging infectious diseases constitute zoonotic
and over 50 % of human pathogens can infect other vertebrates as well. A spill-over
event from a wildlife animal to humans can either occur directly or indirectly via
domestic animals (TAYLOR et al. 2001). There is growing opportunity for
transmission events due to an increased contact between humans and domestic or
wildlife animals. Reasons for this include global travel and trade, agricultural
expansion, deforestation, urbanization, and habit fragmentation (DASZAK et al.
2000, 2001; TAYLOR et al. 2001).
RNA viruses like CoVs are extremely variable and mutable because they have only a
few or no proofreading mechanisms which lead to mutations, recombination or reassortment. These events lead to alterations of viral phenotypes and may support the
virus during interspecies transmission or the adaptation to new ecological niches (LAI
& CAVANAGH 1997; HERREWEGH et al. 1998; WEBBY et al. 2004; WOO et al.
2006; WOO et al. 2009). One important determinant for this viral host range is the
receptor-binding-domain (RBD) of CoV S proteins which interfaces with speciesspecific receptors (GRAHAM & BARIC 2010). Studies with MHV display rapid
accumulation of mutations within the S protein during virus persistence (BARIC et al.
1999). An alteration or enhancement of S protein affinity for the receptor may occur,
facilitating the use of orthologous receptor molecules expressed in a different host
species (W. LI et al. 2003; W. LI et al. 2005b; SHEAHAN et al. 2008; GRAHAM &
BARIC 2010). A well-known orthologous receptor within the genus Alphacoronavirus
is APN (TUSELL et al. 2007). In addition, a gene acquisition within the RBD may
result in recognition of a different receptor or co-receptor favoring cross-species
transmission (GRAHAM & BARIC 2010). Regardless, after successful entry the virus
must overcome several more hurdles to efficiently replicate in and emerge from new
hosts. In conjunction, all steps during virus life cycle as well as the immune system of
the host constitute species barriers (HENGEL et al. 2005; SONG et al. 2005;
GARCIA-SASTRE & BIRON 2006; TARENDEAU et al. 2008).
Several human pathogens are believed to have their reservoir in chiropterans. For
example, flying foxes carry filoviruses without showing any clinical symptoms like
hemorrhagic fever in humans (LEROY et al. 2005; BIEK et al. 2006; TOWNER et al.
19
Introduction
2007). Regarding Hendra and Nipah viruses, flying foxes have been identified as
their natural reservoir as well (ENSERINK 2000; YOB et al. 2001; REYNES et al.
2005; HALPIN et al. 2011; RAHMAN et al. 2012; YADAV et al. 2012). The virus
reservoir for SARS-CoVs was found in Chinese horseshoe bats (Rhinolophus sp.)
(W. LI et al. 2005a; GE et al. 2013) and for MERS-CoV bats are implicated as a
reservoir (MEMISH et al. 2013; BARLAN et al. 2014; Y. YANG et al. 2014).
Virus transmission from chiropteran to human can directly occur via bites or
scratches. Desmodus rotundus is known to feed on humans in Latin America, but
only if there are no alternative animal hosts (SCHNEIDER et al. 2001; GONCALVES
et al. 2002). Further transmission routes can be through inhalation of infectious
aerosols of bat secretions or guano as well as transmission via arthropod vectors like
sand flies or mosquitoes which feed on bats and humans (CROSS et al. 1971;
LAMPO et al. 2000; WONG et al. 2007). Chiropterans in Guam, Africa and parts of
Asia, especially flying foxes, are consumed as bush meat (MICKLEBURGH et al.
2009; HAYMAN et al. 2010; KAMINS et al. 2011; WEISS et al. 2012). During the
process of catching and slaughtering the creatures, handlers may come in contact
with the blood or body fluids of the chiropterans by getting scratched or bitten by
them. Other humans may also get infected by eating insufficiently cooked bush meat
(WONG et al. 2007). Another route of virus transmission from chiropterans to
humans may include a secondary vertebrate called an amplifying host. During the
Nipah virus outbreak in Southeast Asia, pigs were identified as an intermediate host,
whereas the Hendra viruses used horses as secondary hosts (CHUA et al. 2000;
WONG et al. 2007). Further, it has been postulated that in the case of SARS-CoV
palm civets can serve as an amplifying host (GUAN et al. 2003; TU et al. 2004; L. F.
WANG & EATON 2007).
1.2.2 Coronaviruses detected in chiropterans
The presence of CoVs in bats seems to be worldwide because they are found in bats
in North Amerika, Africa, Europe, and Asia (D. K. CHU et al. 2006; X. C. TANG et al.
2006; WOO et al. 2006; DOMINGUEZ et al. 2007; LAU et al. 2007; MULLER et al.
2007; GLOZA-RAUSCH et al. 2008). Several viruses of the genus Alpha- and
Betacoronavirus have been detected in bats by PCR targeting fragments of the
ORF1. However, amplicon sizes range from 121 to 404 bp, which is relatively small
20
Introduction
and hinder reconstruction of CoV phylogenetic trees (MOES et al. 2005; DE SOUZA
LUNA et al. 2007; TONG et al. 2009; DA SILVA FILHO et al. 2012; ANTHONY et al.
2013; DREXLER et al. 2014). Only a few full genome sequences of bat CoVs are
characterized like HKU2 (LAU et al. 2007), HKU4 (X. C. TANG et al. 2006; WOO et
al. 2007), HKU5 (WOO et al. 2007), HKU8 (D. K. CHU et al. 2008), HKU9 (WOO et
al. 2007), HKU10 (LAU et al. 2012), and other SARS-related CoV (LAU et al. 2005;
W. LI et al. 2005a; REN et al. 2006; X. C. TANG et al. 2006; DREXLER et al. 2010;
LAU et al. 2010a; J. YUAN et al. 2010; L. YANG et al. 2013). Unfortunately, less
success of CoV isolation from bats makes genomic characterization even more
difficult (DREXLER et al. 2014). Phylogenetic relationships between bats and CoVs
are illustrated in Fig. 1-9. Regarding red-marked bat CoVs, a large number of deep
branches highlights the association of Alpha- and Betacoronaviruses with bat hosts
(DREXLER et al. 2014), (Fig. 1-9). Therefore, many mammalian CoVs appear to
have originated from CoV ancestors which infected bats (SHI & HU 2008; WOO et al.
2009; LIU et al. 2015). In 11 out of 18 extant bat families, CoVs have been detected.
Some bat families were infected only by Alphacoronaviruses (Megadermatidae),
some by Betacoronaviruses (Nycteridae and Mormoopidae), whereas in 8 out of 11
families, both genera have been identified (SIMMONS 2005; DREXLER et al. 2014).
High CoV loads are found in bat feces (DREXLER et al. 2011; ANNAN et al. 2013)
as well as in the small and large intestine of frugivorous bats (WATANABE et al.
2010). These findings indicate the occurrence of CoV replication within the bat
enteric tract (DREXLER et al. 2014). It is supposed that CoVs detected in bats are
bat genus/species specific but one bat genus/species harboring more than one type
of CoVs is possible (WOO et al. 2006). Furthermore, within one bat species located
at different geographical regions (about 1.600 km apart from each other), similar
CoVs have been found to be circulating (X. C. TANG et al. 2006). This may be due to
migration habits of these particular bats (X. C. TANG et al. 2006). Several studies
have shown CoVs in bats year-round, indicating a long-term persistence of these
viruses as it is known for MHV (W. CHEN & BARIC 1996; SCHICKLI et al. 1997; LAU
et al. 2005; W. LI et al. 2005a; POON et al. 2005; X. C. TANG et al. 2006).
Interestingly, high rates of antibodies against CoVs have been observed in bats
although it is unknown if these loads are due to the severity of infection (LAU et al.
2005; MULLER et al. 2007; LAU et al. 2010b; TSUDA et al. 2012; DREXLER et al.
2014).
21
Introduction
Fig. 1-9 Phylogenetic relations between bat hosts and coronaviruses, modified (DREXLER et
al. 2014).
ICTV coronavirus species are shown on the right to the clades. Designations of the viruses contain
strain name, GenBank accession number and host information. Alphacoronaviruses are shown in the
blue box, Betacoronaviruses in the green box. Bat coronaviruses are marked in red. Bat pictograms
represent viruses only found in bats.
22
Introduction
1.3 Aims of the study
During the last years, new human CoVs emerged, resulting in outbreaks of severe
respiratory diseases (like SARS and MERS). Both virus species – responsible for
SARS and MERS – originated from animal hosts. Thus, mechanisms of the species
transfer from animals to humans are important to investigate. The CoV life cycle is a
complex and not fully understood topic. Having more detailed information about CoV
replication strategies and involved viral and cellular factors would be an achievement
in the field of host-CoV interaction.
This thesis is divided into three parts helping to understand mechanisms of entry,
assembly and release of Alphacoronaviruses in mammalian cell lines. The first
section focuses on TGEV entry into chiropteran cells and the release of infectious
virus particles:
1a) Are chiropteran cell lines susceptible for TGEV infection?
1b) Is TGEV able to successfully replicate within chiropteran cell lines
resulting in the release of infectious progeny?
Within the second part cellular components involved in Alphacoronavirus life cycle
are investigated. The main focus lays on the interaction between CoV S proteins and
host factors:
2a) Which
host
factors
interact
with
the
cytoplasmic
domain
of
Alphacoronavirus S proteins?
2b) Does the tyrosine-based signal within the cytoplasmic tail of the S protein
influence the interaction?
2c) Characterization of identified host interaction partners
The third study emphasizes virus-virus interaction. Here the interaction between CoV
S and M proteins are examined:
3a) Is the cytoplasmic domain of CoV S proteins crucial for the interaction with
the M protein?
3b) Does a tyrosine-based motif influence this protein association?
3c) Are the tyrosines existing in the TGEV M cytoplasmic domain responsible
for S retention?
23
Introduction
1.4 References
ALMEIDA, J. D. B., D.M.; CUNNINGHAM, C.H.; HAMRE, D.; HOFSTADT, M.S.; MALLUCCI, L.;
MCINTOSH, K. (1968): Virology - Coronaviruses. Nature 220, 650
ALONSO, C., J. MISKIN, B. HERNAEZ, P. FERNANDEZ-ZAPATERO, L. SOTO, C. CANTO, I.
RODRIGUEZ-CRESPO, L. DIXON & J. M. ESCRIBANO (2001): African swine fever virus protein
p54 interacts with the microtubular motor complex through direct binding to light-chain dynein.
J Virol 75, 9819-9827
ANNAN, A., H. J. BALDWIN, V. M. CORMAN, S. M. KLOSE, M. OWUSU, E. E. NKRUMAH, E. K.
BADU, P. ANTI, O. AGBENYEGA, B. MEYER, S. OPPONG, Y. A. SARKODIE, E. K. KALKO, P. H.
LINA, E. V. GODLEVSKA, C. REUSKEN, A. SEEBENS, F. GLOZA-RAUSCH, P. VALLO, M.
TSCHAPKA, C. DROSTEN & J. F. DREXLER (2013): Human betacoronavirus 2c EMC/2012related viruses in bats, Ghana and Europe. Emerg Infect Dis 19, 456-459
ANTHONY, S. J., R. OJEDA-FLORES, O. RICO-CHAVEZ, I. NAVARRETE-MACIAS, C. M.
ZAMBRANA-TORRELIO, M. K. ROSTAL, J. H. EPSTEIN, T. TIPPS, E. LIANG, M. SANCHEZ-LEON,
J. SOTOMAYOR-BONILLA, A. A. AGUIRRE, R. AVILA-FLORES, R. A. MEDELLIN, T. GOLDSTEIN,
G. SUZAN, P. DASZAK & W. I. LIPKIN (2013): Coronaviruses in bats from Mexico. J Gen Virol 94,
1028-1038
BABISS, L. E., R. B. LUFTIG, J. A. WEATHERBEE, R. R. WEIHING, U. R. RAY & B. N. FIELDS
(1979): Reovirus serotypes 1 and 3 differ in their in vitro association with microtubules. J Virol
30, 863-874
BANERJEE, S., S. AN, A. ZHOU, R. H. SILVERMAN & S. MAKINO (2000): RNase L-independent
specific 28S rRNA cleavage in murine coronavirus-infected cells. J Virol 74, 8793-8802
BANERJEE, S., K. NARAYANAN, T. MIZUTANI & S. MAKINO (2002): Murine coronavirus
replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6
production and virus replication in cultured cells. J Virol 76, 5937-5948
BARIC, R. S., E. SULLIVAN, L. HENSLEY, B. YOUNT & W. CHEN (1999): Persistent infection
promotes cross-species transmissibility of mouse hepatitis virus. J Virol 73, 638-649
BARLAN, A., J. ZHAO, M. K. SARKAR, K. LI, P. B. MCCRAY, JR., S. PERLMAN & T. GALLAGHER
(2014): Receptor variation and susceptibility to Middle East respiratory syndrome coronavirus
infection. J Virol 88, 4953-4961
BAUDOUX, P., C. CARRAT, L. BESNARDEAU, B. CHARLEY & H. LAUDE (1998): Coronavirus
pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis
by leukocytes. J Virol 72, 8636-8643
BEAUCHEMIN, N., P. DRABER, G. DVEKSLER, P. GOLD, S. GRAY-OWEN, F. GRUNERT, S.
HAMMARSTROM, K. V. HOLMES, A. KARLSSON, M. KUROKI, S. H. LIN, L. LUCKA, S. M. NAJJAR,
M. NEUMAIER, B. OBRINK, J. E. SHIVELY, K. M. SKUBITZ, C. P. STANNERS, P. THOMAS, J. A.
THOMPSON, M. VIRJI, S. VON KLEIST, C. WAGENER, S. WATT & W. ZIMMERMANN (1999):
Redefined nomenclature for members of the carcinoembryonic antigen family. Exp Cell Res
252, 243-249
BECHILL, J., Z. CHEN, J. W. BREWER & S. C. BAKER (2008): Coronavirus infection modulates
the unfolded protein response and mediates sustained translational repression. J Virol 82,
4492-4501
BELOV, G. A., N. ALTAN-BONNET, G. KOVTUNOVYCH, C. L. JACKSON, J. LIPPINCOTTSCHWARTZ & E. EHRENFELD (2007): Hijacking components of the cellular secretory pathway
for replication of poliovirus RNA. J Virol 81, 558-567
24
Introduction
BENBOUDJEMA, L., M. MULVEY, Y. GAO, S. W. PIMPLIKAR & I. MOHR (2003): Association of
the herpes simplex virus type 1 Us11 gene product with the cellular kinesin light-chain-related
protein PAT1 results in the redistribution of both polypeptides. J Virol 77, 9192-9203
BESKE, O., M. REICHELT, M. P. TAYLOR, K. KIRKEGAARD & R. ANDINO (2007): Poliovirus
infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of
the Golgi complex. J Cell Sci 120, 3207-3218
BHARDWAJ, K., P. LIU, J. L. LEIBOWITZ & C. C. KAO (2012): The coronavirus endoribonuclease
Nsp15 interacts with retinoblastoma tumor suppressor protein. J Virol 86, 4294-4304
BHATTACHARYA, B., R. J. NOAD & P. ROY (2007): Interaction between Bluetongue virus outer
capsid protein VP2 and vimentin is necessary for virus egress. Virol J 4, 7
BIEK, R., P. D. WALSH, E. M. LEROY & L. A. REAL (2006): Recent common ancestry of Ebola
Zaire virus found in a bat reservoir. PLoS Pathog 2, e90
BISWAS, K. & J. DAS SARMA (2014): Effect of microtubule disruption on neuronal spread and
replication of demyelinating and nondemyelinating strains of mouse hepatitis virus in vitro. J
Virol 88, 3043-3047
BOSCH, B. J., C. A. DE HAAN, S. L. SMITS & P. J. ROTTIER (2005): Spike protein assembly into
the coronavirion: exploring the limits of its sequence requirements. Virology 334, 306-318
BRIAN, D. A. & R. S. BARIC (2005): Coronavirus genome structure and replication. Curr Top
Microbiol Immunol 287, 1-30
BUTLER, J. E., N. WERTZ, Y. ZHAO, S. ZHANG, Y. BAO, S. BRATSCH, T. H. KUNZ, J. O.
WHITAKER, JR. & T. SCHOUNTZ (2011): The two suborders of chiropterans have the canonical
heavy-chain immunoglobulin (Ig) gene repertoire of eutherian mammals. Dev Comp Immunol 35,
273-284
CALISHER, C. H., J. E. CHILDS, H. E. FIELD, K. V. HOLMES & T. SCHOUNTZ (2006): Bats:
important reservoir hosts of emerging viruses. Clin Microbiol Rev 19, 531-545
CALLEBAUT, P., I. CORREA, M. PENSAERT, G. JIMENEZ & L. ENJUANES (1988): Antigenic
differentiation between transmissible gastroenteritis virus of swine and a related porcine
respiratory coronavirus. J Gen Virol 69 ( Pt 7), 1725-1730
CALVO, E., D. ESCORS, J. A. LOPEZ, J. M. GONZALEZ, A. ALVAREZ, E. ARZA & L. ENJUANES
(2005): Phosphorylation and subcellular localization of transmissible gastroenteritis virus
nucleocapsid protein in infected cells. J Gen Virol 86, 2255-2267
CANN, A. (2005): Principles of Molecular Virology. Elsevier Academic Press, Waltham, USA,
CASAIS, R., M. DAVIES, D. CAVANAGH & P. BRITTON (2005): Gene 5 of the avian coronavirus
infectious bronchitis virus is not essential for replication. J Virol 79, 8065-8078
CAVANAGH, D. (1995): The Coronavirus Surface Glycoprotein. In: S. SIDDELL "The
Coronaviridae" Plenum Press, New York, pp. 73-113
CAVANAGH, D. (2007): Coronavirus avian infectious bronchitis virus. Vet Res 38, 281-297
CHAKRAVARTY, A. K. &
S. K. SARKAR (1994): Immunofluorescence analysis of
immunoglobulin bearing lymphocytes in the Indian fruit bat: Pteropus giganteus. Lymphology
27, 97-104
CHAN, J. F., S. K. LAU, K. K. TO, V. C. CHENG, P. C. WOO & K. Y. YUEN (2015): Middle East
respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like
disease. Clin Microbiol Rev 28, 465-522
25
Introduction
CHANG, C. K., M. H. HOU, C. F. CHANG, C. D. HSIAO & T. H. HUANG (2014): The SARS
coronavirus nucleocapsid protein--forms and functions. Antiviral Res 103, 39-50
CHANG, C. K., Y. L. HSU, Y. H. CHANG, F. A. CHAO, M. C. WU, Y. S. HUANG, C. K. HU & T. H.
HUANG (2009): Multiple nucleic acid binding sites and intrinsic disorder of severe acute
respiratory syndrome coronavirus nucleocapsid protein: implications for ribonucleocapsid
protein packaging. J Virol 83, 2255-2264
CHEN, H., T. WURM, P. BRITTON, G. BROOKS & J. A. HISCOX (2002): Interaction of the
coronavirus nucleoprotein with nucleolar antigens and the host cell. J Virol 76, 5233-5250
CHEN, W. & R. S. BARIC (1996): Molecular anatomy of mouse hepatitis virus persistence:
coevolution of increased host cell resistance and virus virulence. J Virol 70, 3947-3960
CHEN, Y., C. SU, M. KE, X. JIN, L. XU, Z. ZHANG, A. WU, Y. SUN, Z. YANG, P. TIEN, T. AHOLA, Y.
LIANG, X. LIU & D. GUO (2011): Biochemical and structural insights into the mechanisms of
SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex. PLoS Pathog
7, e1002294
CHOE, S. S., D. A. DODD & K. KIRKEGAARD (2005): Inhibition of cellular protein secretion by
picornaviral 3A proteins. Virology 337, 18-29
CHU, D. K., J. S. PEIRIS, H. CHEN, Y. GUAN & L. L. POON (2008): Genomic characterizations of
bat coronaviruses (1A, 1B and HKU8) and evidence for co-infections in Miniopterus bats. J Gen
Virol 89, 1282-1287
CHU, D. K., L. L. POON, K. H. CHAN, H. CHEN, Y. GUAN, K. Y. YUEN & J. S. PEIRIS (2006):
Coronaviruses in bent-winged bats (Miniopterus spp.). J Gen Virol 87, 2461-2466
CHU, J. J. & M. L. NG (2004): Infectious entry of West Nile virus occurs through a clathrinmediated endocytic pathway. J Virol 78, 10543-10555
CHUA, K. B., W. J. BELLINI, P. A. ROTA, B. H. HARCOURT, A. TAMIN, S. K. LAM, T. G. KSIAZEK,
P. E. ROLLIN, S. R. ZAKI, W. SHIEH, C. S. GOLDSMITH, D. J. GUBLER, J. T. ROEHRIG, B.
EATON, A. R. GOULD, J. OLSON, H. FIELD, P. DANIELS, A. E. LING, C. J. PETERS, L. J.
ANDERSON & B. W. MAHY (2000): Nipah virus: a recently emergent deadly paramyxovirus.
Science 288, 1432-1435
CORSE, E. & C. E. MACHAMER (2000): Infectious bronchitis virus E protein is targeted to the
Golgi complex and directs release of virus-like particles. J Virol 74, 4319-4326
COTTAM, E. M., H. J. MAIER, M. MANIFAVA, L. C. VAUX, P. CHANDRA-SCHOENFELDER, W.
GERNER, P. BRITTON, N. T. KTISTAKIS & T. WILEMAN (2011): Coronavirus nsp6 proteins
generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate.
Autophagy 7, 1335-1347
COWLED, C., M. BAKER, M. TACHEDJIAN, P. ZHOU, D. BULACH & L. F. WANG (2011): Molecular
characterisation of Toll-like receptors in the black flying fox Pteropus alecto. Dev Comp
Immunol 35, 7-18
COX, E., J. HOOYBERGHS & M. B. PENSAERT (1990): Sites of replication of a porcine
respiratory coronavirus related to transmissible gastroenteritis virus. Res Vet Sci 48, 165-169
CROSS, J. H., J. C. LIEN, W. C. HUANG, S. C. LIEN & S. F. CHIU (1971): Japanese encephalitis
virus surveillance in Taiwan. II. Isolations from mosquitoes and bats in Taipei area 1969-1970.
Taiwan Yi Xue Hui Za Zhi 70, 681-686
DA SILVA FILHO, L. V., R. M. ZERBINATI, A. F. TATENO, L. V. BOAS, M. B. DE ALMEIDA, J. E.
LEVI, J. F. DREXLER, C. DROSTEN & C. S. PANNUTI (2012): The differential clinical impact of
human coronavirus species in children with cystic fibrosis. J Infect Dis 206, 384-388
26
Introduction
DALES, S. & Y. CHARDONNET (1973): Early events in the interaction of adenoviruses with
HeLa cells. IV. Association with microtubules and the nuclear pore complex during vectorial
movement of the inoculum. Virology 56, 465-483
DASZAK, P., A. A. CUNNINGHAM & A. D. HYATT (2000): Emerging infectious diseases of
wildlife--threats to biodiversity and human health. Science 287, 443-449
DASZAK, P., A. A. CUNNINGHAM & A. D. HYATT (2001): Anthropogenic environmental change
and the emergence of infectious diseases in wildlife. Acta tropica 78, 103-116
DE HAAN, C. A., P. S. MASTERS, X. SHEN, S. WEISS & P. J. ROTTIER (2002): The groupspecific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is
attenuating in the natural host. Virology 296, 177-189
DE HAAN, C. A. & F. REGGIORI (2008): Are nidoviruses hijacking the autophagy machinery?
Autophagy 4, 276-279
DE HAAN, C. A., P. ROESTENBERG, M. DE WIT, A. A. DE VRIES, T. NILSSON, H. VENNEMA & P.
J. ROTTIER (1998): Structural requirements for O-glycosylation of the mouse hepatitis virus
membrane protein. J Biol Chem 273, 29905-29914
DE HAAN, C. A. & P. J. ROTTIER (2005): Molecular interactions in the assembly of
coronaviruses. Adv Virus Res 64, 165-230
DE HAAN, C. A., K. STADLER, G. J. GODEKE, B. J. BOSCH & P. J. ROTTIER (2004): Cleavage
inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but
not virus-cell fusion. J Virol 78, 6048-6054
DE SOULTRAIT, V. R., A. CAUMONT, P. DURRENS, C. CALMELS, V. PARISSI, P. RECORDON, E.
BON, C. DESJOBERT, L. TARRAGO-LITVAK & M. FOURNIER (2002): HIV-1 integrase interacts
with yeast microtubule-associated proteins. Biochim Biophys Acta 1575, 40-48
DE SOUZA LUNA, L. K., V. HEISER, N. REGAMEY, M. PANNING, J. F. DREXLER, S. MULANGU, L.
POON, S. BAUMGARTE, B. J. HAIJEMA, L. KAISER & C. DROSTEN (2007): Generic detection of
coronaviruses and differentiation at the prototype strain level by reverse transcription-PCR and
nonfluorescent low-density microarray. J Clin Microbiol 45, 1049-1052
DECROLY, E., I. IMBERT, B. COUTARD, M. BOUVET, B. SELISKO, K. ALVAREZ, A. E.
GORBALENYA, E. J. SNIJDER & B. CANARD (2008): Coronavirus nonstructural protein 16 is a
cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity. J Virol 82, 80718084
DEDEURWAERDER, A., D. A. OLYSLAEGERS, L. M. DESMARETS, I. D. ROUKAERTS, S. THEUNS
& H. J. NAUWYNCK (2014): ORF7-encoded accessory protein 7a of feline infectious peritonitis
virus as a counteragent against IFN-alpha-induced antiviral response. J Gen Virol 95, 393-402
DELMAS, B., J. GELFI, E. KUT, H. SJOSTROM, O. NOREN & H. LAUDE (1994): Determinants
essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain
of aminopeptidase-N that is distinct from the enzymatic site. J Virol 68, 5216-5224
DELMAS, B., J. GELFI, R. L'HARIDON, L. K. VOGEL, H. SJOSTROM, O. NOREN & H. LAUDE
(1992): Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV.
Nature 357, 417-420
DEWERCHIN, H. L., L. M. DESMARETS, Y. NOPPE & H. J. NAUWYNCK (2014): Myosins 1 and 6,
myosin light chain kinase, actin and microtubules cooperate during antibody-mediated
internalisation and trafficking of membrane-expressed viral antigens in feline infectious
peritonitis virus infected monocytes. Vet Res 45, 17
27
Introduction
DIEFENBACH, R. J., M. MIRANDA-SAKSENA, E. DIEFENBACH, D. J. HOLLAND, R. A. BOADLE, P.
J. ARMATI & A. L. CUNNINGHAM (2002): Herpes simplex virus tegument protein US11 interacts
with conventional kinesin heavy chain. J Virol 76, 3282-3291
DOHERTY, G. J. & H. T. MCMAHON (2009): Mechanisms of endocytosis. Annu Rev Biochem 78,
857-902
DOMINGUEZ, S. R., T. J. O'SHEA, L. M. OKO & K. V. HOLMES (2007): Detection of group 1
coronaviruses in bats in North America. Emerg Infect Dis 13, 1295-1300
DOYLE, L. P. & L. M. HUTCHINGS (1946): A transmissible gastroenteritis in pigs. J Am Vet Med
Assoc 108, 257-259
DRÁBER, P. & E. DRÁBEROVÁ (2012): Microtubules. In: M. KAVALLARIS "Cytoskeleton and
Human Disease" Humana Press, Springer, New York, pp. 29-53
DREXLER, J. F., V. M. CORMAN & C. DROSTEN (2014): Ecology, evolution and classification of
bat coronaviruses in the aftermath of SARS. Antiviral Res 101, 45-56
DREXLER, J. F., V. M. CORMAN, T. WEGNER, A. F. TATENO, R. M. ZERBINATI, F. GLOZARAUSCH, A. SEEBENS, M. A. MULLER & C. DROSTEN (2011): Amplification of emerging
viruses in a bat colony. Emerg Infect Dis 17, 449-456
DREXLER, J. F., F. GLOZA-RAUSCH, J. GLENDE, V. M. CORMAN, D. MUTH, M. GOETTSCHE, A.
SEEBENS, M. NIEDRIG, S. PFEFFERLE, S. YORDANOV, L. ZHELYAZKOV, U. HERMANNS, P.
VALLO, A. LUKASHEV, M. A. MULLER, H. DENG, G. HERRLER & C. DROSTEN (2010): Genomic
characterization of severe acute respiratory syndrome-related coronavirus in European bats
and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene
sequences. J Virol 84, 11336-11349
DU, L., Y. HE, Y. ZHOU, S. LIU, B. J. ZHENG & S. JIANG (2009): The spike protein of SARS-CoV-a target for vaccine and therapeutic development. Nat Rev Microbiol 7, 226-236
DVEKSLER, G. S., M. N. PENSIERO, C. B. CARDELLICHIO, R. K. WILLIAMS, G. S. JIANG, K. V.
HOLMES & C. W. DIEFFENBACH (1991): Cloning of the mouse hepatitis virus (MHV) receptor:
expression in human and hamster cell lines confers susceptibility to MHV. J Virol 65, 6881-6891
ENJUANES, L., F. ALMAZAN, I. SOLA & S. ZUNIGA (2006): Biochemical aspects of coronavirus
replication and virus-host interaction. Annu Rev Microbiol 60, 211-230
ENJUANES, L. & B. A. M. VAN DER ZEIJST (1995): Molecular basis of transmissible
gastroenteritis coronavirus (TGEV) epidemiology. In: S. G. SIDDELL "The Coronaviridae" Plenum
Press, New York, pp. 337–376
ENSERINK, M. (2000): Emerging diseases. Malaysian researchers trace Nipah virus outbreak to
bats. Science 289, 518-519
ESCORS, D., E. CAMAFEITA, J. ORTEGO, H. LAUDE & L. ENJUANES (2001): Organization of
two transmissible gastroenteritis coronavirus membrane protein topologies within the virion
and core. J Virol 75, 12228-12240
FIELD, H., B. MCCALL & J. BARRETT (1999): Australian bat lyssavirus infection in a captive
juvenile black flying fox. Emerg Infect Dis 5, 438-440
FLEMING, T. H. & P. EBY (2003): Ecology of bat migration. In: T. H. KUNZ &M. B. FENTON "Bat
Ecology" The University of Chicago Press, Chicago, pp. 156-208
FUNG, T. S., M. HUANG & D. X. LIU (2014): Coronavirus-induced ER stress response and its
involvement in regulation of coronavirus-host interactions. Virus Res 194, 110-123
28
Introduction
GALAN, C., I. SOLA, A. NOGALES, B. THOMAS, A. AKOULITCHEV, L. ENJUANES & F. ALMAZAN
(2009): Host cell proteins interacting with the 3' end of TGEV coronavirus genome influence
virus replication. Virology 391, 304-314
GALLAGHER, T. M. & M. J. BUCHMEIER (2001): Coronavirus spike proteins in viral entry and
pathogenesis. Virology 279, 371-374
GARCIA-SASTRE, A. & C. A. BIRON (2006): Type 1 interferons and the virus-host relationship: a
lesson in detente. Science 312, 879-882
GAUDIN, R., B. C. DE ALENCAR, N. ARHEL & P. BENAROCH (2013): HIV trafficking in host cells:
motors wanted! Trends in cell biology 23, 652-662
GE, X. Y., J. L. LI, X. L. YANG, A. A. CHMURA, G. ZHU, J. H. EPSTEIN, J. K. MAZET, B. HU, W.
ZHANG, C. PENG, Y. J. ZHANG, C. M. LUO, B. TAN, N. WANG, Y. ZHU, G. CRAMERI, S. Y.
ZHANG, L. F. WANG, P. DASZAK & Z. L. SHI (2013): Isolation and characterization of a bat
SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535-538
GELHAUS, S., B. THAA, K. ESCHKE, M. VEIT &
C. SCHWEGMANN-WESSELS (2014):
Palmitoylation of the Alphacoronavirus TGEV spike protein S is essential for incorporation into
virus-like particles but dispensable for S-M interaction. Virology 464-465, 397-405
GLOZA-RAUSCH, F., A. IPSEN, A. SEEBENS, M. GOTTSCHE, M. PANNING, J. F. DREXLER, N.
PETERSEN, A. ANNAN, K. GRYWNA, M. MULLER, S. PFEFFERLE & C. DROSTEN (2008):
Detection and prevalence patterns of group I coronaviruses in bats, northern Germany. Emerg
Infect Dis 14, 626-631
GODEKE, G. J., C. A. DE HAAN, J. W. ROSSEN, H. VENNEMA & P. J. ROTTIER (2000): Assembly
of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike
protein. J Virol 74, 1566-1571
GONCALVES, M. A., R. J. SA-NETO & T. K. BRAZIL (2002): Outbreak of aggressions and
transmission of rabies in human beings by vampire bats in northeastern Brazil. Rev Soc Bras
Med Trop 35, 461-464
GORBALENYA, A. E., L. ENJUANES, J. ZIEBUHR & E. J. SNIJDER (2006): Nidovirales: evolving
the largest RNA virus genome. Virus Res 117, 17-37
GOSERT, R., A. KANJANAHALUETHAI, D. EGGER, K. BIENZ & S. C. BAKER (2002): RNA
replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol 76, 36973708
GRAHAM, R. L. & R. S. BARIC (2010): Recombination, reservoirs, and the modular spike:
mechanisms of coronavirus cross-species transmission. J Virol 84, 3134-3146
GUAN, Y., B. J. ZHENG, Y. Q. HE, X. L. LIU, Z. X. ZHUANG, C. L. CHEUNG, S. W. LUO, P. H. LI, L.
J. ZHANG, Y. J. GUAN, K. M. BUTT, K. L. WONG, K. W. CHAN, W. LIM, K. F. SHORTRIDGE, K. Y.
YUEN, J. S. PEIRIS & L. L. POON (2003): Isolation and characterization of viruses related to the
SARS coronavirus from animals in southern China. Science 302, 276-278
HALPIN, K., A. D. HYATT, R. FOGARTY, D. MIDDLETON, J. BINGHAM, J. H. EPSTEIN, S. A.
RAHMAN, T. HUGHES, C. SMITH, H. E. FIELD & P. DASZAK (2011): Pteropid bats are confirmed
as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus
transmission. Am J Trop Med Hyg 85, 946-951
HALPIN, K., P. L. YOUNG, H. E. FIELD & J. S. MACKENZIE (2000): Isolation of Hendra virus from
pteropid bats: a natural reservoir of Hendra virus. J Gen Virol 81, 1927-1932
HANSEN, G. H., B. DELMAS, L. BESNARDEAU, L. K. VOGEL, H. LAUDE, H. SJOSTROM & O.
NOREN (1998): The coronavirus transmissible gastroenteritis virus causes infection after
29
Introduction
receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment. J
Virol 72, 527-534
HAYMAN, D. T., P. EMMERICH, M. YU, L. F. WANG, R. SUU-IRE, A. R. FOOKS, A. A.
CUNNINGHAM & J. L. WOOD (2010): Long-term survival of an urban fruit bat seropositive for
Ebola and Lagos bat viruses. PLoS One 5, e11978
HE, Y. & R. SMITH (2009): Nuclear functions of heterogeneous nuclear ribonucleoproteins A/B.
Cell Mol Life Sci 66, 1239-1256
HENGEL, H., U. H. KOSZINOWSKI & K. K. CONZELMANN (2005): Viruses know it all: new
insights into IFN networks. Trends Immunol 26, 396-401
HENRY SUM, M. S. (2015): The involvement of microtubules and actin during the infection of
Japanese encephalitis virus in neuroblastoma cell line, IMR32. Biomed Res Int 2015, 695283
HERREWEGH, A. A., I. SMEENK, M. C. HORZINEK, P. J. ROTTIER & R. J. DE GROOT (1998):
Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination
between feline coronavirus type I and canine coronavirus. J Virol 72, 4508-4514
HILLER, G., K. WEBER, L. SCHNEIDER, C. PARAJSZ & C. JUNGWIRTH (1979): Interaction of
assembled progeny pox viruses with the cellular cytoskeleton. Virology 98, 142-153
HILSER, V. J. & E. B. THOMPSON (2007): Intrinsic disorder as a mechanism to optimize
allosteric coupling in proteins. Proc Natl Acad Sci U S A 104, 8311-8315
HIROKAWA, N. & R. TAKEMURA (2005): Molecular motors and mechanisms of directional
transport in neurons. Nat Rev Neurosci 6, 201-214
HISCOX, J. A., T. WURM, L. WILSON, P. BRITTON, D. CAVANAGH & G. BROOKS (2001): The
coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus. J Virol 75, 506512
HODGSON, T., P. BRITTON & D. CAVANAGH (2006): Neither the RNA nor the proteins of open
reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for
replication. J Virol 80, 296-305
HOGUE, B. G. & C. E. MACHAMER (2008): Coronavirus Structural Proteins and Virus Assembly.
In: S. PERLMAN, T. GALLAGHER &E. J. SNIJDER "Nidoviruses" American Society of Microbiology,
Washington, DC, pp. 179-200
HUANG, Y., Z. Y. YANG, W. P. KONG & G. J. NABEL (2004): Generation of synthetic severe
acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and
vaccine production. J Virol 78, 12557-12565
IHA, K., T. OMATSU, S. WATANABE, N. UEDA, S. TANIGUCHI, H. FUJII, Y. ISHII, S. KYUWA, H.
AKASHI & Y. YOSHIKAWA (2009): Molecular cloning and sequencing of the cDNAs encoding
the bat interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p40, and tumor necrosis factor-alpha. J Vet Med
Sci 71, 1691-1695
IHA, K., T. OMATSU, S. WATANABE, N. UEDA, S. TANIGUCHI, H. FUJII, Y. ISHII, S. KYUWA, H.
AKASHI & Y. YOSHIKAWA (2010): Molecular cloning and expression analysis of bat toll-like
receptors 3, 7 and 9. J Vet Med Sci 72, 217-220
KAMINS, A. O., O. RESTIF, Y. NTIAMOA-BAIDU, R. SUU-IRE, D. T. HAYMAN, A. A. CUNNINGHAM,
J. L. WOOD & J. M. ROWCLIFFE (2011): Uncovering the fruit bat bushmeat commodity chain
and the true extent of fruit bat hunting in Ghana, West Africa. Biol Conserv 144, 3000-3008
30
Introduction
KANLAYA, R., S. N. PATTANAKITSAKUL, S. SINCHAIKUL, S. T. CHEN & V. THONGBOONKERD
(2010): Vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue
nonstructural protein 1 and is important for viral replication and release. Mol Biosyst 6, 795-806
KIM, L., J. HAYES, P. LEWIS, A. V. PARWANI, K. O. CHANG & L. J. SAIF (2000): Molecular
characterization and pathogenesis of transmissible gastroenteritis coronavirus (TGEV) and
porcine respiratory coronavirus (PRCV) field isolates co-circulating in a swine herd. Arch Virol
145, 1133-1147
KLUMPERMAN, J., J. K. LOCKER, A. MEIJER, M. C. HORZINEK, H. J. GEUZE & P. J. ROTTIER
(1994): Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion
budding. J Virol 68, 6523-6534
KNOOPS, K., M. KIKKERT, S. H. WORM, J. C. ZEVENHOVEN-DOBBE, Y. VAN DER MEER, A. J.
KOSTER, A. M. MOMMAAS & E. J. SNIJDER (2008): SARS-coronavirus replication is supported
by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6, e226
KONAN, K. V., T. H. GIDDINGS, JR., M. IKEDA, K. LI, S. M. LEMON & K. KIRKEGAARD (2003):
Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and
ultrastructure of host secretory apparatus. J Virol 77, 7843-7855
KOSHIZUKA, T., Y. KAWAGUCHI & Y. NISHIYAMA (2005): Herpes simplex virus type 2
membrane protein UL56 associates with the kinesin motor protein KIF1A. J Gen Virol 86, 527533
KOUDELKA, K. J., G. DESTITO, E. M. PLUMMER, S. A. TRAUGER, G. SIUZDAK & M.
MANCHESTER (2009): Endothelial targeting of cowpea mosaic virus (CPMV) via surface
vimentin. PLoS Pathog 5, e1000417
KRIJNSE-LOCKER, J., M. ERICSSON, P. J. ROTTIER & G. GRIFFITHS (1994): Characterization of
the budding compartment of mouse hepatitis virus: evidence that transport from the RER to
the Golgi complex requires only one vesicular transport step. J Cell Biol 124, 55-70
KUNZ, T. H. & L. F. LUMSDEN (2003): Ecology of cavity and foliage roosting bats. In: T. H. KUNZ
&M. B. FENTON "Bat Ecology" The University of Chicago Press, Chicago, pp. 3-89
KYUWA, S., M. COHEN, G. NELSON, S. M. TAHARA & S. A. STOHLMAN (1994): Modulation of
cellular macromolecular synthesis by coronavirus: implication for pathogenesis. J Virol 68,
6815-6819
LAI, M. M. & D. CAVANAGH (1997): The molecular biology of coronaviruses. Adv Virus Res 48, 1100
LAI, M. M., C. D. PATTON & S. A. STOHLMAN (1982): Further characterization of mRNA's of
mouse hepatitis virus: presence of common 5'-end nucleotides. J Virol 41, 557-565
LAI, M. M. & S. A. STOHLMAN (1981): Comparative analysis of RNA genomes of mouse
hepatitis viruses. J Virol 38, 661-670
LAMPO, M., M. D. FELICIANGELI, L. M. MARQUEZ, C. BASTIDAS & P. LAU (2000): A possible
role of bats as a blood source for the Leishmania vector Lutzomyia longipalpis (Diptera:
Psychodidae). Am J Trop Med Hyg 62, 718-719
LANSER, J. A. & C. R. HOWARD (1980): The polypeptides of infectious bronchitis virus (IBV-41
strain). J Gen Virol 46, 349-361
LAU, S. K., K. S. LI, Y. HUANG, C. T. SHEK, H. TSE, M. WANG, G. K. CHOI, H. XU, C. S. LAM, R.
GUO, K. H. CHAN, B. J. ZHENG, P. C. WOO & K. Y. YUEN (2010a): Ecoepidemiology and
complete genome comparison of different strains of severe acute respiratory syndrome-related
31
Introduction
Rhinolophus bat coronavirus in China reveal bats as a reservoir for acute, self-limiting
infection that allows recombination events. J Virol 84, 2808-2819
LAU, S. K., K. S. LI, A. K. TSANG, C. T. SHEK, M. WANG, G. K. CHOI, R. GUO, B. H. WONG, R. W.
POON, C. S. LAM, S. Y. WANG, R. Y. FAN, K. H. CHAN, B. J. ZHENG, P. C. WOO & K. Y. YUEN
(2012): Recent transmission of a novel alphacoronavirus, bat coronavirus HKU10, from
Leschenault's rousettes to pomona leaf-nosed bats: first evidence of interspecies transmission
of coronavirus between bats of different suborders. J Virol 86, 11906-11918
LAU, S. K., R. W. POON, B. H. WONG, M. WANG, Y. HUANG, H. XU, R. GUO, K. S. LI, K. GAO, K.
H. CHAN, B. J. ZHENG, P. C. WOO & K. Y. YUEN (2010b): Coexistence of different genotypes in
the same bat and serological characterization of Rousettus bat coronavirus HKU9 belonging to
a novel Betacoronavirus subgroup. J Virol 84, 11385-11394
LAU, S. K., P. C. WOO, K. S. LI, Y. HUANG, H. W. TSOI, B. H. WONG, S. S. WONG, S. Y. LEUNG,
K. H. CHAN & K. Y. YUEN (2005): Severe acute respiratory syndrome coronavirus-like virus in
Chinese horseshoe bats. Proc Natl Acad Sci U S A 102, 14040-14045
LAU, S. K., P. C. WOO, K. S. LI, Y. HUANG, M. WANG, C. S. LAM, H. XU, R. GUO, K. H. CHAN, B.
J. ZHENG & K. Y. YUEN (2007): Complete genome sequence of bat coronavirus HKU2 from
Chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary
lineage from the rest of the genome. Virology 367, 428-439
LAUDE, H., K. VAN REETH & M. PENSAERT (1993): Porcine respiratory coronavirus: molecular
features and virus-host interactions. Vet Res 24, 125-150
LAZARIDES, E. (1980): Intermediate filaments as mechanical integrators of cellular space.
Nature 283, 249-256
LENK, R. & S. PENMAN (1979): The cytoskeletal framework and poliovirus metabolism. Cell 16,
289 301
LEONG, W. F., H. C. TAN, E. E. OOI, D. R. KOH & V. T. CHOW (2005): Microarray and real-time
RT-PCR analyses of differential human gene expression patterns induced by severe acute
respiratory syndrome (SARS) coronavirus infection of Vero cells. Microbes Infect 7, 248-259
LEOPOLD, P. L. & K. K. PFISTER (2006): Viral strategies for intracellular trafficking: motors and
microtubules. Traffic 7, 516-523
LEROY, E. M., A. EPELBOIN, V. MONDONGE, X. POURRUT, J. P. GONZALEZ, J. J. MUYEMBETAMFUM & P. FORMENTY (2009): Human Ebola outbreak resulting from direct exposure to
fruit bats in Luebo, Democratic Republic of Congo, 2007. Vector Borne Zoonotic Dis 9, 723-728
LEROY, E. M., B. KUMULUNGUI, X. POURRUT, P. ROUQUET, A. HASSANIN, P. YABA, A.
DELICAT, J. T. PAWESKA, J. P. GONZALEZ & R. SWANEPOEL (2005): Fruit bats as reservoirs of
Ebola virus. Nature 438, 575-576
LI, F. Q., J. P. TAM & D. X. LIU (2007): Cell cycle arrest and apoptosis induced by the
coronavirus infectious bronchitis virus in the absence of p53. Virology 365, 435-445
LI, H. P., X. ZHANG, R. DUNCAN, L. COMAI & M. M. LAI (1997): Heterogeneous nuclear
ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus
RNA. Proc Natl Acad Sci U S A 94, 9544-9549
LI, W., M. J. MOORE, N. VASILIEVA, J. SUI, S. K. WONG, M. A. BERNE, M. SOMASUNDARAN, J.
L. SULLIVAN, K. LUZURIAGA, T. C. GREENOUGH, H. CHOE & M. FARZAN (2003): Angiotensinconverting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450-454
32
Introduction
LI, W., Z. SHI, M. YU, W. REN, C. SMITH, J. H. EPSTEIN, H. WANG, G. CRAMERI, Z. HU, H.
ZHANG, J. ZHANG, J. MCEACHERN, H. FIELD, P. DASZAK, B. T. EATON, S. ZHANG & L. F.
WANG (2005a): Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679
LI, W., C. ZHANG, J. SUI, J. H. KUHN, M. J. MOORE, S. LUO, S. K. WONG, I. C. HUANG, K. XU, N.
VASILIEVA, A. MURAKAMI, Y. HE, W. A. MARASCO, Y. GUAN, H. CHOE & M. FARZAN (2005b):
Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J 24,
1634-1643
LIANG, J. J., C. Y. YU, C. L. LIAO & Y. L. LIN (2011): Vimentin binding is critical for infection by
the virulent strain of Japanese encephalitis virus. Cell Microbiol 13, 1358-1370
LIM, K. P. & D. X. LIU (2001): The missing link in coronavirus assembly. Retention of the avian
coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and
physical interaction between the envelope and membrane proteins. J Biol Chem 276, 1751517523
LIU, C., J. TANG, Y. MA, X. LIANG, Y. YANG, G. PENG, Q. QI, S. JIANG, J. LI, L. DU & F. LI (2015):
Receptor usage and cell entry of porcine epidemic diarrhea coronavirus. J Virol 89, 6121-6125
LOCKER, J. K., D.-J. E. OPSTELTEN, M. ERICSSON, M. C. HORZINEK & P. J. M. ROTTIER (1995):
Oligomerization of a trans-Golgi/ trans-Golgi Network Retained Protein Occurs in the Golgi
Complex and May Be Part of Its Retention. Journal of Biological Chemistry 270, 8815-8821
LOCKER, J. K., J. K. ROSE, M. C. HORZINEK & P. J. ROTTIER (1992): Membrane assembly of
the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred
orientation. J Biol Chem 267, 21911-21918
LONTOK, E., E. CORSE & C. E. MACHAMER (2004): Intracellular targeting signals contribute to
localization of coronavirus spike proteins near the virus assembly site. J Virol 78, 5913-5922
LUFTIG, R. B. (1982): Does the cytoskeleton play a significant role in animal virus replication? J
Theor Biol 99, 173-191
LUO, H., Q. CHEN, J. CHEN, K. CHEN, X. SHEN & H. JIANG (2005): The nucleocapsid protein of
SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear
ribonucleoprotein A1. FEBS Lett 579, 2623-2628
LUYTJES, W., L. S. STURMAN, P. J. BREDENBEEK, J. CHARITE, B. A. VAN DER ZEIJST, M. C.
HORZINEK & W. J. SPAAN (1987): Primary structure of the glycoprotein E2 of coronavirus
MHV-A59 and identification of the trypsin cleavage site. Virology 161, 479-487
MABIT, H., M. Y. NAKANO, U. PRANK, B. SAAM, K. DOHNER, B. SODEIK & U. F. GREBER (2002):
Intact microtubules support adenovirus and herpes simplex virus infections. J Virol 76, 99629971
MARTINEZ-MORENO, M., I. NAVARRO-LERIDA, F. RONCAL, J. P. ALBAR, C. ALONSO, F.
GAVILANES & I. RODRIGUEZ-CRESPO (2003): Recognition of novel viral sequences that
associate with the dynein light chain LC8 identified through a pepscan technique. FEBS Lett
544, 262-267
MASTERS, P. S., L. KUO, R. YE, K. R. HURST, C. A. KOETZNER & B. HSUE (2006): Genetic and
molecular biological analysis of protein-protein interactions in coronavirus assembly. Adv Exp
Med Biol 581, 163-173
MCBRIDE, C. E., J. LI & C. E. MACHAMER (2007): The cytoplasmic tail of the severe acute
respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum
retrieval signal that binds COPI and promotes interaction with membrane protein. J Virol 81,
2418-2428
33
Introduction
MCCOLL, K. A., T. CHAMBERLAIN, R. A. LUNT, K. M. NEWBERRY, D. MIDDLETON & H. A.
WESTBURY (2002): Pathogenesis studies with Australian bat lyssavirus in grey-headed flying
foxes (Pteropus poliocephalus). Aust Vet J 80, 636-641
MCMURRAY, D. N., J. STROUD, J. J. MURPHY, M. A. CARLOMAGNO & D. L. GREER (1982): Role
of immunoglobulin classes in experimental histoplasmosis in bats. Dev Comp Immunol 6, 557567
MEMISH, Z. A., N. MISHRA, K. J. OLIVAL, S. F. FAGBO, V. KAPOOR, J. H. EPSTEIN, R.
ALHAKEEM, A. DUROSINLOUN, M. AL ASMARI, A. ISLAM, A. KAPOOR, T. BRIESE, P. DASZAK,
A. A. AL RABEEAH & W. I. LIPKIN (2013): Middle East respiratory syndrome coronavirus in bats,
Saudi Arabia. Emerg Infect Dis 19, 1819-1823
MICKLEBURGH, S., K. WAYLEN & P. A. RACEY (2009): "Bats as bushmeat: a global review.".
Fauna & Flora International, Oryx 43, 217-234
MIDDLETON, D. J., C. J. MORRISSY, B. M. VAN DER HEIDE, G. M. RUSSELL, M. A. BRAUN, H. A.
WESTBURY, K. HALPIN & P. W. DANIELS (2007): Experimental Nipah virus infection in pteropid
bats (Pteropus poliocephalus). J Comp Pathol 136, 266-272
MILES, B. D., R. B. LUFTIG, J. A. WEATHERBEE, R. R. WEIHING & J. WEBER (1980):
Quantitation of the interaction between adenovirus types 2 and 5 and microtubules inside
infected cells. Virology 105, 265-269
MILLER, L. C. & J. M. FOX (2004): Apoptosis and porcine reproductive and respiratory
syndrome virus. Vet Immunol Immunopathol 102, 131-142
MILLER, M. S. & L. HERTEL (2009): Onset of human cytomegalovirus replication in fibroblasts
requires the presence of an intact vimentin cytoskeleton. J Virol 83, 7015-7028
MOES, E., L. VIJGEN, E. KEYAERTS, K. ZLATEVA, S. LI, P. MAES, K. PYRC, B. BERKHOUT, L.
VAN DER HOEK & M. VAN RANST (2005): A novel pancoronavirus RT-PCR assay: frequent
detection of human coronavirus NL63 in children hospitalized with respiratory tract infections
in Belgium. BMC Infect Dis 5, 6
MOFFAT, K., C. KNOX, G. HOWELL, S. J. CLARK, H. YANG, G. J. BELSHAM, M. RYAN & T.
WILEMAN (2007): Inhibition of the secretory pathway by foot-and-mouth disease virus 2BC
protein is reproduced by coexpression of 2B with 2C, and the site of inhibition is determined
by the subcellular location of 2C. J Virol 81, 1129-1139
MOU, H., V. S. RAJ, F. J. VAN KUPPEVELD, P. J. ROTTIER, B. L. HAAGMANS & B. J. BOSCH
(2013): The receptor binding domain of the new Middle East respiratory syndrome coronavirus
maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies.
J Virol 87, 9379-9383
MULLER, M. A., J. T. PAWESKA, P. A. LEMAN, C. DROSTEN, K. GRYWNA, A. KEMP, L. BRAACK,
K. SONNENBERG, M. NIEDRIG & R. SWANEPOEL (2007): Coronavirus antibodies in African bat
species. Emerg Infect Dis 13, 1367-1370
NARAYANAN, K., A. MAEDA, J. MAEDA & S. MAKINO (2000): Characterization of the
coronavirus M protein and nucleocapsid interaction in infected cells. J Virol 74, 8127-8134
NAVAS, S., S. H. SEO, M. M. CHUA, J. DAS SARMA, S. T. HINGLEY, E. LAVI & S. R. WEISS
(2001): Role of the spike protein in murine coronavirus induced hepatitis: an in vivo study
using targeted RNA recombination. Adv Exp Med Biol 494, 139-144
NAVAS, S. & S. R. WEISS (2003): Murine coronavirus-induced hepatitis: JHM genetic
background eliminates A59 spike-determined hepatotropism. J Virol 77, 4972-4978
NEUWEILER, G. (2000): The Biology of Bats. Oxford University Press, New York,
34
Introduction
NGUYEN, V. P. & B. G. HOGUE (1997): Protein interactions during coronavirus assembly. J
Virol 71, 9278-9284
NIEMANN, H., R. GEYER, H. D. KLENK, D. LINDER, S. STIRM & M. WIRTH (1984): The
carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked
oligosaccharides of glycoprotein E1. EMBO J 3, 665-670
NIEMEYER, D., T. ZILLINGER, D. MUTH, F. ZIELECKI, G. HORVATH, T. SULIMAN, W. BARCHET,
F. WEBER, C. DROSTEN & M. A. MULLER (2013): Middle East respiratory syndrome
coronavirus accessory protein 4a is a type I interferon antagonist. J Virol 87, 12489-12495
OKUNOLA, H. L. & A. R. KRAINER (2009): Cooperative-binding and splicing-repressive
properties of hnRNP A1. Mol Cell Biol 29, 5620-5631
OOSTRA, M., C. A. DE HAAN, R. J. DE GROOT & P. J. ROTTIER (2006): Glycosylation of the
severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M. J
Virol 80, 2326-2336
OOSTRA, M., E. G. TE LINTELO, M. DEIJS, M. H. VERHEIJE, P. J. ROTTIER & C. A. DE HAAN
(2007): Localization and membrane topology of coronavirus nonstructural protein 4:
involvement of the early secretory pathway in replication. J Virol 81, 12323-12336
OP DE BEECK, A. & P. CAILLET-FAUQUET (1997): Viruses and the cell cycle. In: L. MEIJER, S.
GUIDET &M. PHILIPPE "Progress in Cell Cycle Research" Plenum Press, New York, pp. 1-19
OPSTELTEN, D. J., M. J. RAAMSMAN, K. WOLFS, M. C. HORZINEK & P. J. ROTTIER (1995):
Envelope glycoprotein interactions in coronavirus assembly. The Journal of cell biology 131,
339-349
PASICK, J. M., K. KALICHARRAN & S. DALES (1994): Distribution and trafficking of JHM
coronavirus structural proteins and virions in primary neurons and the OBL-21 neuronal cell
line. J Virol 68, 2915-2928
PAUL, P. S., E. M. VAUGHN & P. G. HALBUR (1997): Pathogenicity and sequence analysis
studies suggest potential role of gene 3 in virulence of swine enteric and respiratory
coronaviruses. Adv Exp Med Biol 412, 317-321
PENSAERT, M., P. CALLEBAUT & J. VERGOTE (1986): Isolation of a porcine respiratory, nonenteric coronavirus related to transmissible gastroenteritis. Vet Q 8, 257-261
PERLMAN, S. & J. NETLAND (2009): Coronaviruses post-SARS: update on replication and
pathogenesis. Nat Rev Microbiol 7, 439-450
PETIT, C., M. L. GIRON, J. TOBALY-TAPIERO, P. BITTOUN, E. REAL, Y. JACOB, N. TORDO, H. DE
THE & A. SAIB (2003): Targeting of incoming retroviral Gag to the centrosome involves a direct
interaction with the dynein light chain 8. J Cell Sci 116, 3433-3442
PETIT, C. M., V. N. CHOULJENKO, A. IYER, R. COLGROVE, M. FARZAN, D. M. KNIPE & K. G.
KOUSOULAS (2007): Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus
spike glycoprotein is important for spike-mediated cell fusion. Virology 360, 264-274
PICARD-JEAN, F., M. TREMBLAY-LÉTOURNEAU, E. SERRA, C. DIMECH, H. SCHULZ, M.
ANSELIN, V. DUTILLY & M. BISAILLON (2013): RNA 5′-end Maturation: A Crucial Step in the
Replication of Viral Genomes. In: V. ROMANOWSKI "Current Issues in Molecular Virology - Viral
Genetics and Biotechnological Applications" InTech, pp.
POISSON, N., E. REAL, Y. GAUDIN, M. C. VANEY, S. KING, Y. JACOB, N. TORDO & D. BLONDEL
(2001): Molecular basis for the interaction between rabies virus phosphoprotein P and the
dynein light chain LC8: dissociation of dynein-binding properties and transcriptional
functionality of P. J Gen Virol 82, 2691-2696
35
Introduction
POLLARD, T. D. & J. A. COOPER (2009): Actin, a central player in cell shape and movement.
Science 326, 1208-1212
POON, L. L., D. K. CHU, K. H. CHAN, O. K. WONG, T. M. ELLIS, Y. H. LEUNG, S. K. LAU, P. C.
WOO, K. Y. SUEN, K. Y. YUEN, Y. GUAN & J. S. PEIRIS (2005): Identification of a novel
coronavirus in bats. J Virol 79, 2001-2009
PYRC, K., B. BERKHOUT & L. VAN DER HOEK (2007): The novel human coronaviruses NL63
and HKU1. J Virol 81, 3051-3057
RAAMSMAN, M. J., J. K. LOCKER, A. DE HOOGE, A. A. DE VRIES, G. GRIFFITHS, H. VENNEMA &
P. J. ROTTIER (2000): Characterization of the coronavirus mouse hepatitis virus strain A59
small membrane protein E. J Virol 74, 2333-2342
RADTKE, K., K. DOHNER & B. SODEIK (2006): Viral interactions with the cytoskeleton: a
hitchhiker's guide to the cell. Cell Microbiol 8, 387-400
RAHMAN, M. A., M. J. HOSSAIN, S. SULTANA, N. HOMAIRA, S. U. KHAN, M. RAHMAN, E. S.
GURLEY, P. E. ROLLIN, M. K. LO, J. A. COMER, L. LOWE, P. A. ROTA, T. G. KSIAZEK, E. KENAH,
Y. SHARKER & S. P. LUBY (2012): Date palm sap linked to Nipah virus outbreak in Bangladesh,
2008. Vector Borne Zoonotic Dis 12, 65-72
RAJ, V. S., H. MOU, S. L. SMITS, D. H. DEKKERS, M. A. MULLER, R. DIJKMAN, D. MUTH, J. A.
DEMMERS, A. ZAKI, R. A. FOUCHIER, V. THIEL, C. DROSTEN, P. J. ROTTIER, A. D.
OSTERHAUS, B. J. BOSCH & B. L. HAAGMANS (2013): Dipeptidyl peptidase 4 is a functional
receptor for the emerging human coronavirus-EMC. Nature 495, 251-254
RAO, P. V. & T. M. GALLAGHER (1998): Mouse hepatitis virus receptor levels influence virusinduced cytopathology. Adv Exp Med Biol 440, 549-555
RASSCHAERT, D., M. DUARTE & H. LAUDE (1990): Porcine respiratory coronavirus differs from
transmissible gastroenteritis virus by a few genomic deletions. J Gen Virol 71 ( Pt 11), 25992607
RAUX, H., A. FLAMAND & D. BLONDEL (2000): Interaction of the rabies virus P protein with the
LC8 dynein light chain. J Virol 74, 10212-10216
REN, W., W. LI, M. YU, P. HAO, Y. ZHANG, P. ZHOU, S. ZHANG, G. ZHAO, Y. ZHONG, S. WANG,
L. F. WANG & Z. SHI (2006): Full-length genome sequences of two SARS-like coronaviruses in
horseshoe bats and genetic variation analysis. J Gen Virol 87, 3355-3359
REYNES, J. M., D. COUNOR, S. ONG, C. FAURE, V. SENG, S. MOLIA, J. WALSTON, M. C.
GEORGES-COURBOT, V. DEUBEL & J. L. SARTHOU (2005): Nipah virus in Lyle's flying foxes,
Cambodia. Emerg Infect Dis 11, 1042-1047
RIDLEY, A. J., M. A. SCHWARTZ, K. BURRIDGE, R. A. FIRTEL, M. H. GINSBERG, G. BORISY, J. T.
PARSONS & A. R. HORWITZ (2003): Cell migration: integrating signals from front to back.
Science 302, 1704-1709
RISCO, C., I. M. ANTON, C. SUNE, A. M. PEDREGOSA, J. M. MARTIN-ALONSO, F. PARRA, J. L.
CARRASCOSA &
L. ENJUANES (1995): Membrane protein molecules of transmissible
gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of
the virion. J Virol 69, 5269-5277
RODRIGUEZ-CRESPO, I., B. YELAMOS, F. RONCAL, J. P. ALBAR, P. R. ORTIZ DE MONTELLANO
& F. GAVILANES (2001): Identification of novel cellular proteins that bind to the LC8 dynein
light chain using a pepscan technique. FEBS Lett 503, 135-141
ROTTIER, P. J., M. C. HORZINEK & B. A. VAN DER ZEIJST (1981): Viral protein synthesis in
mouse hepatitis virus strain A59-infected cells: effect of tunicamycin. J Virol 40, 350-357
36
Introduction
ROTTIER, P. J. & J. K. ROSE (1987): Coronavirus E1 glycoprotein expressed from cloned cDNA
localizes in the Golgi region. J Virol 61, 2042-2045
SAIF, L. J., M. B. PENSAERT, K. SESTAK, S. G. YEO & K. JUNG (2012): Coronaviruses. In: J. J.
ZIMMERMAN, L. A. KARRIKER, A. RAMIREZ, K. J. SCHWARTZ &G. W. STEVENSON "Diseases of
Swine" Wiley, pp. 501-524
SAKAGUCHI, T., G. P. LESER & R. A. LAMB (1996): The ion channel activity of the influenza
virus M2 protein affects transport through the Golgi apparatus. J Cell Biol 133, 733-747
SALANUEVA, I. J., J. L. CARRASCOSA & C. RISCO (1999): Structural maturation of the
transmissible gastroenteritis coronavirus. J Virol 73, 7952-7964
SANCHEZ, C. M., F. GEBAUER, C. SUNE, A. MENDEZ, J. DOPAZO & L. ENJUANES (1992):
Genetic evolution and tropism of transmissible gastroenteritis coronaviruses. Virology 190, 92105
SARKAR, S. K. & A. K. CHAKRAVARTY (1991): Analysis of immunocompetent cells in the bat,
Pteropus giganteus: isolation and scanning electron microscopic characterization. Dev Comp
Immunol 15, 423-430
SATAKE, M., P. N. MCMILLAN & R. B. LUFTIG (1981): Effect of vinblastine on distribution of
murine leukemia virus-derived membrane-associated antigens. Proc Natl Acad Sci U S A 78,
6266-6270
SAWICKI, S. G. & D. L. SAWICKI (1998): A new model for coronavirus transcription. Adv Exp
Med Biol 440, 215-219
SCHICKLI, J. H., B. D. ZELUS, D. E. WENTWORTH, S. G. SAWICKI & K. V. HOLMES (1997): The
murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells
exhibits an extended host range. J Virol 71, 9499-9507
SCHLIWA, M. (1981): Proteins associated with cytoplasmic actin. Cell 25, 587-590
SCHNEIDER, M. C., J. ARON, C. SANTOS-BURGOA, W. UIEDA & S. RUIZ-VELAZCO (2001):
Common vampire bat attacks on humans in a village of the Amazon region of Brazil. Cad Saude
Publica 17, 1531-1536
SCHWEGMANN-WESSELS, C., M. AL-FALAH, D. ESCORS, Z. WANG, G. ZIMMER, H. DENG, L.
ENJUANES, H. Y. NAIM & G. HERRLER (2004): A novel sorting signal for intracellular
localization is present in the S protein of a porcine coronavirus but absent from severe acute
respiratory syndrome-associated coronavirus. J Biol Chem 279, 43661-43666
SETHNA, P. B. & D. A. BRIAN (1997): Coronavirus genomic and subgenomic minus-strand
RNAs copartition in membrane-protected replication complexes. J Virol 71, 7744-7749
SHARP, D. J., G. C. ROGERS & J. M. SCHOLEY (2000): Microtubule motors in mitosis. Nature
407, 41-47
SHARPE, A. H., L. B. CHEN & B. N. FIELDS (1982): The interaction of mammalian reoviruses
with the cytoskeleton of monkey kidney CV-1 cells. Virology 120, 399-411
SHEAHAN, T., B. ROCKX, E. DONALDSON, D. CORTI & R. BARIC (2008): Pathways of crossspecies transmission of synthetically reconstructed zoonotic severe acute respiratory
syndrome coronavirus. J Virol 82, 8721-8732
SHEN, S., Z. L. WEN & D. X. LIU (2003): Emergence of a coronavirus infectious bronchitis virus
mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis
and replication. Virology 311, 16-27
37
Introduction
SHI, Z. & Z. HU (2008): A review of studies on animal reservoirs of the SARS coronavirus. Virus
Res 133, 74-87
SHIRATO, K., M. MAEJIMA, S. MATSUYAMA, M. UJIKE, A. MIYAZAKI, N. TAKEYAMA, H. IKEDA &
F. TAGUCHI (2011): Mutation in the cytoplasmic retrieval signal of porcine epidemic diarrhea
virus spike (S) protein is responsible for enhanced fusion activity. Virus Res 161, 188-193
SIMMONS, N. B. (2005): Evolution. An Eocene big bang for bats. Science 307, 527-528
SMITH, H. C. (1956): Advances made in swine practice. IX. Transmissible gastroenteritis.
Veterinary Medicine 51, 425-429
SNIJDER, E. J., Y. VAN DER MEER, J. ZEVENHOVEN-DOBBE, J. J. ONDERWATER, J. VAN DER
MEULEN, H. K. KOERTEN & A. M. MOMMAAS (2006): Ultrastructure and origin of membrane
vesicles associated with the severe acute respiratory syndrome coronavirus replication
complex. J Virol 80, 5927-5940
SONG, H. D., C. C. TU, G. W. ZHANG, S. Y. WANG, K. ZHENG, L. C. LEI, Q. X. CHEN, Y. W. GAO,
H. Q. ZHOU, H. XIANG, H. J. ZHENG, S. W. CHERN, F. CHENG, C. M. PAN, H. XUAN, S. J. CHEN,
H. M. LUO, D. H. ZHOU, Y. F. LIU, J. F. HE, P. Z. QIN, L. H. LI, Y. Q. REN, W. J. LIANG, Y. D. YU, L.
ANDERSON, M. WANG, R. H. XU, X. W. WU, H. Y. ZHENG, J. D. CHEN, G. LIANG, Y. GAO, M.
LIAO, L. FANG, L. Y. JIANG, H. LI, F. CHEN, B. DI, L. J. HE, J. Y. LIN, S. TONG, X. KONG, L. DU, P.
HAO, H. TANG, A. BERNINI, X. J. YU, O. SPIGA, Z. M. GUO, H. Y. PAN, W. Z. HE, J. C.
MANUGUERRA, A. FONTANET, A. DANCHIN, N. NICCOLAI, Y. X. LI, C. I. WU & G. P. ZHAO
(2005): Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet
and human. Proc Natl Acad Sci U S A 102, 2430-2435
SPAAN, W., D. CAVANAGH & M. C. HORZINEK (1988): Coronaviruses: structure and genome
expression. J Gen Virol 69 ( Pt 12), 2939-2952
STERN, D. F. & B. M. SEFTON (1982): Coronavirus proteins: structure and function of the
oligosaccharides of the avian infectious bronchitis virus glycoproteins. J Virol 44, 804-812
STOKES, G. V. (1976): High-voltage electron microscope study of the release of vaccinia virus
from whole cells. J Virol 18, 636-643
STURMAN, L. S., K. V. HOLMES & J. BEHNKE (1980): Isolation of coronavirus envelope
glycoproteins and interaction with the viral nucleocapsid. J Virol 33, 449-462
SULKIN, S. E., R. ALLEN & R. SIMS (1966): Studies of arthropod-borne virus infections in
Chiroptera. 3. Influence of environmental temperature on experimental infection with Japanese
B and St. Louis encephalitis viruses. Am J Trop Med Hyg 15, 406-417
SUZUKI, H. & F. TAGUCHI (1996): Analysis of the receptor-binding site of murine coronavirus
spike protein. J Virol 70, 2632-2636
SWANEPOEL, R., P. A. LEMAN, F. J. BURT, N. A. ZACHARIADES, L. E. BRAACK, T. G. KSIAZEK,
P. E. ROLLIN, S. R. ZAKI & C. J. PETERS (1996): Experimental inoculation of plants and animals
with Ebola virus. Emerg Infect Dis 2, 321-325
TAHARA, S. M., T. A. DIETLIN, C. C. BERGMANN, G. W. NELSON, S. KYUWA, R. P. ANTHONY &
S. A. STOHLMAN (1994): Coronavirus translational regulation: leader affects mRNA efficiency.
Virology 202, 621-630
TAKEDA, M., C. K. CHANG, T. IKEYA, P. GUNTERT, Y. H. CHANG, Y. L. HSU, T. H. HUANG & M.
KAINOSHO (2008): Solution structure of the c-terminal dimerization domain of SARS
coronavirus nucleocapsid protein solved by the SAIL-NMR method. J Mol Biol 380, 608-622
38
Introduction
TAN, Y. W., W. HONG & D. X. LIU (2012): Binding of the 5'-untranslated region of coronavirus
RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and
transcription. Nucleic Acids Res 40, 5065-5077
TANG, B. S., K. H. CHAN, V. C. CHENG, P. C. WOO, S. K. LAU, C. C. LAM, T. L. CHAN, A. K. WU, I.
F. HUNG, S. Y. LEUNG & K. Y. YUEN (2005): Comparative host gene transcription by microarray
analysis early after infection of the Huh7 cell line by severe acute respiratory syndrome
coronavirus and human coronavirus 229E. J Virol 79, 6180-6193
TANG, X. C., J. X. ZHANG, S. Y. ZHANG, P. WANG, X. H. FAN, L. F. LI, G. LI, B. Q. DONG, W. LIU,
C. L. CHEUNG, K. M. XU, W. J. SONG, D. VIJAYKRISHNA, L. L. POON, J. S. PEIRIS, G. J. SMITH,
H. CHEN & Y. GUAN (2006): Prevalence and genetic diversity of coronaviruses in bats from
China. J Virol 80, 7481-7490
TARENDEAU, F., T. CREPIN, D. GUILLIGAY, R. W. RUIGROK, S. CUSACK & D. J. HART (2008):
Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of
influenza virus polymerase PB2 subunit. PLoS Pathog 4, e1000136
TAYLOR, L. H., S. M. LATHAM & M. E. WOOLHOUSE (2001): Risk factors for human disease
emergence. Philos Trans R Soc Lond B Biol Sci 356, 983-989
TEELING, E. C., M. S. SPRINGER, O. MADSEN, P. BATES, J. O'BRIEN S & W. J. MURPHY (2005):
A molecular phylogeny for bats illuminates biogeography and the fossil record. Science 307,
580-584
THORP, E. B., J. A. BOSCARINO, H. L. LOGAN, J. T. GOLETZ & T. M. GALLAGHER (2006):
Palmitoylations on murine coronavirus spike proteins are essential for virion assembly and
infectivity. J Virol 80, 1280-1289
TONG, S., C. CONRARDY, S. RUONE, I. V. KUZMIN, X. GUO, Y. TAO, M. NIEZGODA, L. HAYNES,
B. AGWANDA, R. F. BREIMAN, L. J. ANDERSON & C. E. RUPPRECHT (2009): Detection of novel
SARS-like and other coronaviruses in bats from Kenya. Emerg Infect Dis 15, 482-485
TOOZE, J., S. TOOZE & G. WARREN (1984): Replication of coronavirus MHV-A59 in sac- cells:
determination of the first site of budding of progeny virions. European journal of cell biology 33,
281-293
TOWNER, J. S., B. R. AMMAN, T. K. SEALY, S. A. CARROLL, J. A. COMER, A. KEMP, R.
SWANEPOEL, C. D. PADDOCK, S. BALINANDI, M. L. KHRISTOVA, P. B. FORMENTY, C. G.
ALBARINO, D. M. MILLER, Z. D. REED, J. T. KAYIWA, J. N. MILLS, D. L. CANNON, P. W. GREER,
E. BYARUHANGA, E. C. FARNON, P. ATIMNEDI, S. OKWARE, E. KATONGOLE-MBIDDE, R.
DOWNING, J. W. TAPPERO, S. R. ZAKI, T. G. KSIAZEK, S. T. NICHOL & P. E. ROLLIN (2009):
Isolation of genetically diverse Marburg viruses from Egyptian fruit bats. PLoS Pathog 5,
e1000536
TOWNER, J. S., X. POURRUT, C. G. ALBARINO, C. N. NKOGUE, B. H. BIRD, G. GRARD, T. G.
KSIAZEK, J. P. GONZALEZ, S. T. NICHOL & E. M. LEROY (2007): Marburg virus infection
detected in a common African bat. PLoS One 2, e764
TRESNAN, D. B., R. LEVIS & K. V. HOLMES (1996): Feline aminopeptidase N serves as a
receptor for feline, canine, porcine, and human coronaviruses in serogroup I. J Virol 70, 86698674
TSUDA, S., S. WATANABE, J. S. MASANGKAY, T. MIZUTANI, P. ALVIOLA, N. UEDA, K. IHA, S.
TANIGUCHI, H. FUJII, K. KATO, T. HORIMOTO, S. KYUWA, Y. YOSHIKAWA & H. AKASHI (2012):
Genomic and serological detection of bat coronavirus from bats in the Philippines. Arch Virol
157, 2349-2355
39
Introduction
TU, C., G. CRAMERI, X. KONG, J. CHEN, Y. SUN, M. YU, H. XIANG, X. XIA, S. LIU, T. REN, Y. YU,
B. T. EATON, H. XUAN & L. F. WANG (2004): Antibodies to SARS coronavirus in civets. Emerg
Infect Dis 10, 2244-2248
TURMELLE, A. S., J. A. ELLISON, M. T. MENDONCA & G. F. MCCRACKEN (2010): Histological
assessment of cellular immune response to the phytohemagglutinin skin test in Brazilian freetailed bats (Tadarida brasiliensis). J Comp Physiol B 180, 1155-1164
TUSELL, S. M., S. A. SCHITTONE &
K. V. HOLMES (2007): Mutational analysis of
aminopeptidase N, a receptor for several group 1 coronaviruses, identifies key determinants of
viral host range. J Virol 81, 1261-1273
UJIKE, M. & F. TAGUCHI (2015): Incorporation of spike and membrane glycoproteins into
coronavirus virions. Viruses 7, 1700-1725
VABRET, A., J. DINA, E. BRISON, J. BROUARD & F. FREYMUTH (2009): [Human coronaviruses].
Pathologie-biologie 57, 149-160
VALLEE, R. B., J. C. WILLIAMS, D. VARMA & L. E. BARNHART (2004): Dynein: An ancient motor
protein involved in multiple modes of transport. J Neurobiol 58, 189-200
VAN DER POEL, W. H., P. H. LINA & J. A. KRAMPS (2006): Public health awareness of emerging
zoonotic viruses of bats: a European perspective. Vector Borne Zoonotic Dis 6, 315-324
VENNEMA, H., G. J. GODEKE, J. W. ROSSEN, W. F. VOORHOUT, M. C. HORZINEK, D. J.
OPSTELTEN & P. J. ROTTIER (1996): Nucleocapsid-independent assembly of coronavirus-like
particles by co-expression of viral envelope protein genes. EMBO J 15, 2020-2028
VOGELS, M. W., B. W. VAN BALKOM, D. V. KALOYANOVA, J. J. BATENBURG, A. J. HECK, J. B.
HELMS, P. J. ROTTIER & C. A. DE HAAN (2011): Identification of host factors involved in
coronavirus replication by quantitative proteomics analysis. Proteomics 11, 64-80
WANG, J., S. FANG, H. XIAO, B. CHEN, J. P. TAM & D. X. LIU (2009): Interaction of the
coronavirus infectious bronchitis virus membrane protein with beta-actin and its implication in
virion assembly and budding. PLoS One 4, e4908
WANG, L. F. & B. T. EATON (2007): Bats, civets and the emergence of SARS. Curr Top Microbiol
Immunol 315, 325-344
WANG, Y. & X. ZHANG (1999): The nucleocapsid protein of coronavirus mouse hepatitis virus
interacts with the cellular heterogeneous nuclear ribonucleoprotein A1 in vitro and in vivo.
Virology 265, 96-109
WARD, B. M. & B. MOSS (2004): Vaccinia virus A36R membrane protein provides a direct link
between intracellular enveloped virions and the microtubule motor kinesin. J Virol 78, 24862493
WATANABE, S., J. S. MASANGKAY, N. NAGATA, S. MORIKAWA, T. MIZUTANI, S. FUKUSHI, P.
ALVIOLA, T. OMATSU, N. UEDA, K. IHA, S. TANIGUCHI, H. FUJII, S. TSUDA, M. ENDOH, K. KATO,
Y. TOHYA, S. KYUWA, Y. YOSHIKAWA & H. AKASHI (2010): Bat coronaviruses and experimental
infection of bats, the Philippines. Emerg Infect Dis 16, 1217-1223
WEATHERBEE, J. A. (1981): Membranes and cell movement: interactions of membranes with
the proteins of the cytoskeleton. Int Rev Cytol Suppl 12, 113-176
WEBBY, R., E. HOFFMANN & R. WEBSTER (2004): Molecular constraints to interspecies
transmission of viral pathogens. Nat Med 10, S77-81
40
Introduction
WEISS, S., K. NOWAK, J. FAHR, G. WIBBELT, J. V. MOMBOULI, H. J. PARRA, N. D. WOLFE, B. S.
SCHNEIDER & F. H. LEENDERTZ (2012): Henipavirus-related sequences in fruit bat bushmeat,
Republic of Congo. Emerg Infect Dis 18, 1536-1537
WENTWORTH, D. E. & K. V. HOLMES (2007): Coronavirus Binding and Entry. In: V. THIEL
"Coronaviruses: Molecular and Cellular Biology" Caister Academic Press, Norfolk, UK, pp. 3-31
WESLEY, R. D., R. D. WOODS & A. K. CHEUNG (1990a): Genetic basis for the pathogenesis of
transmissible gastroenteritis virus. J Virol 64, 4761-4766
WESLEY, R. D., R. D. WOODS & A. K. CHEUNG (1991): Genetic analysis of porcine respiratory
coronavirus, an attenuated variant of transmissible gastroenteritis virus. J Virol 65, 3369-3373
WESLEY, R. D., R. D. WOODS, H. T. HILL & J. D. BIWER (1990b): Evidence for a porcine
respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the
United States. Journal of veterinary diagnostic investigation : official publication of the American
Association of Veterinary Laboratory Diagnosticians, Inc 2, 312-317
WHO-World Health Organization (2015): Zoonoses.
http://www.who.int/topics/zoonoses/en/ (20 Dec 2015)
WILLIAMSON, M. M., P. T. HOOPER, P. W. SELLECK, L. J. GLEESON, P. W. DANIELS, H. A.
WESTBURY &
P. K. MURRAY (1998): Transmission studies of Hendra virus (equine
morbillivirus) in fruit bats, horses and cats. Aust Vet J 76, 813-818
WILLIAMSON, M. M., P. T. HOOPER, P. W. SELLECK, H. A. WESTBURY & R. F. SLOCOMBE
(2000): Experimental hendra virus infectionin pregnant guinea-pigs and fruit Bats (Pteropus
poliocephalus). J Comp Pathol 122, 201-207
WINDER, S. J. & K. R. AYSCOUGH (2005): Actin-binding proteins. J Cell Sci 118, 651-654
WINTER, C., C. SCHWEGMANN-WESSELS, U. NEUMANN & G. HERRLER (2008): The spike
protein of infectious bronchitis virus is retained intracellularly by a tyrosine motif. J Virol 82,
2765-2771
WONG, S., S. LAU, P. WOO & K. Y. YUEN (2007): Bats as a continuing source of emerging
infections in humans. Rev Med Virol 17, 67-91
WOO, P. C., S. K. LAU, Y. HUANG & K. Y. YUEN (2009): Coronavirus diversity, phylogeny and
interspecies jumping. Exp Biol Med (Maywood) 234, 1117-1127
WOO, P. C., S. K. LAU, C. S. LAM, C. C. LAU, A. K. TSANG, J. H. LAU, R. BAI, J. L. TENG, C. C.
TSANG, M. WANG, B. J. ZHENG, K. H. CHAN & K. Y. YUEN (2012): Discovery of seven novel
Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat
coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian
coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol 86, 39954008
WOO, P. C., S. K. LAU, K. S. LI, R. W. POON, B. H. WONG, H. W. TSOI, B. C. YIP, Y. HUANG, K. H.
CHAN & K. Y. YUEN (2006): Molecular diversity of coronaviruses in bats. Virology 351, 180-187
WOO, P. C., M. WANG, S. K. LAU, H. XU, R. W. POON, R. GUO, B. H. WONG, K. GAO, H. W. TSOI,
Y. HUANG, K. S. LI, C. S. LAM, K. H. CHAN, B. J. ZHENG & K. Y. YUEN (2007): Comparative
analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals
unique group and subgroup features. J Virol 81, 1574-1585
WURM, T., H. CHEN, T. HODGSON, P. BRITTON, G. BROOKS & J. A. HISCOX (2001):
Localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the
protein may disrupt host cell division. J Virol 75, 9345-9356
41
Introduction
XU, L., S. KHADIJAH, S. FANG, L. WANG, F. P. TAY & D. X. LIU (2010): The cellular RNA helicase
DDX1 interacts with coronavirus nonstructural protein 14 and enhances viral replication. J Virol
84, 8571-8583
YADAV, P. D., C. G. RAUT, A. M. SHETE, A. C. MISHRA, J. S. TOWNER, S. T. NICHOL & D. T.
MOURYA (2012): Detection of Nipah virus RNA in fruit bat (Pteropus giganteus) from India. Am
J Trop Med Hyg 87, 576-578
YAMADA, Y. & D. X. LIU (2009): Proteolytic activation of the spike protein at a novel RRRR/S
motif is implicated in furin-dependent entry, syncytium formation, and infectivity of
coronavirus infectious bronchitis virus in cultured cells. J Virol 83, 8744-8758
YANG, L., Z. WU, X. REN, F. YANG, G. HE, J. ZHANG, J. DONG, L. SUN, Y. ZHU, J. DU, S. ZHANG
& Q. JIN (2013): Novel SARS-like betacoronaviruses in bats, China, 2011. Emerg Infect Dis 19,
989-991
YANG, Y., L. DU, C. LIU, L. WANG, C. MA, J. TANG, R. S. BARIC, S. JIANG & F. LI (2014):
Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human
transmission of MERS coronavirus. Proc Natl Acad Sci U S A 111, 12516-12521
YE, G. J., K. T. VAUGHAN, R. B. VALLEE & B. ROIZMAN (2000): The herpes simplex virus 1
U(L)34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear
membrane. J Virol 74, 1355-1363
YEAGER, C. L., R. A. ASHMUN, R. K. WILLIAMS, C. B. CARDELLICHIO, L. H. SHAPIRO, A. T.
LOOK & K. V. HOLMES (1992): Human aminopeptidase N is a receptor for human coronavirus
229E. Nature 357, 420-422
YOB, J. M., H. FIELD, A. M. RASHDI, C. MORRISSY, B. VAN DER HEIDE, P. ROTA, A. BIN
ADZHAR, J. WHITE, P. DANIELS, A. JAMALUDDIN & T. KSIAZEK (2001): Nipah virus infection in
bats (order Chiroptera) in peninsular Malaysia. Emerg Infect Dis 7, 439-441
YOGO, Y., N. HIRANO, S. HINO, H. SHIBUTA & M. MATUMOTO (1977): Polyadenylate in the
virion RNA of mouse hepatitis virus. Journal of biochemistry 82, 1103-1108
YOO, D. W., M. D. PARKER & L. A. BABIUK (1991): The S2 subunit of the spike glycoprotein of
bovine coronavirus mediates membrane fusion in insect cells. Virology 180, 395-399
YUAN, J., C. C. HON, Y. LI, D. WANG, G. XU, H. ZHANG, P. ZHOU, L. L. POON, T. T. LAM, F. C.
LEUNG & Z. SHI (2010): Intraspecies diversity of SARS-like coronaviruses in Rhinolophus
sinicus and its implications for the origin of SARS coronaviruses in humans. J Gen Virol 91,
1058-1062
YUAN, X., Z. YAO, Y. SHAN, B. CHEN, Z. YANG, J. WU, Z. ZHAO, J. CHEN & Y. CONG (2005):
Nucleolar localization of non-structural protein 3b, a protein specifically encoded by the severe
acute respiratory syndrome coronavirus. Virus Res 114, 70-79
ZHANG, X., H. SHI, J. CHEN, D. SHI, H. DONG & L. FENG (2015): Identification of the interaction
between vimentin and nucleocapsid protein of transmissible gastroenteritis virus. Virus Res
200, 56-63
ZHANG, X., H. Y. SHI, J. F. CHEN, D. SHI, H. W. LANG, Z. T. WANG & L. FENG (2013):
Identification of cellular proteome using two-dimensional difference gel electrophoresis in ST
cells infected with transmissible gastroenteritis coronavirus. Proteome science 11, 31
ZHAO, S., J. GAO, L. ZHU & Q. YANG (2014): Transmissible gastroenteritis virus and porcine
epidemic diarrhoea virus infection induces dramatic changes in the tight junctions and
microfilaments of polarized IPEC-J2 cells. Virus Res 192, 34-45
42
Introduction
ZHONG, Y., Y. W. TAN & D. X. LIU (2012): Recent progress in studies of arterivirus- and
coronavirus-host interactions. Viruses 4, 980-1010
ZIEBUHR, J. & E. J. SNIJDER (2007): The Coronavirus Replicase Gene: Special Enzymes for
Special Viruses. In: V. THIEL "Coronaviruses: Molecular and Cellular Biology" Caister Academic
Press, Norfolk, UK, pp. 33-63
ZIEBUHR, J., E. J. SNIJDER & A. E. GORBALENYA (2000): Virus-encoded proteinases and
proteolytic processing in the Nidovirales. J Gen Virol 81, 853-879
43
44
Manuscript I
2 Manuscript I
Release of infectious virus particles as well as spike and
membrane protein expression pattern differ within
chiropteran cell lines infected by the Alphacoronavirus
TGEV
Anna-Theresa Rüdiger1, Marcel Alexander Müller2, Christel Schwegmann-Weßels1
1
Institute of Virology, University of Veterinary Medicine Hannover, Bünteweg 17,
30559 Hannover, Germany
2
University of Bonn Medical Center, Institute of Virology, Bonn, Germany
The extend of ATR’s contribution to the article is evaluated according to the following
scale:
A. Has contributed to collaboration (0-33 %).
B. Has contributed significantly (34-66 %).
C. Has essentially performed this study independently (67-100 %).
1. Design of the project including design of individual experiments: C
2. Performing of the experimental part of the study: C
3. Analysis of the experiments: C
4. Presentation and discussion of the study in article form: C
45
Manuscript I
2.1 Introduction
Coronaviruses (CoV) belong to the order Nidovirales and are enveloped viruses with
a positive single-stranded RNA genome of about 30 kb (SPAAN et al. 1988). In its
lipid bilayer the membrane protein (M), spike protein (S), envelope protein (E) and in
some cases the hemagglutinin esterase protein (HE) are embedded. Within the virion
the nucleocapsid protein (N) associates with the viral RNA. In general, viruses can
only replicate successfully in host cells when using the host cell machinery.
Therefore, each virus has developed different patterns how to interfere the host for its
own advantage. Cells infected by CoV show alterations in their cell cycle,
transcription and translation pattern, in their cytoskeleton as well as apoptosis. The
balance of positively and negatively regulated host genes may explain pathogenesis
and determine pathogenicity (ENJUANES et al. 2006). The coronavirus S protein
mediates host-cell-attachment and virus entry. The receptor binding domain is an
important part for host range determination because it interacts with the viral receptor
(GRAHAM & BARIC 2010). It has been shown that the exchange of the S
ectodomain among CoVs leads to host range alteration (KUO et al. 2000; HAIJEMA
et al. 2003).During translation, the viral proteins S, M, and E are embedded in the
endoplasmatic reticulum. In contrast, the nucleocapsid protein remains in the
cytoplasm and binds to the viral genomic RNA. The interaction between the N
proteins and the M, E, and S proteins at the membrane of the endoplasmatic
reticulum-Golgi intermediate compartment (ERGIC) results in virus assembly and
budding (KLUMPERMAN et al. 1994; KRIJNSE-LOCKER et al. 1994; VENNEMA et
al. 1996; DE VRIES et al. 1997). Viral particles are transported via the exocytotic
pathway to the plasma membrane and are released via exocytosis.
Coronaviruses show a narrow host range but their high recombination and mutation
rate promote spill-over events to new hosts as well as ecological niches (LAI &
CAVANAGH 1997; HERREWEGH et al. 1998; WOO et al. 2006b; ZENG et al. 2008;
WOO et al. 2009; LAU et al. 2011). Several host-shifting events from animal-tohuman and from animal-to-animal are described for CoVs (GUAN et al. 2003;
CONSORTIUM 2004; LAU et al. 2005). Studies on bats as potential zoonotic
reservoir were intensified after the severe acute respiratory syndrome (SARS)
outbreak in China in 2002/2003, when almost 800 people were killed by the SARSCoV (PEIRIS et al. 2004; LI et al. 2005). Until now more than 930 bat species are
46
Manuscript I
known which exist all over the world apart from arctic and antarctic. They are
nocturnal and hide in caves, rock crevices, trees, old mines or buildings during the
day (WONG et al. 2007). Depending on the species, the dietary of bats comprises
insects, small mammals, fish, blood, fruits or pollen. Most bats roost in very large
populations which facilitate the spread of pathogens. More than 80 different viruses
(Rhabdoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Flaviviridae,
Arenaviridae, Herpesviridae Picornaviridae, etc.) were isolated from bats, yet. The
high species diversity of bats, their wide geographical distribution and their ability to
fly are considered risk factors for zoonotic transmission (CALISHER et al. 2006).
Until now, several Alpha- as well as Betacoronaviruses were identified in bats (WOO
et al. 2006a; DREXLER et al. 2010). To date, no viable Alphacoronavirus could be
isolated from bats directly.
The
transmissible
gastroenteritis
virus
(TGEV)
belongs
to
the
genus
Alphacoronavirus and infects pigs. It replicates within villous epithelial cells of the
small intestine and in lung cells (SAIF & BOHL 1986; ENJUANES & VAN DER
ZEIJST 1995). The specific receptor of TGEV is the porcine aminopeptidase N
(pAPN). The viral spike protein is essential for entry and membrane fusion, while the
membrane protein M is necessary for virus assembly (ROTTIER 1995).
To understand the alphacoronaviral replication in bats, a model virus like TGEV may
be highly useful. We investigated the entry and replication as well as the amount of
released infectious virus particles of TGEV as a representative of the genus
Alphacoronavirus in different bat cell species. For comparison we used swine
influenza A virus (SIV) of the H3N2 subtype, the vesicular stomatitis virus (VSV) as
well as the Gammacoronavirus infectious bronchitis virus (IBV).
2.2 Material & Methods
2.2.1 Cell lines and virus strains
ST (swine testicular cells), BHK-21 (Baby hamster kidney cells), HypNi/1.1
(Hypsignathus monstrosus kidney cells) (KUHL et al. 2011), MyDauDa/46 (Myotis
daubentonii intestine cells), Tb1Lu (Tadarida brasiliensis lung cells; provided by
Friedrich-Loeffler-Institut, Insel Riems, Germany), PipNi/1 (Pipistrellus pipistrellus
kidney cells) (MULLER et al. 2012), MDCK II (Madin-Darby canine kidney) and Vero
(African green monkey kidney cells) cells were grown in Dulbecco's modified Eagle
47
Manuscript I
medium supplemented with fetal calf serum (10 % for ST, 5 % for BHK-21,
HypNi/1.1, MyDauDa/46, Tb1Lu, PipNi/1, MDCK II, Vero).
Trapping, sampling, and testing of animals were approved by the Landesamt für
Naturschutz und Umwelt (LANUV); ethics permit: LANU 314/5327.74.1.6, Ghana
(Ministry of Food and Agriculture, Wildlife Division, Forestry Commission, Accra;
research permit A04957; ethics permit: CHRPE49/09; export permit: state contract
between Ghana and Germany; Gabon (Ministry of Water and Forest, Libreville;
ethics and export permit: 00021/MEFEPA/SG/DGEF/DFC). Bats were handled
according to national and European legislation for the protection of animals (EU
council directive 86/609/EEC). Maximum efforts were made to leave animals
unharmed or to minimize suffering of animals. All bats were caught with mist nets
and handled by trained personnel. Any surgical procedure was performed under
sodium pentobarbital/ketamine anesthesia. In case of Myotis daubentonii and
Pipstrellus pipistrellus, dead animals, that were delivered to the Noctalis - Bat Center
(Bad Segeberg, Germany), were used for cell culture preparations.
The Purdue strain of TGEV (PUR46-MAD, provided by L. Enjuanes) was propagated
on ST cells, VSV strain Indiana on BHK-21 cells, swine influenza A virus of the H3N2
subtype (strain A/sw/Bissendorf/IDT1864/2003; provided by Ralf Dürrwald, IDT
Biologika GmbH) on MDCK II cells and IBV Beaudette on Vero cells. After 24 h of
incubation at 37 °C the supernatants were harvested, centrifuged and after addition
of 1 % fetal calf serum stored at -80 °C.
2.2.2 Plasmids
The pAPN gene was cloned into the pEGFP-C1 plasmid (Clonetech) by BamHI and
XhoI restriction sites (Fermentas, St. Leon-Rot) using standard PCR techniques.
DNA sequencing was done by Eurofins MWG Operon.
2.2.3 Immunofluorescence analysis
2.2.3.1
Viral infection
Cells were seeded in 24-well plates. The next day cells were infected with TGEV,
SIV-H3N2, VSV or IBV with a MOI of 1.5 for 1 h at 37 °C. One day post infection cells
were fixed with 3 % paraformaldehyde, permeabilized with 0.2 % Triton-X-100 and
48
Manuscript I
treated with antibodies against viral proteins. The TGEV S protein was detected by
mouse monoclonal antibody (6A.C3, provided by Luis Enjuanes) at a dilution of 1:200
(GEBAUER et al. 1991) followed by a polyclonal Cy3-labeled sheep-anti-mouse
antibody (1:500, Sigma). For the detection of the SIV-H3N2, a monoclonal antibody
against the influenza A virus nucleoprotein (1:750, AbDSeroTec, Düsseldorf) and for
the VSV G protein detection the monoclonal antibodies I-1 and I-14 were used
(1:100) (HANIKA et al. 2005). For fluorescence-labeling, FITC-conjugated antimouse antibody (1:100, Sigma) was used. The IBV antigen was stained with
polyclonal anti-IBV Beaudette serum produced in rabbits (WINTER et al. 2008a)
followed by FITC-conjugated anti-rabbit antibody (1:100, Sigma). Nuclei were stained
with DAPI (4',6-diamidino-2-phenylindole). The Nikon Eclipse Ti microscope was
used for analysis.
Regarding the TGEV infection study the same experiment was done with the cells
transfected by the help of Lipofectamine® (Life Technologies) with pEGFP-pAPN
(porcine aminopeptidase N) or the empty vector pEGFP-C1 one day before infection,
except from ST cells. The expression of endogenous pAPN in ST cells was detected
with a monoclonal mouse anti-G43 antibody (1:200, provided by Hubert Laude)
followed by a FITC-labeled anti-mouse antibody (1:100, Sigma).
2.2.3.2
Viral protein localization after TGEV infection
Cells were seeded in 24-well plates. After 24 h cells were transfected with pCG1pAPN or the empty pCG1 vector by the help of Lipofectamine ® (except from ST
cells). The next day cells were infected with TGEV with a MOI of 1.5 for 1 h at 37 °C.
One day post infection cells were fixed with 3 % paraformaldehyde, permeabilized
with 0.2 % Triton-X-100 and treated with antibodies against viral proteins. The TGEV
S protein was detected with monoclonal antibody 6A.C3 (1:200), TGEV M protein
with monoclonal antibody 9D.B4 (1:200, provided by Luis Enjuanes), TGEV N protein
with anti-coronavirus monoclonal antibody FIPV3-70 (1:1000, Thermo Scientific), and
the TGEV E protein with a monoclonal mouse anti-TGEV E (1:100, provided by Luis
Enjuanes). Fluorescence-labeling was done with a Cy3-conjugated anti-mouse
antibody (1:500, Sigma). To visualize the Golgi apparatus the galactosyltransferase
gene fused to GFP (provided by Eric Snijder) was used. For the detection of the
ERGIC the cDNA of ERGIC-53 (kindly provided by Hans-Peter Hauri) was fused to
49
Manuscript I
GFP (WINTER et al. 2008b; PAUL et al. 2014). Nuclei were stained with DAPI (4',6diamidino-2-phenylindole). The Nikon Eclipse Ti microscope was used for
fluorescence analysis.
2.2.4 Growth curve
Cells were seeded on 6-well plates. Cells which were infected with TGEV were
transfected with pEGFP-pAPN one day before infection. After infection cells were
washed three times with medium and then cultured with medium containing 3 % fetal
calf serum at 37 °C. Supernatants were collected at different time points (0, 6, 12, 18,
24, 30, and 36 h) post infection. The collected supernatants from TGEV infected cells
were incubated on ST cells, from VSV infected cells on BHK-21 cells, from SIV-H3N2
infected cells on MDCK cells and from IBV infected cells on Vero cells for 1-2 h at
37 °C. These cells were seeded on 96-well plates and inoculated with 40 µl of the
collected supernatants. Afterwards, the cells were treated with methylcellulose
overnight. The next day cells were fixed with 3 % PFA and treated with antibodies
against viral proteins (same as above). By using the Nikon Eclipse Ti microscope
plaque forming units were counted for each time point and the change in virus titer
over time was visualized in growth curves.
2.2.5 Determination of receptor expression levels by flow cytometry
2.2.5.1
Determination of pAPN expression
Cells were seeded on 6-well plates. One day later, cells were transfected with
pEGFP-pAPN or with the empty vector pCG1. The next day cells were trypsinized for
5 min and after addition of fetal calf serum centrifuged at 956 x g, 4 °C for 5 min. Cell
pellets were washed with 1 % bovine serum albumin (BSA), centrifuged again, and
diluted in 1 ml of 1 % BSA in phosphate buffered saline (PBS). The expression rate
of pAPN-GFP was measured by flow cytometry.
2.2.5.2
Determination of alpha-2,6-linked sialic acids
Cells were seeded on 6-well plates. The next day cells were trypsinized for 5 min and
after addition of fetal calf serum centrifuged at 956 x g at 4 °C for 5 min. Cell pellets
were washed with 1 % BSA/PBS and treated with SNA (Sambucus Nigra, 1:200,
50
Manuscript I
Vector laboratories) lectin conjugated to FITC for 1 h at 4 °C. Afterwards, cells were
washed three times with 1 % BSA/PBS. By flow cytometry analysis the expression
rate of alpha-2,6 sialic acids was measured.
2.2.5.3
Determination of low density lipoprotein receptor (LDL) receptor expression
To quantify the LDL receptors on the different cell lines, cells were treated as
described above and incubated with anti-rabbit LDL (1:200, Biorbyt) for 1 h at 4 °C.
Afterwards, cells were washed three times with 1 % BSA/PBS. After treatment with
anti-rabbit-FITC for 1 h at 4 °C cells were washed with 1 % BSA and then analyzed
by flow cytometry.
2.2.5.4
Determination of alpha 2,3-linked sialic acids
Cells were seeded on 6-well plates. The next day cells were trypsinized for 5 min and
after addition of fetal calf serum centrifuged at 956 x g, 4 °C for 5 min. Cell pellets
were washed with 1 % BSA/PBS and treated with biotinylated MAA (Maackia
Amurensis) lectin II (1:1000, Vector laboratories) for 1 h at 4 °C. Afterwards, cells
were washed three times with 1 % BSA/PBS. For detection of MAA binding, DyLight®
488 Streptavidin (1:100; Vector) was used for 1 h at 4 °C. Then, cells were washed
again for 3 times. The expression rate of alpha 2,3-linked sialic acids was measured
by flow cytometry.
2.3 Results
2.3.1 TGEV infection is restricted to pAPN expressing cells although the
receptor expression level does not fully correlate with the amount of
released infectious viral particles
In order to compare TGEV growth porcine cell line ST, BHK-21 as well as the
chiropteran cells HypNi/1.1 (Hypsignathus monstrosus kidney cells), MyDauDa/46
(Myotis daubentonii intestine cells), Tb1Lu (Tadarida brasiliensis lung cells), and
PipNi/1 (Pipistrellus pipistrellus kidney cells) were infected (Fig. 2-1). ST cells
endogenously express pAPN and are susceptible to TGEV. Regarding the other
tested cell lines, no infected cells were detectable. When BHK-21 cells and all
chiropteran cells of interest were transfected with pEGFP-pAPN, TGEV was able to
51
Manuscript I
enter the cells. These results confirm a previous study and show that the expression
of pAPN, the specific receptor for TGEV, is essential for this virus to enter the cells
(HOFFMANN et al. 2013).
To measure the release of infectious viral particles the cells were transfected with
pEGFP-pAPN (except for ST cells) and infected by TGEV. At several time points
post-infection, samples of the supernatant were collected. Virus titers were
determined by plaque assay (Fig. 2-2, Tab. 1).
Upon receptor expression, TGEV was able to replicate in all tested cell lines (Fig. 2-1
& Tab.1). The highest amount of infectious released virus particles was measured for
ST cells (virus titer: 7.5x106 pfu/ml) 24 h after TGEV infection. Regarding chiropteran
cells, HypNi/1.1 cells showed the highest amount of released infectious virus
particles (virus titer: 2.2x105 pfu/ml), while MyDauDa/46 cells showed the lowest
amount with a viral titer of 1.3x103 pfu/ml.
52
Manuscript I
Fig. 2-1 TGEV infection study: Immunofluorescence analysis of pAPN and TGEV S protein
expression.
Cells were infected by TGEV after transfection with the empty vector pEGFP-C1 or with pEGFPpAPN. ST cells were not transfected with pEGFP-pAPN, here the expression of the endogenous
pAPN was detected. All cells were treated with Triton-X-100; red: TGEV S protein; green: pEGFPpAPN; blue: nuclei. PFA fixation 24 hpi.
53
Manuscript I
Fig. 2-2 TGEV infection study: Growth curve.
Supernatants were collected after TGEV infection of chiropteran cells, BHK-21, and ST cells and
propagated on new seeded ST cells. All cell lines (except from ST cells) were transfected with
pEGFP-pAPN. n=3.
Tab. 1 TGEV titer measured by plaque assay 24 hpi.
After virus infection collected supernatants of chiropteran cells, BHK-21, and ST cells were
propagated on ST cells. All cell lines (except from ST cells) were transfected with pEGFP-pAPN. n=3.
pfu/ml
TGEV
24 hpi
MyDauDa
PipNi
HypNi
Tb1Lu
BHK-21
ST
Log(Mean)
3.12
4.08
5.34
4.93
4.36
6.87
Log(SD)
0.31
0.66
0.69
0.74
0.48
0.63
Mean
1.3x10
3
4
5
1.2x10
2.2x10
8.6x10
4
4
2.3x10
6
7.5x10
To find out if the different amounts of virus titers are due to the receptor expression
level on the host cells the amount of pAPN-positive cells was determined by flow
cytometry (Fig. 2-3).
BHK-21 cells showed the highest pAPN receptor expression level of about 70 % on
their cell surface 1 day after pEGFP-pAPN transfection. PipNi/1, HypNi/1.1, and
54
Manuscript I
Tb1Lu cells showed comparable results with values ranging from 21-25 %.
MyDauDa/46 cells showed the lowest amount of pAPN expression on their surfaces
with a value of nearly 14 %. Although, the pAPN expression on PipNi/1 cell surfaces
was slightly higher compared to HypNi/1.1 cells the amount of released virus
particles was higher in HypNi/1.1 cells compared to the virus titers measured in
PipNi/1 cells.
Fig. 2-3 pAPN-GFP expression level: Flow cytometry
Measurement of pAPN-GFP expression level on cell surfaces of tested chiropteran cells and BHK-21
cells. n=3.
These results indicate that the TGEV infection rate in tested host cells is dependent
on pAPN expression but does not exclusively correspond with the pAPN expression
level on cell surfaces. Further specific host factors or viral factors seem to influence
the infection rate. To find out if, for example, the higher infection rate in HypNi/1.1
cells is due to the abundance of important proteins promoting virus replication which
may not be expressed by BHK-21 cells or MyDauDa/46 cells, other virus species
were included in the study. Cells of interest were infected by an SIV of the H3N2
subtype (strain A/sw/Bissendorf/IDT1864/2003), VSV (strain Indiana), or IBV (strain
Beaudette) and viral infection was visualized by immunofluorescent staining of cells.
Virus titers as well as the endogenous receptor expression level on the cell surfaces
were determined by plaque assay and flow cytometry, respectively.
55
Manuscript I
2.3.2 The infection rate of SIV-H3N2 was similar in all tested chiropteran cell
lines despite different expression levels of the receptor determinant
The chiropteran cell lines HypNi/1.1, MyDauDa/46, Tb1Lu, and PipNi/1 as well as ST
and BHK21 cells were infected by SIV-H3N2. MDCKII cells were used as a positive
control.
All tested cell lines were susceptible to H3N2 virus (Fig. 2-4) and expressed alpha2,6-linked sialic acids on their cell surfaces, which is the preferred receptor
determinant for most mammalian influenza viruses (Fig. 2-6). The infection rate of
SIV-H3N2 in chiropteran cells, BHK-21 cells, ST cells, and MDCKII cells was
measured by plaque assay (Fig. 2-5; Tab. 2).
56
Manuscript I
Fig. 2-4 SIV-H3N2 infection study: Immunofluorescence of SIV-H3N2 nucleocapsid protein.
SIV-H3N2 uninfected/infected chiropteran cells as well as BHK-21 and ST cells. Green: SIV
nucleoprotein; blue: nuclei. PFA fixation 24 hpi.
57
Manuscript I
Fig. 2-5 SIV-H3N2 infection study: Growth curve.
Supernatants were collected after SIV infection of chiropteran cells, MDCK II, BHK-21, and ST cells
and titrated on new seeded MDCKII cells. n=3.
pfu/ml
Tab. 2 SIV-H3N2 titer measured by plaque assay 24 hpi.
After virus infection collected supernatants of chiropteran cells, BHK-21, ST, MDCKII or Vero cells
were propagated on MDCKII cells. n=3
SIV
24 hpi
MyDauDa
PipNi
HypNi
Tb1Lu
BHK-21
ST
MDCKII
Log(Mean)
5.32
6.00
5.43
5.32
4.75
5.68
6.17
Log(SD)
0.46
0.66
0.83
0.64
0.46
0.24
0.40
Mean
2.0x10
5
5
9.9x10
5
2.7x10
58
5
2.1x10
4
5.6x10
5
4.8x10
6
1.5x10
Manuscript I
Fig. 2-6 Alpha-2,6-linked sialic acid expression level: Flow cytometry.
Measurement of alpha-2,6-linked sialic acid expression levels on cell surfaces of tested chiropteran
cells, BHK-21 cells, ST cells, and MDCK II cells. n=3.
The receptor determinant of SIV-H3N2 was expressed differently in the analyzed bat
cell lines which is not reflected by infectious titers from supernatants of these cells.
PipNi/1 cells are the ones with the highest amount of alpha-2,6-linked sialic acids on
their surface (50 %) while MyDauDa/46 showed the lowest amount (12 %).
Nevertheless, the amount of released virus particles of MyDauDa/46 cells with
2.0x105 pfu/ml was slightly lower compared to PipNi/1 cells with an infectious titer of
9.9x105 pfu/ml.
The differences between HypNi/1.1 and MyDauDa/46 cells found for TGEV infection
were not detected in SIV infection.
2.3.3 Highest VSV infection rate in TB1Lu cells while receptor expression
levels were similar on bat cell surfaces
To include further viruses in our analysis, the above mentioned chiropteran cells, as
well as BHK-21 cells and ST cells were infected by VSV (Fig. 2-7, Fig. 2-8, Fig. 2-9).
In this experiment BHK-21 cells served as positive control.
59
Manuscript I
All tested cell lines were susceptible to VSV and expressed the low-density
lipoprotein (LDL) receptor on their plasma membrane (Fig. 2-7, Fig. 2-9). Respective
virus titers were determined by a plaque assay and calculated (Fig. 2-8; Tab. 3).
Regarding bat cell lines, Tb1Lu cells showed the highest amount of released
infectious virus particles 10 hours post-infection (hpi) with a viral titer of about
3.5x107 pfu/ml, while PipNi/1 cells were the ones with the lowest infection rate of
1.6x106 pfu/ml. Nevertheless, PipNi/1 cells and Tb1Lu cells both showed the highest
amounts of LDL on their surfaces (18.5-20 %). All in all, the amount of receptor
expression was similar in the analyzed chiropteran cell lines and VSV efficiently
replicated in all of them.
60
Manuscript I
Fig. 2-7 VSV infection study: Immunofluorescence of VSV glycoprotein.
VSV uninfected/ infected chiropteran cells, BHK-21 as well as ST cells. PFA fixation 24 hpi.
61
Manuscript I
Fig. 2-8 VSV infection study: Growth curve.
Supernatants were collected at 0, 4, 10, and 20 h after VSV infection of chiropteran cells, BHK-21, and
ST cells and titrated on new seeded BHK-21 cells. n=3.
pfu/ml
Tab. 3 VSV titer measured by plaque assay 24 hpi.
After virus infection collected supernatants of chiropteran cells, BHK-21, ST, MDCKII or Vero cells
were propagated on BHK-21 cells. n=3.
VSV
10 hpi
MyDauDa
PipNi
HypNi
Tb1Lu
BHK-21
ST
Log(Mean)
6.60
6.20
7.07
7.54
6.02
3.61
Log(SD)
0.69
0.51
0.78
0.39
0.59
0.31
Mean
3.9x10
6
6
1.6x10
7
1.2x10
62
3.5x10
7
6
1.0x10
3
4.1x10
Manuscript I
Fig. 2-9 LDL expression level: Flow cytometry.
Measurement of LDL expression level on cell surfaces of tested chiropteran cells, BHK-21 cells, and
ST cells. n=3.
2.3.4 The Gammacoronavirus IBV was able to enter chiropteran cell lines
To include another coronavirus species the different chiropteran cell lines as well as
ST and BHK21 cells were infected by IBV Beaudette (Fig. 2-10, Fig. 2-11, Fig. 2-12).
Vero cells served as positive control.
IBV Beaudette was able to infect all tested cell lines although the amount of bat cells
positive for viral antigen was quite low in the immunofluorescence assay (Fig. 2-10).
As for the above analyzed viruses the infection rate was measured by plaque assay
(Fig. 2-11, Tab. 4). BHK-21 cells showed the highest amount of released infectious
virus particles 24 hpi with a virus titer of about 2x107 pfu/ml. Vero cells showed the
second highest amount of released viral particles (2.1x10 5 pfu/ml). Regarding
chiropteran cells only Tb1Lu cells showed a measurable amount of released IBV
particles with a titer of about 3.7x101 pfu/ml.
63
Manuscript I
Fig. 2-10 IBV infection study: Immunofluorescence of IBV.
IBV uninfected/ infected chiropteran cells, BHK-21, ST, and Vero cells. PFA fixation 24 hpi.
64
Manuscript I
Fig. 2-11 IBV infection study: Growth curve.
Supernatants were collected after IBV infection of chiropteran cells, BHK-21, ST, and Vero
cells and titrated on new seeded Vero cells. n=3.
pfu/ml
Tab. 4 IBV titer measured by plaque assay 24 hpi.
After virus infection collected supernatants of chiropteran cells, BHK-21, ST, MDCKII or Vero cells
were propagated on Vero cells. n=3.
IBV
24 hpi
MyDauDa
PipNi
HypNi
Tb1Lu
BHK-21
ST
Vero
Log(Mean)
0.00
0.00
0.00
1.56
7.30
0.12
5.32
Log(SD)
0.00
0.00
0.00
0.22
0.33
0.24
0.77
Mean
0.0x10
0
0
0.0x10
0
0.0x10
1
3.7x10
7
2.0x10
0
1.3x10
5
2.1x10
The receptor of IBV on its natural host cells is not known yet. However, it was shown
that alpha-2,3-linked sialic acids represent receptor determinants for IBV infection
(WINTER et al. 2006). Therefore, the expression level of alpha-2,3-linked sialic acids
was determined by flow cytometry using Maackia Amurensis lectin. Interestingly, all
bat cells expressed high levels of alpha-2,3-linked sialic acids between 75 and 96 %
whereas Vero cells showed a quite low expression level of about 10 % (Fig. 2-12).
65
Manuscript I
These results demonstrate that for IBV in addition to the receptor determinant other
factors are important for successful virus entry and replication.
Fig. 2-12 Alpha-2,3-sialic acid expression level: Flow cytometry.
Measurement of alpha-2,3-sialic acid expression level on cell surfaces of tested chiropteran cells,
BHK-21, ST and Vero cells. n=3.
All tested chiropteran cell lines were susceptible for SIV H3N2, VSV and IBV, while
for successful TGEV entry the transfection of pAPN was necessary. HypNi/1.1 cells
showed the highest infection rate for TGEV but not for SIV-H3N2, VSV and IBV. In
summary, no specific chiropteran cell line was observed in which all tested virus
species had the highest replication efficiency. The TGEV receptor expression level
did not completely correlate with the measured virus titers. Accordingly to these
findings, in addition to receptor expression other cellular as well as viral factors may
influence the replication, maturation and budding process of viruses in chiropteran
cells.
The focus of this work is on the replication study of an alphacoronavirus within
chiropteran cell lines. To get more information about the virus itself after host cell
entry, TGEV proteins were localized within chiropteran cell lines. These viral factors
would give an explanation for the higher efficiency of TGEV replication in HypNi/1.1
cells compared to the other chiropteran cell lines.
66
Manuscript I
2.3.5 TGEV S and M protein expression pattern differed in the tested
chiropteran cell lines
To study the expression pattern of TGEV S, M, N and E proteins, chiropteran cells
were co-transfected with pAPN and compartment marker cDNAs fused to GFP for
the ERGIC (endoplasmatic reticulum-Golgi intermediate compartment) and Golgi
compartment (Fig. 2-13, Fig. 2-14, Fig. 2-15, Fig. 2-16). One day later, cells were
infected by TGEV. Immunofluorescence studies were done to localize the viral
proteins intracellularly 24 hpi.
The TGEV S protein was expressed differently in the examined chiropteran cell lines
(Fig. 2-13). In HypNi/1.1 and Tb1Lu cells, S was expressed within the whole
cytoplasm as well as near the ERGIC and Golgi compartment. Regarding the PipNi/1
and MyDauDa/46 cells, S was accumulated near the nucleus, ERGIC and Golgi
co-transfected with pAPN and compartment marker cDNA. Cells were treated with Triton-X-100.
r ERGIC/Golgi: green; spike protein: red; nucleus: blue. PFA fixation 24 hpi.
n of TGEV spike (S) protein 1 dpi.
compartment. No distribution over the whole cytoplasm could be observed.
The TGEV M protein was expressed similar to the S protein concerning the
examined chiropteran cell lines (Fig. 2-14).
67
The TGEV N protein was expressed similarly in all tested chiropteran cell lines (Fig.
2-15). It was evenly distributed over the whole cytoplasm whereas no accumulation
could be detected.
68
Chiropteran cells co-transfected with pAPN and compartment marker cDNA. Cells were treated with Triton-X-100. Compartment
marker for ERGIC/Golgi: green; membrane protein: red; nucleus: blue. PFA fixation 24 hpi.
Fig. 2-14 Localization of TGEV membrane (M) protein 1 dpi.
Manuscript I
Like the N protein, the TGEV E protein was expressed similarly in all tested
chiropteran cell lines (Fig. 2-16). The E protein accumulated near the nucleus,
ERGIC and Golgi compartment.
69
Chiropteran cells co-transfected with pAPN and compartment marker cDNA. Cells were treated with Triton-X-100. Compartment marker
for ERGIC/Golgi: green; nucleocapsid protein: red; nucleus: blue. PFA fixation 24 hpi.
Fig. 2-15 Localization of TGEV nucleocapsid (N) protein 1 dpi.
Manuscript I
2.4 Discussion
Chiropteran cells co-transfected with pAPN and compartment marker cDNA. Cells were treated with Triton-X-100. Compartment marker
for ERGIC/Golgi: green; envelope protein: red; nucleus: blue. PFA fixation 24 hpi.
Fig. 2-16 Localization of TGEV envelope (E) protein 1 dpi.
Manuscript I
2.4.1 Infection of different chiropteran cells by TGEV, SIV, VSV, and IBV
Viral host range depends on the interaction of the viral receptor binding protein and
the presence of the corresponding receptor on the surface of the host cell. In general
coronaviruses have a narrow host range. Most of them infect one or just a few
species (KUO et al. 2000). Porcine aminopeptidase N is the species specific receptor
for TGEV. The TGEV spike protein is essential for pAPN binding (LAUDE et al. 1986;
GODET et al. 1994; KUBO et al. 1994) and membrane fusion (DE GROOT et al.
1989). ST cells endogenously express pAPN, the specific receptor for TGEV, on their
cell surface. After TGEV infection the virus is able to enter the cells and to replicate
within the cytoplasm. We demonstrated that infection of the chiropteran cell lines
HypNi/1.1, MyDauDa/46, Tb1Lu, and PipNi/1 depends on the presence of pAPN.
After pAPN-expression the virus can enter these cells and is able to replicate.
Receptor-dependent entry by TGEV was also shown by Hoffmann et al. using
HypNi/1.1 and Tb1Lu as well as a variety of other chiropteran cell lines (HOFFMANN
70
Manuscript I
et al. 2013). In Myotis daubentonii and Pipistrellus species viral RNA of the genus
Alphacoronavirus has been detected (GLOZA-RAUSCH et al. 2008). Therefore, in
our study we did not only analyze HypNi/1.1 and Tb1Lu cells – like Hoffmann et al. –
but also the PipNi/1 and MyDauDa/46 cell lines. In addition to pAPN-dependent entry
we demonstrated that viral replication in those cell lines was possible although to a
different extent. By connecting the release of infectious TGEV with the measured
expression level of pAPN for each cell line, no relation was observed (e.g. comparing
HypNi/1.1 and PipNi/1 cells). This shows that the receptor expression level does not
correlate with the outcome of infectious virus in every tested cell line. To find out, if
the infection rate of the tested chiropteran cells is similar also when infected with
other viruses (e. g. virus replication is always more successful in HypNi/1.1 cells
compared to the other chiropteran cells), they were infected with SIV-H3N2, VSV,
and IBV. The SIV-H3N2 is an influenza A virus which binds to alpha-2,6-linked sialic
acids to enter the cells. All tested cell lines were susceptible to SIV-H3N2 which is
consistent with the endogenous expression of alpha-2,6-linked sialic acids. PipNi/1
cells showed a higher receptor expression level as well as a higher amount of
released virus particles compared to the other chiropteran cell lines. VSV belongs to
the family Rhabdoviridae and interacts with LDL (Low-Density Lipoprotein) during
virus entry (FINKELSHTEIN et al. 2013). The tested cell lines were all susceptible for
VSV. Tb1Lu and PipNi/1 cells showed a similar density of LDL receptor on their cell
surface although Tb1Lu cells had a higher release (>10 1 pfu/ml) of virus particles.
Regarding VSV the receptor expression level does not correlate with the infection
rate. Concerning IBV, a Gammacoronavirus, all tested cell lines were susceptible.
Infected ST, HypNi/1.1, PipNi/1, and MyDauDa/46 cells were observed via
immunofluorescence but no re-infection of Vero cells was visible when the
supernatants were used for titration although the amount of expressed alpha-2,3linked sialic acids in the bat cells was rather high. Reasons could be on the one hand
that the virus was not able to replicate successfully in these cells or that the virus
assembly, maturation or budding step was impaired. On the other hand the amount
of released infectious virus particles may have been too low for a successful reinfection and/or the presence of a certain – so far unknown – specific protein receptor
is necessary for efficient virus entry. Only in Tb1Lu, BHK-21, and Vero cells, a
measurable amount of IBV was able to replicate and to exit the cells as infectious
virus particles.
71
Manuscript I
The abundance of specific cellular receptors leads to virus species restriction (KUO
et al. 2000). A specific cell line in which all tested virus species can replicate more
successfully than in the other cells was not observed. HypNi/1.1 cells showed the
highest virus titer after TGEV infection compared to the other tested chiropteran cell
lines but not when infected by SIV-H3N2, VSV or IBV. This indicates that beside the
presence of a specific receptor other cellular as well as viral factors may influence
replication, maturation as well as the budding process.
2.4.2 Localization of TGEV proteins within chiropteran cell lines
To further analyze possible factors that influence the efficiency of the TGEV infection
in chiropteran cells the localization of the viral structural proteins S, M, N, and E in
the different cell lines were compared.
Within the tested chiropteran cells different patterns of the TGEV S and M expression
could be observed. In HypNi/1.1 and in Tb1Lu cells, the S protein as well as the M
protein were expressed in the whole cytoplasm whereas in PipNi/1 and MyDauDa/46
cells S and M were accumulated near the nucleus, ERGIC and Golgi compartment. A
difference in distribution of the TGEV S protein was also observed by Hoffmann et al.
(HOFFMANN et al. 2013) after infection of HypNi/1.1 and EpoNi/22.1 cells. As in the
present study, the TGEV S protein was distributed all over the cell in HypNi/1.1 cells
but in EpoNi/22.1 it was restricted to dot-like structures (HOFFMANN et al. 2013).
The TGEV assembly process takes place in the cytoplasm at the ERGIC. After
budding viral particles are transported to the plasma membrane via the secretory
pathway. The restricted localization of the TGEV S and M proteins to the ERGIC 24 h
after infection in PipNi/1 and MyDauDa/46 cells indicates impairment in virus
assembly. Additionally, retention of S and M protein within PipNi/1 and MyDauDa/46
cells due to an interaction with host factors or due to the reduction in interaction with
host factors could be reasons for the reduction in released infectious viral particles.
Within the coronaviral lipid bilayer the TGEV N is associated with the viral genome
and forms a nucleocapsid (BOSCH et al. 2005). The TGEV E protein is involved in
the coordination of virus assembly and release (MCBRIDE & MACHAMER 2010).
Both proteins, N and E, had similar expression patterns in the different chiropteran
cell lines. In all tested cells, the N protein was expressed in the whole cytoplasm
while the E, the TGEV structural protein with the lowest abundance in the virus
72
Manuscript I
particle, was accumulated near the nucleus. These findings suggest that the
observed differences in the efficiency of TGEV replication in chiropteran cells are
restricted to TGEV S and M protein expression as well as viral protein interaction with
host factors.
2.5 Conclusion
In the different investigated chiropteran cell lines various host factors seem to be
involved in the CoV replication cycle resulting in more or less successful replication
as well as virus particle release. The abundance of the species-specific receptor is
essential for virus entry. Within the host cell cytoplasm where the coronavirus
replication takes place host cell factors may influence the expression, transport, and
maturation of viral proteins which leads to different S and M expression patterns. In
this study we showed that the receptor is a primary determinant for species
restriction. The receptor expression level on the cell surface did not in all cases
correlate with the release of infectious virus particles. In further studies host factors
involved in coronaviral replication should be examined. The identification of
intracellular factors which promote the viral life cycle may be interesting for antiviral
target investigation.
Authors’ contributions
ATR and CSW conceived and designed the study. ATR performed the experiments.
ATR and CSW analyzed the data and drafted the manuscript. MAM generated and
provided HypNi/1.1, PipNi/1, and MyDauDa/46 chiropteran cell lines. All authors read
and approved the final manuscript.
Competing interests
The authors have declared that no competing interests exist.
Funding
Financial support was provided by a grant to CSW (SCHW 1408/1.1) from the
German Research Foundation (DFG). The cell cultures (HypNi/1.1, PipNi/1, and
MyDauDa/46) were generated within the framework of EU-FP7 ANTIGONE (no.
73
Manuscript I
278976) and the German Research Council (grants to Christian Drosten, University
of Bonn Medical Center DR 772/10-1, and Marcel A. Müller (MU 3564/1-1)).
CSW is funded by the Emmy Noether Programme from the DFG. ATR is a recipient
of a Georg-Christoph-Lichtenberg PhD fellowship from the Ministry for Science and
Culture of Lower Saxony.
Acknowledgements
This work was performed by ATR in partial fulfillment of the requirements for a Dr.
rer. nat. degree from the University of Veterinary Medicine Hannover.
We are grateful to L. Enjuanes, H. Laude, E. Snijder, R. Dürrwald, and H.-P. Hauri for
providing antibodies, plasmids, and virus.
2.6 References
BOSCH, B. J., C. A. DE HAAN, S. L. SMITS & P. J. ROTTIER (2005): Spike protein assembly into
the coronavirion: exploring the limits of its sequence requirements. Virology 334, 306-318
CALISHER, C. H., J. E. CHILDS, H. E. FIELD, K. V. HOLMES & T. SCHOUNTZ (2006): Bats:
important reservoir hosts of emerging viruses. Clin Microbiol Rev 19, 531-545
CONSORTIUM, C. S. M. E. (2004): Molecular evolution of the SARS coronavirus during the
course of the SARS epidemic in China. Science 303, 1666-1669
DE GROOT, R. J., R. W. VAN LEEN, M. J. DALDERUP, H. VENNEMA, M. C. HORZINEK & W. J.
SPAAN (1989): Stably expressed FIPV peplomer protein induces cell fusion and elicits
neutralizing antibodies in mice. Virology 171, 493-502
DE VRIES, A. A., M. C. HORZINEK, P. J. ROTTIER & R. J. DE GROOT (1997): The genome
organization of the Nidovirales: similarities and differences between arteri-, toro-, and
coronaviruses. In: Seminars in virology, 33-47
DREXLER, J. F., F. GLOZA-RAUSCH, J. GLENDE, V. M. CORMAN, D. MUTH, M. GOETTSCHE, A.
SEEBENS, M. NIEDRIG, S. PFEFFERLE, S. YORDANOV, L. ZHELYAZKOV, U. HERMANNS, P.
VALLO, A. LUKASHEV, M. A. MULLER, H. DENG, G. HERRLER & C. DROSTEN (2010): Genomic
characterization of severe acute respiratory syndrome-related coronavirus in European bats
and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene
sequences. Journal of virology 84, 11336-11349
ENJUANES, L., F. ALMAZAN, I. SOLA & S. ZUNIGA (2006): Biochemical aspects of coronavirus
replication and virus-host interaction. Annu Rev Microbiol 60, 211-230
ENJUANES, L. & B. A. M. VAN DER ZEIJST (1995): Molecular basis of transmissible
gastroenteritis coronavirus (TGEV) epidemiology. In: S. G. SIDDELL "The Coronaviridae" Plenum
Press, New York, pp. 337–376
FINKELSHTEIN, D., A. WERMAN, D. NOVICK, S. BARAK & M. RUBINSTEIN (2013): LDL receptor
and its family members serve as the cellular receptors for vesicular stomatitis virus.
Proceedings of the National Academy of Sciences of the United States of America 110, 7306-7311
74
Manuscript I
GEBAUER, F., W. P. POSTHUMUS, I. CORREA, C. SUNE, C. SMERDOU, C. M. SANCHEZ, J. A.
LENSTRA, R. H. MELOEN & L. ENJUANES (1991): Residues involved in the antigenic sites of
transmissible gastroenteritis coronavirus S glycoprotein. Virology 183, 225-238
GLOZA-RAUSCH, F., A. IPSEN, A. SEEBENS, M. GOTTSCHE, M. PANNING, J. F. DREXLER, N.
PETERSEN, A. ANNAN, K. GRYWNA, M. MULLER, S. PFEFFERLE & C. DROSTEN (2008):
Detection and prevalence patterns of group I coronaviruses in bats, northern Germany. Emerg
Infect Dis 14, 626-631
GODET, M., J. GROSCLAUDE, B. DELMAS & H. LAUDE (1994): Major receptor-binding and
neutralization determinants are located within the same domain of the transmissible
gastroenteritis virus (coronavirus) spike protein. Journal of virology 68, 8008-8016
GRAHAM, R. L. & R. S. BARIC (2010): Recombination, reservoirs, and the modular spike:
mechanisms of coronavirus cross-species transmission. Journal of virology 84, 3134-3146
GUAN, Y., B. J. ZHENG, Y. Q. HE, X. L. LIU, Z. X. ZHUANG, C. L. CHEUNG, S. W. LUO, P. H. LI, L.
J. ZHANG, Y. J. GUAN, K. M. BUTT, K. L. WONG, K. W. CHAN, W. LIM, K. F. SHORTRIDGE, K. Y.
YUEN, J. S. PEIRIS & L. L. POON (2003): Isolation and characterization of viruses related to the
SARS coronavirus from animals in southern China. Science 302, 276-278
HAIJEMA, B. J., H. VOLDERS & P. J. ROTTIER (2003): Switching species tropism: an effective
way to manipulate the feline coronavirus genome. Journal of virology 77, 4528-4538
HANIKA, A., B. LARISCH, E. STEINMANN, C. SCHWEGMANN-WESSELS, G. HERRLER & G.
ZIMMER (2005): Use of influenza C virus glycoprotein HEF for generation of vesicular
stomatitis virus pseudotypes. The Journal of general virology 86, 1455-1465
HERREWEGH, A. A., I. SMEENK, M. C. HORZINEK, P. J. ROTTIER & R. J. DE GROOT (1998):
Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination
between feline coronavirus type I and canine coronavirus. Journal of virology 72, 4508-4514
HOFFMANN, M., M. A. MULLER, J. F. DREXLER, J. GLENDE, M. ERDT, T. GUTZKOW, C.
LOSEMANN, T. BINGER, H. DENG, C. SCHWEGMANN-WESSELS, K. H. ESSER, C. DROSTEN &
G. HERRLER (2013): Differential sensitivity of bat cells to infection by enveloped RNA viruses:
coronaviruses, paramyxoviruses, filoviruses, and influenza viruses. PloS one 8, e72942
KLUMPERMAN, J., J. K. LOCKER, A. MEIJER, M. C. HORZINEK, H. J. GEUZE & P. J. ROTTIER
(1994): Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion
budding. Journal of virology 68, 6523-6534
KRIJNSE-LOCKER, J., M. ERICSSON, P. J. ROTTIER & G. GRIFFITHS (1994): Characterization of
the budding compartment of mouse hepatitis virus: evidence that transport from the RER to
the Golgi complex requires only one vesicular transport step. The Journal of cell biology 124, 5570
KUBO, H., Y. K. YAMADA & F. TAGUCHI (1994): Localization of neutralizing epitopes and the
receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus
spike protein. Journal of virology 68, 5403-5410
KUHL, A., M. HOFFMANN, M. A. MULLER, V. J. MUNSTER, K. GNIRSS, M. KIENE, T. S. TSEGAYE,
G. BEHRENS, G. HERRLER, H. FELDMANN, C. DROSTEN & S. POHLMANN (2011): Comparative
analysis of Ebola virus glycoprotein interactions with human and bat cells. J Infect Dis 204
Suppl 3, S840-849
KUO, L., G. J. GODEKE, M. J. RAAMSMAN, P. S. MASTERS & P. J. ROTTIER (2000): Retargeting
of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell
species barrier. Journal of virology 74, 1393-1406
75
Manuscript I
LAI, M. M. & D. CAVANAGH (1997): The molecular biology of coronaviruses. Adv Virus Res 48, 1100
LAU, S. K., P. LEE, A. K. TSANG, C. C. YIP, H. TSE, R. A. LEE, L. Y. SO, Y. L. LAU, K. H. CHAN, P.
C. WOO & K. Y. YUEN (2011): Molecular epidemiology of human coronavirus OC43 reveals
evolution of different genotypes over time and recent emergence of a novel genotype due to
natural recombination. Journal of virology 85, 11325-11337
LAU, S. K., P. C. WOO, K. S. LI, Y. HUANG, H. W. TSOI, B. H. WONG, S. S. WONG, S. Y. LEUNG,
K. H. CHAN & K. Y. YUEN (2005): Severe acute respiratory syndrome coronavirus-like virus in
Chinese horseshoe bats. Proceedings of the National Academy of Sciences of the United States of
America 102, 14040-14045
LAUDE, H., J. M. CHAPSAL, J. GELFI, S. LABIAU & J. GROSCLAUDE (1986): Antigenic structure
of transmissible gastroenteritis virus. I. Properties of monoclonal antibodies directed against
virion proteins. The Journal of general virology 67 ( Pt 1), 119-130
LI, W., Z. SHI, M. YU, W. REN, C. SMITH, J. H. EPSTEIN, H. WANG, G. CRAMERI, Z. HU, H.
ZHANG, J. ZHANG, J. MCEACHERN, H. FIELD, P. DASZAK, B. T. EATON, S. ZHANG & L. F.
WANG (2005): Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679
MCBRIDE, C. E. & C. E. MACHAMER (2010): A single tyrosine in the severe acute respiratory
syndrome coronavirus membrane protein cytoplasmic tail is important for efficient interaction
with spike protein. Journal of virology 84, 1891-1901
MULLER, M. A., V. S. RAJ, D. MUTH, B. MEYER, S. KALLIES, S. L. SMITS, R. WOLLNY, T. M.
BESTEBROER, S. SPECHT, T. SULIMAN, K. ZIMMERMANN, T. BINGER, I. ECKERLE, M.
TSCHAPKA, A. M. ZAKI, A. D. OSTERHAUS, R. A. FOUCHIER, B. L. HAAGMANS & C. DROSTEN
(2012): Human coronavirus EMC does not require the SARS-coronavirus receptor and
maintains broad replicative capability in mammalian cell lines. mBio 3,
PAUL, A., A. TRINCONE, S. SIEWERT, G. HERRLER & C. SCHWEGMANN-WESSELS (2014): A
lysine-methionine exchange in a coronavirus surface protein transforms a retention motif into
an endocytosis signal. Biological chemistry 395, 657-665
PEIRIS, J. S., Y. GUAN & K. Y. YUEN (2004): Severe acute respiratory syndrome. Nat Med 10,
S88-97
ROTTIER, P. J. M. (1995): The coronavirus membrane glycoprotein. In: S. G. SIDDELL "The
Coronaviridae" Plenum Press, New York, pp. 115-139
SAIF, L. J. & E. BOHL (1986): Transmissible gastroenteritis.
SPAAN, W., D. CAVANAGH & M. C. HORZINEK (1988): Coronaviruses: structure and genome
expression. The Journal of general virology 69 ( Pt 12), 2939-2952
VENNEMA, H., G. J. GODEKE, J. W. ROSSEN, W. F. VOORHOUT, M. C. HORZINEK, D. J.
OPSTELTEN & P. J. ROTTIER (1996): Nucleocapsid-independent assembly of coronavirus-like
particles by co-expression of viral envelope protein genes. The EMBO journal 15, 2020-2028
WINTER, C., G. HERRLER & U. NEUMANN (2008a): Infection of the tracheal epithelium by
infectious bronchitis virus is sialic acid dependent. Microbes Infect 10, 367-373
WINTER, C., C. SCHWEGMANN-WESSELS, D. CAVANAGH, U. NEUMANN & G. HERRLER (2006):
Sialic acid is a receptor determinant for infection of cells by avian Infectious bronchitis virus. J
Gen Virol 87, 1209-1216
WINTER, C., C. SCHWEGMANN-WESSELS, U. NEUMANN & G. HERRLER (2008b): The spike
protein of infectious bronchitis virus is retained intracellularly by a tyrosine motif. J Virol 82,
2765-2771
76
Manuscript I
WONG, S., S. LAU, P. WOO & K. Y. YUEN (2007): Bats as a continuing source of emerging
infections in humans. Rev Med Virol 17, 67-91
WOO, P. C., S. K. LAU, Y. HUANG & K. Y. YUEN (2009): Coronavirus diversity, phylogeny and
interspecies jumping. Exp Biol Med (Maywood) 234, 1117-1127
WOO, P. C., S. K. LAU, K. S. LI, R. W. POON, B. H. WONG, H. W. TSOI, B. C. YIP, Y. HUANG, K. H.
CHAN & K. Y. YUEN (2006a): Molecular diversity of coronaviruses in bats. Virology 351, 180-187
WOO, P. C., S. K. LAU, C. C. YIP, Y. HUANG, H. W. TSOI, K. H. CHAN & K. Y. YUEN (2006b):
Comparative analysis of 22 coronavirus HKU1 genomes reveals a novel genotype and
evidence of natural recombination in coronavirus HKU1. Journal of virology 80, 7136-7145
ZENG, Q., M. A. LANGEREIS, A. L. VAN VLIET, E. G. HUIZINGA & R. J. DE GROOT (2008):
Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus
evolution. Proceedings of the National Academy of Sciences of the United States of America 105,
9065-9069
77
Manuscript II
3 Manuscript II
Tubulins interact with porcine & human S proteins of the
genus
Alphacoronavirus
and
support
successful
assembly and release of infectious viral particles
Anna- Theresa Rüdiger1§, Peter Mayrhofer2§, Yue Ma-Lauer2, Gottfried Pohlentz3,
Johannes Müthing3, Albrecht von Brunn2,4,, Christel Schwegmann-Weßels1
1
Institute of Virology, University of Veterinary Medicine Hannover, Bünteweg 17,
30559 Hannover, Germany
2
Virology Department, Max-von-Pettenkofer Institute, Ludwig-Maximilians University
Munich, Pettenkoferstraße 9a, 80336 Munich, Germany
3
Institute
for
Hygiene,
University
of
Münster,
Robert-Koch-Straße
41,
48149 Münster, Germany
4
German Centers for Infection Research (DZIF), Ludwig-Maximilians-University
Munich, Germany
§
contributed equally to the work
The extend of ATR’s contribution to the article is evaluated according to the following
scale:
A. Has contributed to collaboration (0-33 %).
B. Has contributed significantly (34-66 %).
C. Has essentially performed this study independently (67-100 %).
1. Design of the project including design of individual experiments: B
2. Performing of the experimental part of the study: B
3. Analysis of the experiments: B
4. Presentation and discussion of the study in article form: C
78
Manuscript II
3.1 Introduction
Coronaviruses (CoVs) belong to the family Coronaviridae and are positive singlestranded RNA viruses (KRIJNSE-LOCKER et al. 1994). They infect birds and
mammals, especially their respiratory and gastrointestinal systems. Due to high
mutation and recombination rates in coronaviruses frequent host-shifting events from
animal-to-animal and animal-to-human occurred (GUAN et al. 2003; CHINESE 2004;
LAU et al. 2005; WOO et al. 2009). Regarding the severe acute respiratory syndrome
(SARS) outbreak in China in the year 2002/2003, bats were identified as natural
reservoir for SARS-CoVs (LI et al. 2005; GE et al. 2013). Concerning the human CoV
229E (HCoV 229E) a close relative was found in bats in Ghana (PFEFFERLE et al.
2009). Thus, it is speculated that several human and animal CoVs originated from
bats (SHI & HU 2008; WOO et al. 2009; LIU et al. 2015).
Bats show great species diversity and a worldwide distribution, they have the ability
to fly and most of them live in colonies with large population sizes and densities. Due
to these characteristics bats represent high potential as reservoirs for animal and
human pathogens along with high risk factors for zoonotic transmission (CALISHER
et al. 2006). The CoV spike (S) glycoprotein – the key for determining host range – is
necessary for receptor binding and membrane fusion (GRAHAM & BARIC 2010).
The S protein contains a large ectodomain, a transmembrane domain, and a Cterminal cytoplasmic tail. The cytoplasmic domain consists of a cysteine-rich and a
charge-rich region and mediates S incorporation into virions resulting in infectious
virus particles (GODEKE et al. 2000; BOSCH et al. 2005). Some CoVs like the
transmissible gastroenteritis virus (TGEV), as well as the HCoVs NL63 and 229E
contain a tyrosine-based sorting signal within their charge-rich region which – in the
case of the TGEV S protein – was shown to be important for intracellular retention
(SCHWEGMANN-WESSELS et al. 2004).
Free movement of virus particles through the host cell cytoplasm is restricted. The
cytosol is highly viscose and contains structural barriers like organelles as well as
cytoskeletal elements (LUBY-PHELPS 2000; VERKMAN 2002; LEOPOLD &
PFISTER 2006). Therefore, diffusion of virus-sized particles is very unlikely and the
arrival at specific cellular regions or compartments is nearly impossible (SODEIK
2000). Consequently, many viruses are using the cytoskeleton network of the host
cell as transport system to reach the compartment were replication processes take
79
Manuscript II
place or to find their way out of the cell (PLOUBIDOU & WAY 2001; LEOPOLD &
PFISTER 2006; RADTKE et al. 2006). Microtubules are used for the retrograde
transport of adenovirus serotype 2 to get from the cell periphery towards the nucleus
or of influenza viruses to reach the microtubule organizing center (SUOMALAINEN et
al. 1999; LAKADAMYALI et al. 2003). Herpes simplex virus type 1 utilizes
microtubules for its anterograde transport towards the cell periphery (MIRANDASAKSENA et al. 2000). It is known that the cytoskeleton plays a crucial role during
virus attachment, internalization, endocytosis, transcription, replication, assembly,
exocytosis as well as cell-to-cell spread. After rearrangements of cellular filaments by
the virus itself it uses the filaments as tracks (RADTKE et al. 2006). Regarding this,
virus uptake of retrovirus pseudotyped with glycoproteins of ebola virus and
adenovirus type 2 is dependent on dynamic actin filaments (NAKANO et al. 2000;
MEIER et al. 2002; YONEZAWA et al. 2005). Concerning genome replication, human
parainfluenza virus type 3 RNA synthesis is inhibited after actin depolymerization
(GUPTA et al. 1998). Furthermore, depolymerization of actin filaments is known to
inhibit budding of human immunodeficiency virus type 1 as well (SASAKI et al. 1995).
One major component of the dynamic cytoskeletal matrix is represented by
microtubules. Those polarized structures are built of α/β-tubulin heterodimers and are
important for cell shape, transport, motility and cell division (NOGALES 2000; HEALD
& NOGALES 2002). Several viruses are known to interact with tubulin or their
molecular motors like kinesin or dynein (HARA et al. 2009; HSIEH et al. 2010; HAN
et al. 2012; HYDE et al. 2012; BISWAS & DAS SARMA 2014; HENRY SUM 2015).
Coronaviruses (CoVs) like a demyelinating strain of mouse hepatitis virus (MHV)
uses microtubules for neuronal spread and the feline infectious peritonitis virus
(FIPV) is transported via microtubules towards the microtubule organizing center
(BISWAS & DAS SARMA 2014; DEWERCHIN et al. 2014). For TGEV an upregulation of microtubule-associated α- and β-tubulin was detected in swine testis
(ST) cells after infection (ZHANG et al. 2013). In this work the focus is laying on the
interaction of tubulin with S proteins of Alphacoronaviruses like TGEV, HCoV NL63,
and 229E especially with the interaction of the last 39 aa stretches of the S
cytoplasmic tail. Our results show that tubulins interact with the cytoplasmic domain
of Alphacoronavirus S proteins. Reduced releases of infectious virus particles as well
as differentially distributed S proteins were observed after drug-induced tubulin
80
Manuscript II
depolymerization. Therefore, tubulin may help the S protein to be properly
transported, localized, and assembled into virions.
3.2 Material & Methods
3.2.1 Cell lines and virus strains
Human embryonic kidney cells (HEK-293) were used for co-immunoprecipitation via
GFP Trap®
pull down assay and ST cells were used for immunofluorescence
analysis and plaque assay. Both cell lines were grown in Dulbecco's modified Eagle
medium (DMEM) supplemented with 10 % fetal calf serum. Hypsignathus
monstrosus kidney cells (HypNi/1.1) (KUHL et al. 2011) and Pipistrellus pipistrellus
kidney cells (PipNi/1) (MULLER et al. 2012) (provided by Marcel Müller) were used
for immunofluorescence analysis and were grown in DMEM supplemented with 5 %
fetal calf serum.
The Purdue strain of TGEV (PUR46-MAD, provided by L. Enjuanes) was propagated
on ST cells. After 24 h of incubation at 37 °C the supernatants were harvested,
centrifuged and after addition of 1 % fetal calf serum stored at -80 °C.
3.2.2 Plasmids
Fusion proteins of the last 39 amino acid (aa) stretches of TGEV-S, SARS-CoV-S,
HCoV NL63 S, and HCoV 229E S cytoplasmic domains with GFP were constructed
(S-39aa-GFP-CT and S-39aa-GFP-NT) by using Invitrogen/ Life Technologies
Gateway cloning (Tab. 5).
Tab. 5 Last 39 amino acid stretches of coronavirus cytoplasmic domains linked to GFP.
Transmissible gastroenteritis virus (TGEV), human coronavirus (HCoV), severe acute respiratory
syndrome coronavirus (SARS-CoV); stop codon (*)
TGEV
CCCSTGCCGCIGCLGSCCHISCSRRQFENYEPIEKVHVH*
HCoV NL63
CLSTGCCGCCNCLTSSMRGCCDCGSTKLPYYEFEKVHVQ*
HCoV 229E
LCCCSTGCCGFFSCFASSIRGCCESTKLPYYDVEKIHIQ*
SARS-CoV
CCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT*
Full length TGEV S wildtype was fused to GFP named TGEV Swt-GFP. The full
length mutant of TGEV S where the tyrosine at position 1440 is exchanged by an
81
Manuscript II
alanine fused to GFP is named TGEV S Y/A-GFP. Tubulins C-terminally tagged with
an HA (YPYDVPDYA) peptide (TUBB2-HA, TUBB4A-HA, TUBB6-HA, TUBA4A-HA)
were used for co-immunoprecipitation. As compartment markers a GFP-tagged
ERGIC-53 and galactosyltransferase were used (WINTER et al. 2008).
ST cells used for immunofluorescence were transfected by the help of ICAfectin™
441 (Eurogentec) following the manufacturer’s instructions; in contrast to the
instructions an amount of 0.75 µl ICAfectin™ 441 per well was used. For
immunofluorescence analyses, HypNi/1.1 and PipNi/1 cells were seeded on 24-well
plates and transfected by Lipofectamine® 2000 (Life Technologies) following the
manufacturer’s instructions. Cells seeded on 10 cm dishes for GFP Trap® pull down
were transfected by using Polyethylenimine (PEI 1 µg/µl, Polysciences). 24 µg DNA
were mixed with 3 ml Opti-MEM (Life Technologies) and incubated for 5 min.
Afterwards, 20 µl of PEI were added, incubated for 15 min and then the mixture was
pipetted onto the cells and left there overnight.
3.2.3 GFP Trap® pull down assay & SDS-PAGE
For the search for interaction partners of the S protein cytoplasmic domain, HEK-293
cells were seeded in 10 cm dishes and transfected with empty GFP vector or with S39aa-GFP-CT/NT fusion protein one day later. For co-immunoprecipitation, the cells
were additionally transfected with the tubulin candidates tagged to HA. HEK-293 cells
were lysed in NP-40 lysis buffer plus cOmplete™ (protease inhibitor cocktail, Roche
Diagnostics, Mannheim) for 30 min on ice followed by centrifugation for 10 min at
20,000 x g and 4 °C. ChromoTek GFP-Trap® which consists of agarose beads
coated with antibodies derived from alpaca against GFP was used for both, the
general screening method as well as specific interaction studies between tubulins
and S proteins. The purification was done as described in the manufacturer’s
protocol. For the interaction experiments, the NaCl concentration of the washing
buffer was increased from 150 mM to 300 mM to avoid unspecific pull down of
tubulins. Instead of 100 µl 2x SDS sample buffer 25 µl 5x SDS sample buffer was
used. Cell lysates and eluates were subjected to SDS-PAGE. By a semi-dry
technique (KYHSE-ANDERSEN 1984) the proteins in the gel were transferred to
nitrocellulose membranes (GE Healthcare) which were subsequently blocked in 5 %
milk powder in TBS for one hour and then incubated with the first antibody overnight
82
Manuscript II
at 4 °C (rat-anti-HA antibody 1:300 provided by E. Kremmer; rat-anti-GFP antibody
1:1,000, Chromotek). The next day, the membrane was washed 3 times with TBS-T
for 10 min and treated with the secondary antibody (HRP-conjugated donkey-anti-rat
antibody 1:10,000, Sigma-Aldrich) for 1 to 2 h. Afterwards, the membrane was
washed 3 times in TBS-T for 10 min and once in TBS for 5 min. The blot was
exposed to Millipore Immobilon™ Western Chemiluminescent HRP substrate
(Fischer Scientific) and visualized in a GelDoc documentation system. Regarding the
general screening for interaction partners of the S protein, the SDS gel was fixed and
then stained with Coomassie solution. After destaining of the gel, visible bands were
cut out, digested and analyzed by mass spectrometry.
3.2.4 In gel digestion and mass spectrometry
The excised gel pieces were equilibrated with 25 mM NH4HCO3 and subsequently
incubated for 16 h with trypsin (25 ng/µl) as described previously (THYROCK et al.
2013). The resulting proteolytic peptides were extracted by sequential agitation with
200 µl of 25 mM NH4HCO3, 50 % acetonitrile (ACN)/0.1 % trifluoroacetic acid (TFA),
80 % ACN/ 0.1 % TFA, and neat ACN. The supernatants were combined and dried in
vacuo. Desalting was performed by use of ZipTip C18-tips as per description
(THYROCK et al. 2013).
Analyses of purified peptides were performed by use of Synapt GS2 instrument
(Waters, Manchester, UK) equipped with a Z-spray source in the positive ion
sensitivity mode. Source parameters were: source temperature: 80 °C, capillary
voltage: 0.8 kV, sampling cone voltage: 20 V, and source offset voltage: 50 V.
Peptide sequences were deduced from fragment ion spectra derived from nanoESI
ion mobility spectrometry (IMS) low energy CID experiments (THYROCK et al. 2013).
3.2.5 Immunofluorescence analysis
ST cells were transfected with TGEV Swt-GFP, TGEV S Y/A-GFP or with the S39aa-GFP-CT fusion protein of TGEV, NL63, 229E or SARS-CoV. HypNi/1.1 and
PipNi/1 cells were transfected with TGEV-Swt-GFP or S-39aa-GFP-CT of TGEV and
SARS-CoV. Three hours post transfection (hpt) the cells were mock-treated with
0.2 % dimethylsulfoxid (DMSO) or with 10 µg/ml Nocodazole (NOC, 5 mg diluted in
1 ml DMSO) for 3 h, following medium change. One hour later, i.e. 7 hpt, the cells
83
Manuscript II
were fixed with 3 % paraformaldehyde (PFA) in PBS. Cells were permeabilized with
0.2 % Triton/PBS for 5 min. To detect the authentic tubulin a monoclonal Cy3
conjugated anti-β-tubulin antibody produced in mice (1:500, Sigma-Aldrich) was
used.
ST, HypNi/1.1, and PipNi/1 cells were infected by TGEV (MOI of 1.5) for 1 h at 37 °C.
In the case of HypNi/1.1 and PipNi/1 cells, transfection with cDNA encoding the
cellular receptor for TGEV porcine aminopeptidase N (pAPN) was performed one day
prior to infection. One hour post infection (hpi), the cells were mock-treated with
0.2 % DMSO or with 10 µg/ml NOC for 3 h followed by medium change. Seven hpi
the cells were fixed with 3 % PFA and permeabilized. TGEV S protein was detected
with monoclonal antibody 6A.C3 (1:200, provided by L. Enjuanes). As secondary
antibody Alexa
Fluor® 568
or Alexa
Fluor®488
anti-mouse
(1:1000,
Life
Technologies) was used. Nuclei were stained with DAPI. ST cells expressing the
compartment markers were transfected with the specific cDNAs one day before
infection.
Immunofluorescence analyses were done by confocal microscopy using Leica TCS
SP5.
3.2.6 Plaque assay
ST, HypNi/1.1, and PipNi/1 cells were seeded on 6-well plates and infected by TGEV
(MOI 1.5 for 1 h at 37 °C). The chiropteran cells were transfected with cDNA
encoding for pAPN one day prior to infection. Transfected cells were washed tree
times with medium and then cultured with DMEM plus 3 % fetal calf serum at 37 °C.
Supernatants (100 µl) were collected at different time points (0, 4, 24 hpi).
Additionally, the cells were either treated with 0.2 % DMSO or incubated with
10 µg/ml NOC at different time points (1 h before infection, during infection, directly
after infection). Regarding HypNi/1.1 and PipNi/1 cells, NOC treatment was only
done after infection. ST cells were seeded on 96-well plates and inoculated with 40 µl
of the collected supernatants for 1-2 h at 37 °C. Afterwards, inoculum was discarded
and cells were treated with methylcellulose overnight. The next day cells were fixed
with 3 % PFA and treated with antibodies against viral proteins (same as for IFA). By
using the Nikon Eclipse Ti microscope plaque forming units were counted for each
time point.
84
Manuscript II
3.2.7 Virus particle assay
ST cells were seeded on 6-well plates. One day later they were infected by TGEV
(MOI 1.5 for 1 h at 37 °C). Cell culture supernatant was ultracentrifuged 24 hpi at
150000 x g in a SW 41 rotor (Beckman Coulter) for 1 h at 4 °C. The virus particle
pellet was solubilized in 40 µl 6×SDS sample puffer and subjected to SDS-PAGE. In
parallel, ST cells were lysed in NP-40 lysis puffer and subjected to SDS-PAGE as
well. Separated proteins were transferred to nitrocellulose membranes (GE
Healthcare) which were subsequently blocked with 1 % blocking reagent (Roche) in
blocking buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5) overnight at 4 °C. After
washing the membrane 3 times with phosphate buffered saline + 0.1 % Tween
(PBS+T) and once with PBS the nitrocellulose membrane was treated with the
antibody against the TGEV S protein (mAb 6A.C3, 1:200), the TGEV M protein (mAb
9D.B4, 1:200) or the TGEV N protein (mAb FIPV3-70, 1:1000, Thermo Scientific)
overnight. The next day, blots were washed and treated with anti-mouse peroxidaseconjugated antibody (Dako; 1:1000) for 1 h at 4 °C. Chemiluminescent peroxidase
substrate (Thermo Scientific) and the Chemi Doc system (Biorad) were used for
chemiluminescence signal detection.
3.3 Results
Via GFP Trap® pull down assay and subsequent SDS-PAGE and Coomassie staining
a screening for potential interaction partners of the cytoplasmic domain of TGEV-S
was performed (data not shown). Detected bands were identified by mass
spectrometry and matched to the group of tubulin beta chains (TUBB1, TUBB2A,
TUBB2B, TUBB3, TUBB4A, TUBB4B, TUBB5, TUBB6 and TUBB8). The four genes
TUBB2A, TUBB4A, TUBB6, and TUBA4A were chosen for further analysis, cloned
and tagged with HA.
3.3.1 Co-immunoprecipitation of tubulins with the TGEV S protein cytoplasmic
domain
To validate the interaction of the 39 amino acid stretch of the TGEV S protein
cytoplasmic domain with the different tubulin chain proteins a co-immunoprecipitation
experiment was performed (Fig. 3-1). After co-transfection of HA-tagged tubulins and
TGEV-S-39aa-GFP-NT purification was done via GFP Trap® pull down assay. As
85
Manuscript II
negative control, cells co-expressing tubulin candidates and the empty GFP vector
were used. All four tubulin-HA proteins were detected in the lysates at similar
quantity at the expected sizes of 50 to 55 kDa. Considering the eluates, strong
signals for tubulin-HA were detected in cases of the co-expression of TGEV-S-39aaGFP-NT and tubulins. In the negative control weak bands of unspecific binding were
visible. However, prominent tubulin-HA bands co-purified with TGEV-S-39aa-GFPNT clearly verified the interaction. Similar protein quantities of tubulins tagged to HA
were confirmed in the cell lysates. Also the eluates incubated with anti-GFP
antibodies showed just slight differences in protein quantity. This confirms that the
lack of tubulin in the eluates of the negative controls was due to no interaction and
not because of a lower expression level or reduced purification.
Fig. 3-1 Co-immunoprecipitation of TGEV S-GFP-NT fusion protein and different tubulins via
®
GFP-Trap .
TGEV S constructs was fused to GFP and precipitated by the help of anti-GFP-coated beads and
detected by anti-GFP antibodies. Tubulins were tagged with HA peptide and detected by anti-HA
antibodies in the cell lysates and the precipitated eluate. Co-immunoprecipitation with TGEV-S-GFPNT (TGEV-S) fusion protein.
3.3.2 Co-immunoprecipitation of tubulins with corresponding parts of the
human CoV 229E and CoV NL63 S protein cytoplasmic domains
Similar to the co-immunoprecipitation of tubulins with TGEV-S-39aa-GFP-NT the
39aa stretches of human CoV 229E S and human CoV NL63 S cytoplasmic tail were
86
Manuscript II
fused to GFP. Afterwards, cells were co-transfected with the GFP fusion constructs
and the HA-tagged tubulins followed by GFP Trap® pull down assay, SDS-PAGE and
Western blot (Fig. 3-2). TUBB2A was purified by these methods and clearly
confirmed as interaction partner. TUBB6, TUBB4A, and TUBA4A were coprecipitated in cells co-transfected with 229E S-39aa-GFP-NT but not with NL63 S39aa-GFP-NT. Here, the band for TUBB6-HA, TUBB4A, and TUBA4A in the NL63 S39aa-GFP-NT eluate was as weak as for the negative control.
Fig. 3-2 Co-immunoprecipitation of alphacoronavirus S fusion proteins and different tubulins
®
via GFP-Trap .
S constructs were fused to GFP and precipitated by the help of anti-GFP-coated beads and detected
by anti-GFP antibodies. Tubulins were tagged with HA peptide and detected by anti-HA antibodies in
the cell lysates and the precipitated eluate. Co-immunoprecipitation with human coronavirus NL63-SGFP-NT (NL63-S) or 229E-S-GFP-NT (229E-S) fusion protein.
3.3.3 The TGEV Swt full length protein partly co-localizes with authentic
cellular β-tubulins
As the interaction of the TGEV-S cytoplasmic tail with tubulins was confirmed by coimmunoprecipitation, confocal microscopy was done to localize TGEV Swt full length
protein and host cell β-tubulin (Fig. 3-3). ST cells expressing the TGEV Swt-GFP
were fixed and incubated with an antibody against the authentic β-tubulins. A partial
co-localization of the TGEV Swt-GFP protein with β-tubulin was observed. In some
87
Manuscript II
cases it looked like the S protein is laying on top of the tubulin filaments which could
be a hint for additional indirect interaction (Fig. 3-3, white arrows).
Fig. 3-3 Co-localization study of TGEV Swt proteins and authentic β-tubulin.
ST cells transfected with TGEV-Swt full length fused to GFP, white arrows point at TGEV Swt-GFP
proteins laying on tubulin; Expression of authentic β-tubulin (red), TGEV Swt-GFP (green). The figure
shows three representative image sections out of one experiment. Immunofluorescence analysis was
done by confocal microscopy using Leica TCS SP5.
3.3.4 S proteins are differentially distributed after treatment with Nocodazole
As a next step, a filament depolymerizing drug named Nocodazole (NOC) was used
for functional analysis of the S-tubulin-interaction. ST cells were transfected either
with full length TGEV Swt or TGEV S Y/A mutant both fused to GFP or with the last
39 aa stretches of the S cytoplasmic domain of TGEV, SARS-CoV, 229E or NL63
fused to GFP as well. The TGEV S Y/A mutant contains a destroyed tyrosine-based
retention signal due to the exchange by an alanine. This leads to surface expression
in single transfected cells while TGEV Swt protein is intracellularly retained
(SCHWEGMANN-WESSELS et al. 2004). Three hpt cells were mock-treated or
treated with NOC and 7 hpt cells were fixed. Next, authentic β-tubulins were stained
by using specific antibodies (Fig. 3-4 ). Cells treated with DMSO showed typically
long and filamentous tubulin structures. In NOC treated cells no characteristic
filamentous structures of β-tubulin were detected. Here, β-tubulins looked like
patches or a camouflage net but not like filaments anymore. Differences in the S
protein expression pattern could be observed as well. In DMSO treated cells the S
protein accumulated near the nucleus and was less distributed in the cytosol. In
contrast, the S protein expression in NOC treated cells was scattered throughout the
cytoplasm and looked like vesicles. This phenomenon of differentially distributed S
proteins due to NOC treatment was observed for all tested S constructs. Although,
88
Manuscript II
the S cytoplasmic tail fusion protein of TGEV, SARS-CoV and NL63 showed
additional GFP expression within the nucleus, the accumulation of S itself was clearly
visible as well as the altered localization of the proteins after NOC treatment.
The chiropteran cells HypNi/1.1 and PipNi/1 were also transfected with full length
TGEV-Swt or with the 39 amino acid stretches of TGEV and SARS-CoV-S
cytoplasmic tail fused to GFP and treated with DMSO or NOC (Fig. 3-5). Similar
results as for the ST cells were observed. Without NOC, S protein was more
accumulated near the nucleus whereas in drug-treated cells the S was more
dispersed within the cytoplasm.
89
Manuscript II
Fig. 3-4 S protein expression in untreated and NOC treated ST cells 7 hpt.
ST cells transfected with different S constructs fused to GFP. Expression of authentic β-tubulin (red),
S proteins (green), nuclei stained with DAPI (blue). Cells treated with DMSO (- NOC) or treated with
NOC (+ NOC). Immunofluorescence analysis was done by confocal microscopy using Leica TCS SP5.
90
Manuscript II
Fig. 3-5 S protein expression in untreated and NOC treated chiropteran cells 7 hpt.
HypNi/1.1 cells transfected with different S constructs fused to GFP (a); PipNi/1 cells transfected with
different S constructs fused to GFP (b). Expression of authentic β-tubulin (red), S proteins (green),
nuclei stained with DAPI (blue). Cells treated with DMSO (- NOC) or treated with NOC (+ NOC).
Immunofluorescence analysis was done by confocal microscopy using Leica TCS SP5.
91
Manuscript II
3.3.5 In infected cells, TGEV S protein distribution differs in DMSO and NOC
treated cells, and is expressed near the ERGIC and Golgi compartment
whereby both compartments are scattered throughout the cell after NOC
treatment
ST, HypNi/1.1, and PipNi/1 cells were infected with TGEV (Fig. 3-6). After fixation,
cells were treated with antibodies against the viral protein S. By immunofluorescence
analysis 7 hpi S expression patterns could be observed similar to the S-transfected
cells. In infected, non-treated cells, S accumulated stronger near the nucleus as
compared to S-transfected cells. The S protein was detected in vesicle-like structures
which are distributed all over the cytosol when incubated with NOC.
ST cells, first transfected with markers for the ERGIC or Golgi compartment, were
infected by TGEV and treated with DMSO (mock) or NOC (Fig. 3-7). In mock-treated
cells S accumulated near the ERGIC and Golgi compartment 7 hpi. A partial colocalization with the compartment markers could be observed. In drug-treated cells
the S proteins as well as the ERGIC and the Golgi compartment were scattered
throughout the cytoplasm. They were still expressed close to each other and partially
co-localized. In addition to the effect on the distribution of the TGEV S protein, NOC
had an effect on the cellular organelles themselves.
92
Manuscript II
Fig. 3-6 TGEV-S expression in untreated and NOC treated cells 7hpi.
ST, HypNi/1.1, and PipNi/1 cells were mock-infected or infected by TGEV; S protein was stained in
red or green. Cells treated with DMSO (- NOC), cells treated with NOC (+ NOC), nuclei stained with
DAPI (blue). Immunofluorescence analysis was performed by confocal microscopy using Leica TCS
SP5.
93
ST cells transfected with compartment markers for ERGIC or Golgi compartment (green) were infected by TGEV; TGEV S protein stained in red (b). Cells
treated with DMSO (- NOC), cells treated with NOC (+ NOC), nuclei stained with DAPI (blue). Immunofluorescence analysis was performed by confocal
microscopy using Leica TCS SP5.
Fig. 3-7 TGEV-S expression in untreated and NOC treated ST cells 7hpi.
Manuscript II
3.3.6 Release of infectious virus particles is reduced in NOC treated ST cells
ST cells were infected by TGEV and treated with NOC or DMSO at different time
points (1 h before infection, during infection or directly after infection). Supernatants
were collected (0, 4, 24 hpi) and used for virus quantification on ST cells via plaque
assay (Fig. 3-8a). Infected ST cells which were not treated with the drug showed a
viral titer of about 2x107 pfu/ml 24 hpi. In contrast, for cells treated with NOC at
various time points a titer of about 3x10 5 to 9x105 pfu/ml 24 hpi was measured. A
highly significant difference (p ≤ 0.001) in the virus titer of treated versus untreated
cells was calculated.
Regarding infected HypNi/1.1 cells a six-fold reduced amount of released infectious
virus particles was measured after NOC treatment (~8x10 4 pfu/ml) compared to
untreated (~5x105 pfu/ml) cells (Fig. 3-8b). PipNi/1 cells infected by TGEV showed a
viral titer of about 3x103 pfu/ml, while for infected and NOC treated cells a titer of
2x101 pfu/ml was measured (Fig. 3-8c).
94
Manuscript II
Fig. 3-8 Quantification of released infectious virus particles in mock-treated and NOC treated
cells via plaque assay.
TGEV infected ST cells, NOC treatment at different time points before, while or after infection (a).
TGEV infected HypNi/1.1 cells, NOC treatment after infection (b). TGEV infected PipNi/1 cells, NOC
treatment after infection. * p ≤ 0.05; ** p ≤ 0.01; ***p ≤ 0.001
3.3.7 Less S protein is incorporated into virions after NOC treatment of
infected ST cells
ST cells infected with TGEV were treated with DMSO or NOC. After cell lysis as well
as virus particle concentration by ultracentrifugation the incorporation of S into virions
was examined by SDS-PAGE followed by Western blot (Fig. 3-9). Mock-infected cells
served as negative control. In infected ST cells, not treated with NOC, S protein was
detected in virus particles after ultracentrifugation. In cells treated with NOC a very
weak or nearly no signal was observed for the S protein incorporated into viral
particles. The signal for TGEV S, M and N proteins were similar in the corresponding
cell lysates and comparable amounts of M and N protein were detectable in
95
Manuscript II
concentrated virions irrespective of a Nocodazole treatment. This indicates a reduced
S incorporation into virus particles due to depolymerization of tubulins by NOC
treatment of infected ST cells.
Fig. 3-9 TGEV particle assay 24 hpi.
Detection of viral spike (S), nucleocapsid (N), and membrane (M) proteins in cell lysates and pelleted
supernatants of TGEV- or mock-infected ST cells. Cells were either treated with NOC, DMSO or
medium. Virus particles were concentrated by ultracentrifugation.
3.4 Discussion
Viruses rely on the host cell machinery for successful replication (SAKAGUCHI et al.
1996; KONAN et al. 2003; CHOE et al. 2005; BELOV et al. 2007; BESKE et al. 2007;
MOFFAT et al. 2007; OOSTRA et al. 2007). Nevertheless, more detailed knowledge
on virus-host interaction would raise therapeutic tools for infection control. CoVs use
the host secretory pathway during their replication cycle. The vesicular transport on
secretory pathways is mostly mediated by microtubules and the corresponding motor
proteins (FOKIN et al. 2014). A strong effect on RNA replication was observed when
ER-to-Golgi transport was impaired. In the case of MHV it was shown that the early
secretory pathway is important during the formation of the replication complex
(OOSTRA et al. 2007; VERHEIJE et al. 2008; KNOOPS et al. 2010). However, the
incorporation of CoV S proteins into virus particles is required for the release of
infectious progeny. Information about cellular factors interacting with coronaviral S
proteins would be important to understand the virus infection process.
96
Manuscript II
In this study we searched for interaction partners of the last 39 aa stretches of TGEV,
HCoV NL63 and 229E S cytoplasmic domains (all containing a tyrosine-based motif).
Four different tubulin alpha and beta chains (TUBB2A, TUBB4A, TUBB6 and
TUBA4A) were detected to interact with TGEV-S and HCoV-229E-S. Regarding
HCoV NL63 only an interaction for TUBB2A was noticed. Either NL63-S-39aa-GFPNT does not bind certain tubulins or just with low affinity. A destroyed or impaired
binding of NL63-S to some tubulins due to the GFP-tag could be possible as well.
For many viruses a close association with cytoskeletal elements was shown (LUFTIG
1982). Adenovirus type 2 and 5 particles as well as reovirus particles interact with
microtubules (LUFTIG & WEIHING 1975; BABISS et al. 1979; MILES et al. 1980).
Poliovirus and newly synthesized viral RNA of Simian virus 40 are associated with
actin filaments and intermediate filaments (LENK & PENMAN 1979; BEN-ZE'EV et
al. 1981). An interaction is likely necessary for initial infection, transport of viral
components as well as for the assembly of new viral particles (HSIEH et al. 2010).
Moreover, tubulin was already found packaged into virions of Epstein-Barr virus,
human cytomegalovirus, and murine leukemia virus (WANG et al. 2003;
JOHANNSEN et al. 2004; VARNUM et al. 2004). By confocal microscopy, we
observed a partial co-localization of the full length TGEV S protein with the authentic
β-tubulin in ST cells. However, some S proteins seemed to lay on the tubulin
filaments suggesting an indirect interaction via motor proteins like dynein or kinesin.
For several viruses like adenovirus, African swine fever virus, canine parvovirus,
herpes simplex virus as well as lyssavirus and rabies virus an interaction either of the
whole virus capsid or of single viral proteins with cytoplasmic dynein was examined
(JACOB et al. 2000; RAUX et al. 2000; YE et al. 2000; ALONSO et al. 2001;
SUIKKANEN et al. 2003; DOUGLAS et al. 2004; KELKAR et al. 2004). Viruses may
have evolved microtubule-binding motifs or similar amino acid sequences
complementary to motifs in dynein for successful interaction (PASICK et al. 1994;
LEOPOLD & PFISTER 2006).
To further examine the interaction of the Alphacoronavirus S proteins with tubulin,
nocodazole (NOC) – a drug inducing microtubule depolymerization – was used.
Additionally, the importance of the tyrosine-based motif within the cytoplasmic
domain of the Alphacoronavirus S proteins of interest was examined. Therefore, a
TGEV S mutant with a destroyed tyrosine-based retention signal (TGEV S Y/A) was
used as well as a representative of the genus Betacoronavirus (SARS-CoV S), which
97
Manuscript II
contains no tyrosine motif. ST cells transfected with cDNA encoding the last 39
amino acid stretches of TGEV, NL63, 229E and SARS-CoV S cytoplasmic domain or
with the TGEV S and TGEV S Y/A full length cDNA were treated with DMSO or NOC.
To analyze CoV S protein localization during early assembly events, cells were fixed
7 hpi (RISCO et al. 1998; VOGELS et al. 2011). Mock treated cells showed an
accumulation of the S protein near the nucleus whereas NOC treated cells showed a
scattered pattern within the cytoplasm. All tested constructs showed similar
expression and distribution pattern either in DMSO or in NOC treated cells. Thus, the
tyrosine-based signal is not essential for the association with β-tubulins, whereby the
charge-rich region in general is important. Moreover, the cytoplasmic domain of
Betacoronavirus S proteins (like in this case SARS-CoV S) is equally involved in
tubulin interaction. In TGEV-infected ST cells similar results were obtained 7 hpi.
Also here, instead of an accumulation of S proteins they were distributed in the
cytosol after NOC treatment. CoV assembly occurs at the ERGIC. Thus, S proteins
accumulate near the nucleus in DMSO treated cells 7 hpi. A redistribution in NOC
treated cells was already shown for different viral proteins, like Crimean-Congo
hemorrhagic fever virus nucleocapsid protein (SIMON et al. 2009) or herpes simplex
virus 1 capsids (SODEIK et al. 1997). In the two tested chiropteran cells lines
(HypNi/1.1 and PipNi/1) similar results as for S-transfected or TGEV-infected ST cells
were obtained, suggesting a conserved viral strategy to use the host for its own
advantage during replication. Due to the assumption that many human and animal
CoVs originated from bats and that most eukaryotic cells contain microtubules, a
conserved microtubule-dependent CoV replication strategy is likely.
Furthermore, we demonstrated that NOC leads to a reduced amount of released
infectious virus particles. Less virus yield after cytoskeleton disruption was already
shown for moloney murine leukaemia virus and vaccinia virus (PAYNE &
KRISTENSSON 1982; SATAKE & LUFTIG 1982). To find out which step during The
TGEV replication cycle is mostly influenced by the interaction of S with tubulin, cells
were drug-treated at different time points (before, while, or after infection). A
significantly lower virus titer in the cell culture supernatant of NOC treated cells
compared to untreated cells was detected, whereas no differences in viral titers
concerning the various time points could be observed. CoVs uses the host secretory
pathway to be transported from ER-to-Golgi apparatus as well as during virus
assembly (OOSTRA et al. 2007; VERHEIJE et al. 2008; KNOOPS et al. 2010;
98
Manuscript II
VOGELS et al. 2011). An impaired transport of viral components and an affected
assembly may explain the decrease in virus yield. For MHV, microtubules are
important for neuronal transport, replication and viral spread (BISWAS & DAS
SARMA 2014). Also here, a reduced efficiency of viral infection was observed by
using microtubule disrupting drugs (BISWAS & DAS SARMA 2014). TGEV was able
to enter the cells in the absence or presence of NOC, it was able to replicate and to
egress but the infection was less efficient regarding the amount of newly formed
infectious virions. Due to the fact that depolymerized microtubules did not inhibit
TGEV infection completely, virus components have to use other pathways for
trafficking beside tubulin filaments to finish their replication cycle. Examinations on
adeno-associated, herpes and vaccinia virus showed no complete blocking of
infection after microtubule disruption as well (SODEIK et al. 1997; PLOUBIDOU et al.
2000; SANLIOGLU et al. 2000). Also here, an alternative, microtubule-independent
pathway used by the virus was suggested (XIAO & SAMULSKI 2012). An actindependent strategy for virus movement could be possible (CUDMORE et al. 1997;
LANIER & VOLKMAN 1998; SANLIOGLU et al. 2000). For paramyxoviruses like
measles virus actin seems to be involved in protein movement towards the plasma
membrane as well as the budding process (EHRNST & SUNDQVIST 1975;
STALLCUP et al. 1983; BOHN et al. 1986). Regarding vaccinia virus, microfilaments
affect
intracellular movement
as
well
as
virus
release
(STOKES
1976).
Nucleocapsids of a nucleopolyhedrovirus utilize actin cables for their transport to
and/ or into the nucleus (LANIER & VOLKMAN 1998). However, lower virus titers
may correlate with the differentially distributed S protein in NOC treated cells, too.
Actually, S accumulates near the ERGIC where it interacts with the M protein to be
incorporated into newly assembled virions (OPSTELTEN et al. 1995; NGUYEN &
HOGUE 1997). When microtubules are depolymerized S proteins as well as the
ERGIC and Golgi compartment are scattered throughout the cell. Due to the diffuse
distribution of S and the two compartments which are important for successful TGEV
assembly less interaction of viral proteins during assembly may be possible. An
impaired transport of virus particles from the Golgi compartment to the plasma
membrane may also be a reason for lower viral titers. Concerning this matter, a less
organized cytoplasm with dispersed Golgi stacks in NOC treated ST cells was
already shown by Risco et al. 1998. Additionally, TGEV virions were seen in these
disrupted Golgi stacks whereas only a few were detected on extracellular surfaces
99
Manuscript II
(RISCO et al. 1998). A reason may be a microtubule-dependent transport of vesicles
between the Golgi compartment and the plasma membrane (RISCO et al. 1998). To
find out if S incorporation into virions is influenced by NOC treatment, virus particles
were concentrated out of the supernatant of NOC-treated infected cells and
compared to mock-treated cells. In this case, the amount of S protein was similar in
DMSO and NOC treated cell lysates, whereas S protein quantity was reduced in
pelleted virus particles when infected cells were treated with NOC. This fact fits to the
calculated smaller amount of released infectious virus particles measured in drug
treated cells.
3.5 Conclusion
Our results show that Alpha- and Betacoronavirus S proteins interact with their
charge-rich region of their cytoplasmic domains with tubulin beta chains, regardless if
a tyrosine-based signal is present or not. An interaction with microtubules facilitates
TGEV replication and infection efficiency but the depolymerization of microtubules
did not inhibit it completely. Thus, an interaction of S with tubulin may help the virus
during replication, assembly as well as the budding process and supports viral
particle transport to the plasma membrane.
Authors’ contributions
ATR, AVB and CSW conceived and designed the study. GP and JM performed the
mass
spectrometry
analysis.
PM
and
YML
Technologies Gateway cloning and GFP Trap
®
accomplished
Invitrogen/
Life
pull down assay. ATR performed
immunofluorescence studies and plaque assays. Sandra Bauer performed virus
particle assay. ATR, PM, YML, GP, JM, AVB, and CSW analyzed the data. ATR and
CSW drafted the manuscript. All authors read and approved the final manuscript.
Competing interests
The authors have declared that no competing interests exist.
100
Manuscript II
Funding
Financial support was provided by a grant to CSW (SCHW 1408/1.1) from the
German Research Foundation (DFG) and by the “Bundesministerium fuer Bildung
und Forschung” of the German Government (Zoonosis Network, Consortium on
ecology and pathogenesis of SARS, project code 01KI1005A,F to AvB.
CSW is funded by the Emmy Noether Programme from the DFG. ATR is a recipient
of a Georg-Christoph-Lichtenberg PhD fellowship from the Ministry for Science and
Culture of Lower Saxony.
Acknowledgements
This work was performed by ATR in partial fulfillment of the requirements for a Dr.
rer. nat. degree from the University of Veterinary Medicine Hannover.
We are grateful to L. Enjuanes, E. Snijder, E. Kremmer and H.-P. Hauri for providing
antibodies, plasmids, and virus. We thank M. Müller and C. Drosten for the
chiropteran cell lines (HypNi/1.1, PipNi/1). Thanks to S. Bauer for technical
assistance.
3.6 References
ALONSO, C., J. MISKIN, B. HERNAEZ, P. FERNANDEZ-ZAPATERO, L. SOTO, C. CANTO, I.
RODRIGUEZ-CRESPO, L. DIXON & J. M. ESCRIBANO (2001): African swine fever virus protein
p54 interacts with the microtubular motor complex through direct binding to light-chain dynein.
J Virol 75, 9819-9827
BABISS, L. E., R. B. LUFTIG, J. A. WEATHERBEE, R. R. WEIHING, U. R. RAY & B. N. FIELDS
(1979): Reovirus serotypes 1 and 3 differ in their in vitro association with microtubules. J Virol
30, 863-874
BELOV, G. A., N. ALTAN-BONNET, G. KOVTUNOVYCH, C. L. JACKSON, J. LIPPINCOTTSCHWARTZ & E. EHRENFELD (2007): Hijacking components of the cellular secretory pathway
for replication of poliovirus RNA. J Virol 81, 558-567
BEN-ZE'EV, A., M. HOROWITZ, H. SKOLNIK, R. ABULAFIA, O. LAUB & Y. ALONI (1981): The
metabolism of SV40 RNA is associated with the cytoskeletal framework. Virology 111, 475-487
BESKE, O., M. REICHELT, M. P. TAYLOR, K. KIRKEGAARD & R. ANDINO (2007): Poliovirus
infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of
the Golgi complex. J Cell Sci 120, 3207-3218
BISWAS, K. & J. DAS SARMA (2014): Effect of microtubule disruption on neuronal spread and
replication of demyelinating and nondemyelinating strains of mouse hepatitis virus in vitro. J
Virol 88, 3043-3047
BOHN, W., G. RUTTER, H. HOHENBERG, K. MANNWEILER & P. NOBIS (1986): Involvement of
actin filaments in budding of measles virus: studies on cytoskeletons of infected cells. Virology
149, 91-106
101
Manuscript II
BOSCH, B. J., C. A. DE HAAN, S. L. SMITS & P. J. ROTTIER (2005): Spike protein assembly into
the coronavirion: exploring the limits of its sequence requirements. Virology 334, 306-318
CALISHER, C. H., J. E. CHILDS, H. E. FIELD, K. V. HOLMES & T. SCHOUNTZ (2006): Bats:
important reservoir hosts of emerging viruses. Clin Microbiol Rev 19, 531-545
CHINESE, S. M. E. C. (2004): Molecular evolution of the SARS coronavirus during the course of
the SARS epidemic in China. Science 303, 1666-1669
CHOE, S. S., D. A. DODD & K. KIRKEGAARD (2005): Inhibition of cellular protein secretion by
picornaviral 3A proteins. Virology 337, 18-29
CUDMORE, S., I. RECKMANN & M. WAY (1997): Viral manipulations of the actin cytoskeleton.
Trends in microbiology 5, 142-148
DEWERCHIN, H. L., L. M. DESMARETS, Y. NOPPE & H. J. NAUWYNCK (2014): Myosins 1 and 6,
myosin light chain kinase, actin and microtubules cooperate during antibody-mediated
internalisation and trafficking of membrane-expressed viral antigens in feline infectious
peritonitis virus infected monocytes. Vet Res 45, 17
DOUGLAS, M. W., R. J. DIEFENBACH, F. L. HOMA, M. MIRANDA-SAKSENA, F. J. RIXON, V.
VITTONE, K. BYTH & A. L. CUNNINGHAM (2004): Herpes simplex virus type 1 capsid protein
VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular
transport. J Biol Chem 279, 28522-28530
EHRNST, A. & K. G. SUNDQVIST (1975): Polar appearance and nonligand induced spreading of
measles virus hemagglutinin at the surface of chronically infected cells. Cell 5, 351-359
FOKIN, A. I., I. B. BRODSKY, A. V. BURAKOV & E. S. NADEZHDINA (2014): Interaction of early
secretory pathway and Golgi membranes with microtubules and microtubule motors.
Biochemistry. Biokhimiia 79, 879-893
GE, X. Y., J. L. LI, X. L. YANG, A. A. CHMURA, G. ZHU, J. H. EPSTEIN, J. K. MAZET, B. HU, W.
ZHANG, C. PENG, Y. J. ZHANG, C. M. LUO, B. TAN, N. WANG, Y. ZHU, G. CRAMERI, S. Y.
ZHANG, L. F. WANG, P. DASZAK & Z. L. SHI (2013): Isolation and characterization of a bat
SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535-538
GODEKE, G. J., C. A. DE HAAN, J. W. ROSSEN, H. VENNEMA & P. J. ROTTIER (2000): Assembly
of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike
protein. J Virol 74, 1566-1571
GRAHAM, R. L. & R. S. BARIC (2010): Recombination, reservoirs, and the modular spike:
mechanisms of coronavirus cross-species transmission. J Virol 84, 3134-3146
GUAN, Y., B. J. ZHENG, Y. Q. HE, X. L. LIU, Z. X. ZHUANG, C. L. CHEUNG, S. W. LUO, P. H. LI, L.
J. ZHANG, Y. J. GUAN, K. M. BUTT, K. L. WONG, K. W. CHAN, W. LIM, K. F. SHORTRIDGE, K. Y.
YUEN, J. S. PEIRIS & L. L. POON (2003): Isolation and characterization of viruses related to the
SARS coronavirus from animals in southern China. Science 302, 276-278
GUPTA, S., B. P. DE, J. A. DRAZBA & A. K. BANERJEE (1998): Involvement of actin
microfilaments in the replication of human parainfluenza virus type 3. J Virol 72, 2655-2662
HAN, X., Z. LI, H. CHEN, H. WANG, L. MEI, S. WU, T. ZHANG, B. LIU & X. LIN (2012): Influenza
virus A/Beijing/501/2009(H1N1) NS1 interacts with beta-tubulin and induces disruption of the
microtubule network and apoptosis on A549 cells. PloS one 7, e48340
HARA, Y., R. HASEBE, Y. SUNDEN, K. OCHIAI, E. HONDA, Y. SAKODA & T. UMEMURA (2009):
Propagation of swine hemagglutinating encephalomyelitis virus and pseudorabies virus in
102
Manuscript II
dorsal root ganglia cells. The Journal of veterinary medical science / the Japanese Society of
Veterinary Science 71, 595-601
HEALD, R. & E. NOGALES (2002): Microtubule dynamics. Journal of cell science 115, 3-4
HENRY SUM, M. S. (2015): The involvement of microtubules and actin during the infection of
Japanese encephalitis virus in neuroblastoma cell line, IMR32. Biomed Res Int 2015, 695283
HSIEH, M. J., P. J. WHITE & C. W. POUTON (2010): Interaction of viruses with host cell
molecular motors. Current opinion in biotechnology 21, 633-639
HYDE, J. L., L. K. GILLESPIE & J. M. MACKENZIE (2012): Mouse norovirus 1 utilizes the
cytoskeleton network to establish localization of the replication complex proximal to the
microtubule organizing center. Journal of virology 86, 4110-4122
JACOB, Y., H. BADRANE, P. E. CECCALDI & N. TORDO (2000): Cytoplasmic dynein LC8
interacts with lyssavirus phosphoprotein. J Virol 74, 10217-10222
JOHANNSEN, E., M. LUFTIG, M. R. CHASE, S. WEICKSEL, E. CAHIR-MCFARLAND, D. ILLANES,
D. SARRACINO & E. KIEFF (2004): Proteins of purified Epstein-Barr virus. Proceedings of the
National Academy of Sciences of the United States of America 101, 16286-16291
KELKAR, S. A., K. K. PFISTER, R. G. CRYSTAL & P. L. LEOPOLD (2004): Cytoplasmic dynein
mediates adenovirus binding to microtubules. Journal of virology 78, 10122-10132
KNOOPS, K., C. SWETT-TAPIA, S. H. VAN DEN WORM, A. J. TE VELTHUIS, A. J. KOSTER, A. M.
MOMMAAS, E. J. SNIJDER & M. KIKKERT (2010): Integrity of the early secretory pathway
promotes, but is not required for, severe acute respiratory syndrome coronavirus RNA
synthesis and virus-induced remodeling of endoplasmic reticulum membranes. Journal of
virology 84, 833-846
KONAN, K. V., T. H. GIDDINGS, JR., M. IKEDA, K. LI, S. M. LEMON & K. KIRKEGAARD (2003):
Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and
ultrastructure of host secretory apparatus. J Virol 77, 7843-7855
KRIJNSE-LOCKER, J., M. ERICSSON, P. J. ROTTIER & G. GRIFFITHS (1994): Characterization of
the budding compartment of mouse hepatitis virus: evidence that transport from the RER to
the Golgi complex requires only one vesicular transport step. J Cell Biol 124, 55-70
KUHL, A., M. HOFFMANN, M. A. MULLER, V. J. MUNSTER, K. GNIRSS, M. KIENE, T. S. TSEGAYE,
G. BEHRENS, G. HERRLER, H. FELDMANN, C. DROSTEN & S. POHLMANN (2011): Comparative
analysis of Ebola virus glycoprotein interactions with human and bat cells. J Infect Dis 204
Suppl 3, S840-849
KYHSE-ANDERSEN, J. (1984): Electroblotting of multiple gels: a simple apparatus without
buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. Journal of
biochemical and biophysical methods 10, 203-209
LAKADAMYALI, M., M. J. RUST, H. P. BABCOCK & X. ZHUANG (2003): Visualizing infection of
individual influenza viruses. Proc Natl Acad Sci U S A 100, 9280-9285
LANIER, L. M. & L. E. VOLKMAN (1998): Actin binding and nucleation by Autographa california
M nucleopolyhedrovirus. Virology 243, 167-177
LAU, S. K., P. C. WOO, K. S. LI, Y. HUANG, H. W. TSOI, B. H. WONG, S. S. WONG, S. Y. LEUNG,
K. H. CHAN & K. Y. YUEN (2005): Severe acute respiratory syndrome coronavirus-like virus in
Chinese horseshoe bats. Proc Natl Acad Sci U S A 102, 14040-14045
LENK, R. & S. PENMAN (1979): The cytoskeletal framework and poliovirus metabolism. Cell 16,
289 301
103
Manuscript II
LEOPOLD, P. L. & K. K. PFISTER (2006): Viral strategies for intracellular trafficking: motors and
microtubules. Traffic 7, 516-523
LI, W., Z. SHI, M. YU, W. REN, C. SMITH, J. H. EPSTEIN, H. WANG, G. CRAMERI, Z. HU, H.
ZHANG, J. ZHANG, J. MCEACHERN, H. FIELD, P. DASZAK, B. T. EATON, S. ZHANG & L. F.
WANG (2005): Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679
LIU, C., J. TANG, Y. MA, X. LIANG, Y. YANG, G. PENG, Q. QI, S. JIANG, J. LI, L. DU & F. LI (2015):
Receptor usage and cell entry of porcine epidemic diarrhea coronavirus. J Virol 89, 6121-6125
LUBY-PHELPS, K. (2000): Cytoarchitecture and physical properties of cytoplasm: volume,
viscosity, diffusion, intracellular surface area. International review of cytology 192, 189-221
LUFTIG, R. B. (1982): Does the cytoskeleton play a significant role in animal virus replication? J
Theor Biol 99, 173-191
LUFTIG, R. B. & R. R. WEIHING (1975): Adenovirus binds to rat brain microtubules in vitro. J
Virol 16, 696-706
MEIER, O., K. BOUCKE, S. V. HAMMER, S. KELLER, R. P. STIDWILL, S. HEMMI & U. F. GREBER
(2002): Adenovirus triggers macropinocytosis and endosomal leakage together with its
clathrin-mediated uptake. The Journal of cell biology 158, 1119-1131
MILES, B. D., R. B. LUFTIG, J. A. WEATHERBEE, R. R. WEIHING & J. WEBER (1980):
Quantitation of the interaction between adenovirus types 2 and 5 and microtubules inside
infected cells. Virology 105, 265-269
MIRANDA-SAKSENA, M., P. ARMATI, R. A. BOADLE, D. J. HOLLAND & A. L. CUNNINGHAM
(2000): Anterograde transport of herpes simplex virus type 1 in cultured, dissociated human
and rat dorsal root ganglion neurons. J Virol 74, 1827-1839
MOFFAT, K., C. KNOX, G. HOWELL, S. J. CLARK, H. YANG, G. J. BELSHAM, M. RYAN & T.
WILEMAN (2007): Inhibition of the secretory pathway by foot-and-mouth disease virus 2BC
protein is reproduced by coexpression of 2B with 2C, and the site of inhibition is determined
by the subcellular location of 2C. J Virol 81, 1129-1139
MULLER, M. A., V. S. RAJ, D. MUTH, B. MEYER, S. KALLIES, S. L. SMITS, R. WOLLNY, T. M.
BESTEBROER, S. SPECHT, T. SULIMAN, K. ZIMMERMANN, T. BINGER, I. ECKERLE, M.
TSCHAPKA, A. M. ZAKI, A. D. OSTERHAUS, R. A. FOUCHIER, B. L. HAAGMANS & C. DROSTEN
(2012): Human coronavirus EMC does not require the SARS-coronavirus receptor and
maintains broad replicative capability in mammalian cell lines. mBio 3,
NAKANO, M. Y., K. BOUCKE, M. SUOMALAINEN, R. P. STIDWILL & U. F. GREBER (2000): The
first step of adenovirus type 2 disassembly occurs at the cell surface, independently of
endocytosis and escape to the cytosol. J Virol 74, 7085-7095
NGUYEN, V. P. & B. G. HOGUE (1997): Protein interactions during coronavirus assembly. J
Virol 71, 9278-9284
NOGALES, E. (2000): Structural insights into microtubule function. Annual review of biochemistry
69, 277-302
OOSTRA, M., E. G. TE LINTELO, M. DEIJS, M. H. VERHEIJE, P. J. ROTTIER & C. A. DE HAAN
(2007): Localization and membrane topology of coronavirus nonstructural protein 4:
involvement of the early secretory pathway in replication. J Virol 81, 12323-12336
OPSTELTEN, D. J., M. J. RAAMSMAN, K. WOLFS, M. C. HORZINEK & P. J. ROTTIER (1995):
Envelope glycoprotein interactions in coronavirus assembly. J Cell Biol 131, 339-349
104
Manuscript II
PASICK, J. M., K. KALICHARRAN & S. DALES (1994): Distribution and trafficking of JHM
coronavirus structural proteins and virions in primary neurons and the OBL-21 neuronal cell
line. J Virol 68, 2915-2928
PAYNE, L. G. & K. KRISTENSSON (1982): The effect of cytochalasin D and monensin on
enveloped vaccinia virus release. Archives of virology 74, 11-20
PFEFFERLE, S., S. OPPONG, J. F. DREXLER, F. GLOZA-RAUSCH, A. IPSEN, A. SEEBENS, M. A.
MULLER, A. ANNAN, P. VALLO, Y. ADU-SARKODIE, T. F. KRUPPA & C. DROSTEN (2009):
Distant relatives of severe acute respiratory syndrome coronavirus and close relatives of
human coronavirus 229E in bats, Ghana. Emerg Infect Dis 15, 1377-1384
PLOUBIDOU, A., V. MOREAU, K. ASHMAN, I. RECKMANN, C. GONZALEZ & M. WAY (2000):
Vaccinia virus infection disrupts microtubule organization and centrosome function. The EMBO
journal 19, 3932-3944
PLOUBIDOU, A. & M. WAY (2001): Viral transport and the cytoskeleton. Current opinion in cell
biology 13, 97-105
RADTKE, K., K. DOHNER & B. SODEIK (2006): Viral interactions with the cytoskeleton: a
hitchhiker's guide to the cell. Cell Microbiol 8, 387-400
RAUX, H., A. FLAMAND & D. BLONDEL (2000): Interaction of the rabies virus P protein with the
LC8 dynein light chain. J Virol 74, 10212-10216
RISCO, C., M. MUNTION, L. ENJUANES & J. L. CARRASCOSA (1998): Two types of virus-related
particles are found during transmissible gastroenteritis virus morphogenesis. J Virol 72, 40224031
SAKAGUCHI, T., G. P. LESER & R. A. LAMB (1996): The ion channel activity of the influenza
virus M2 protein affects transport through the Golgi apparatus. J Cell Biol 133, 733-747
SANLIOGLU, S., P. K. BENSON, J. YANG, E. M. ATKINSON, T. REYNOLDS & J. F. ENGELHARDT
(2000): Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by
rac1 and phosphatidylinositol-3 kinase activation. Journal of virology 74, 9184-9196
SASAKI, H., M. NAKAMURA, T. OHNO, Y. MATSUDA, Y. YUDA & Y. NONOMURA (1995): Myosinactin interaction plays an important role in human immunodeficiency virus type 1 release from
host cells. Proc Natl Acad Sci U S A 92, 2026-2030
SATAKE, M. & R. B. LUFTIG (1982): Microtubule-depolymerizing agents inhibit Moloney murine
leukaemia virus production. The Journal of general virology 58, 339-349
SCHWEGMANN-WESSELS, C., M. AL-FALAH, D. ESCORS, Z. WANG, G. ZIMMER, H. DENG, L.
ENJUANES, H. Y. NAIM & G. HERRLER (2004): A novel sorting signal for intracellular
localization is present in the S protein of a porcine coronavirus but absent from severe acute
respiratory syndrome-associated coronavirus. J Biol Chem 279, 43661-43666
SHI, Z. & Z. HU (2008): A review of studies on animal reservoirs of the SARS coronavirus. Virus
Res 133, 74-87
SIMON, M., C. JOHANSSON, A. LUNDKVIST & A. MIRAZIMI (2009): Microtubule-dependent and
microtubule-independent steps in Crimean-Congo hemorrhagic fever virus replication cycle.
Virology 385, 313-322
SODEIK, B. (2000): Mechanisms of viral transport in the cytoplasm. Trends in microbiology 8,
465-472
105
Manuscript II
SODEIK, B., M. W. EBERSOLD & A. HELENIUS (1997): Microtubule-mediated transport of
incoming herpes simplex virus 1 capsids to the nucleus. The Journal of cell biology 136, 10071021
STALLCUP, K. C., C. S. RAINE & B. N. FIELDS (1983): Cytochalasin B inhibits the maturation of
measles virus. Virology 124, 59-74
STOKES, G. V. (1976): High-voltage electron microscope study of the release of vaccinia virus
from whole cells. J Virol 18, 636-643
SUIKKANEN, S., T. AALTONEN, M. NEVALAINEN, O. VALILEHTO, L. LINDHOLM, M. VUENTO &
M. VIHINEN-RANTA (2003): Exploitation of microtubule cytoskeleton and dynein during
parvoviral traffic toward the nucleus. J Virol 77, 10270-10279
SUOMALAINEN, M., M. Y. NAKANO, S. KELLER, K. BOUCKE, R. P. STIDWILL & U. F. GREBER
(1999): Microtubule-dependent plus- and minus end-directed motilities are competing
processes for nuclear targeting of adenovirus. The Journal of cell biology 144, 657-672
THYROCK, A., E. OSSENDORF, M. STEHLING, M. KAIL, T. KURTZ, G. POHLENTZ, D.
WASCHBUSCH, S. EGGERT, E. FORMSTECHER, J. MUTHING, K. DREISEWERD, S. KINS, B.
GOUD & A. BARNEKOW (2013): A new Mint1 isoform, but not the conventional Mint1, interacts
with the small GTPase Rab6. PLoS One 8, e64149
VARNUM, S. M., D. N. STREBLOW, M. E. MONROE, P. SMITH, K. J. AUBERRY, L. PASA-TOLIC, D.
WANG, D. G. CAMP, 2ND, K. RODLAND, S. WILEY, W. BRITT, T. SHENK, R. D. SMITH & J. A.
NELSON (2004): Identification of proteins in human cytomegalovirus (HCMV) particles: the
HCMV proteome. Journal of virology 78, 10960-10966
VERHEIJE, M. H., M. RAABEN, M. MARI, E. G. TE LINTELO, F. REGGIORI, F. J. VAN KUPPEVELD,
P. J. ROTTIER & C. A. DE HAAN (2008): Mouse hepatitis coronavirus RNA replication depends
on GBF1-mediated ARF1 activation. PLoS pathogens 4, e1000088
VERKMAN, A. S. (2002): Solute and macromolecule
compartments. Trends in biochemical sciences 27, 27-33
diffusion
in
cellular
aqueous
VOGELS, M. W., B. W. VAN BALKOM, D. V. KALOYANOVA, J. J. BATENBURG, A. J. HECK, J. B.
HELMS, P. J. ROTTIER & C. A. DE HAAN (2011): Identification of host factors involved in
coronavirus replication by quantitative proteomics analysis. Proteomics 11, 64-80
WANG, M. Q., W. KIM, G. GAO, T. A. TORREY, H. C. MORSE, 3RD, P. DE CAMILLI & S. P. GOFF
(2003): Endophilins interact with Moloney murine leukemia virus Gag and modulate virion
production. Journal of biology 3, 4
WINTER, C., C. SCHWEGMANN-WESSELS, U. NEUMANN & G. HERRLER (2008): The spike
protein of infectious bronchitis virus is retained intracellularly by a tyrosine motif. J Virol 82,
2765-2771
WOO, P. C., S. K. LAU, Y. HUANG & K. Y. YUEN (2009): Coronavirus diversity, phylogeny and
interspecies jumping. Exp Biol Med (Maywood) 234, 1117-1127
XIAO, P. J. & R. J. SAMULSKI (2012): Cytoplasmic trafficking, endosomal escape, and
perinuclear accumulation of adeno-associated virus type 2 particles are facilitated by
microtubule network. Journal of virology 86, 10462-10473
YE, G. J., K. T. VAUGHAN, R. B. VALLEE & B. ROIZMAN (2000): The herpes simplex virus 1
U(L)34 protein interacts with a cytoplasmic dynein intermediate chain and targets nuclear
membrane. J Virol 74, 1355-1363
YONEZAWA, A., M. CAVROIS & W. C. GREENE (2005): Studies of ebola virus glycoproteinmediated entry and fusion by using pseudotyped human immunodeficiency virus type 1
106
Manuscript II
virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha.
J Virol 79, 918-926
ZHANG, X., H. Y. SHI, J. F. CHEN, D. SHI, H. W. LANG, Z. T. WANG & L. FENG (2013):
Identification of cellular proteome using two-dimensional difference gel electrophoresis in ST
cells infected with transmissible gastroenteritis coronavirus. Proteome science 11, 31
107
108
Manuscript III
4 Manuscript III
Analysis of tyrosine motifs within the cytoplasmic
domain of the coronavirus spike (S) and membrane (M)
proteins and their functions during S-M interaction
Anna-Theresa Rüdiger1, Christel Schwegmann-Weßels1
1
Institute of Virology, University of Veterinary Medicine Hannover, Bünteweg 17,
30559 Hannover, Germany
The extend of ATR’s contribution to the article is evaluated according to the following
scale:
A. Has contributed to collaboration (0-33 %).
B. Has contributed significantly (34-66 %).
C. Has essentially performed this study independently (67-100 %).
1. Design of the project including design of individual experiments: C
2. Performing of the experimental part of the study: C
3. Analysis of the experiments: C
4. Presentation and discussion of the study in article form: C
109
Manuscript III
4.1 Introduction
Coronaviruses (CoVs) are single-stranded RNA viruses with positive orientation.
They infect mammals like bats and humans as well as birds. CoVs replicate in the
host cell cytoplasm and bud at the endoplasmatic reticulum-Golgi intermediate
compartment (ERGIC) (TOOZE et al. 1984; KRIJNSE-LOCKER et al. 1994). For
successful assembly of the envelope proteins into virus particles their expression at
the budding site is necessary. Therefore, spike (S), membrane (M), and envelope (E)
proteins contain sorting signals within their amino acid sequence resulting in
localization at the ERGIC or Golgi compartment (MACHAMER & ROSE 1987;
ROTTIER & ROSE 1987; KLUMPERMAN et al. 1994; LOCKER et al. 1995; CORSE
& MACHAMER 2000; LONTOK et al. 2004). The CoV S protein is the determinant for
host range due to its essential function in receptor binding and membrane fusion.
CoV S proteins are type I membrane proteins with a large, highly glycosylated
ectodomain, a transmembrane domain, and a short cytoplasmic tail. The cytoplasmic
domain contains a cysteine-rich and a charge-rich region. Several CoVs show
potential intracellular retention signals within their charge-rich region. Among
Alphacoronaviruses like transmissible gastroenteritis virus (TGEV), feline infectious
peritonitis virus (FIPV), human CoV 229E, and porcine epidemic diarrhea coronavirus
(PEDV) and among Gammacoronaviruses like infectious bronchitis virus (IBV)
potential dibasic ER retrieval signals (KKXX-COOH or KXHXX-COOH) and tyrosinebased localization signals (YXXI or YXXF) were identified (LONTOK et al. 2004;
SCHWEGMANN-WESSELS et al. 2004; WINTER et al. 2008; SHIRATO et al. 2011).
Dibasic signals lead to protein localization at the site of assembly (like ER or ERGIC)
(TEASDALE & JACKSON 1996; LONTOK et al. 2004). For the tyrosine-based motif
several functions have been described. For instance, it regulates the sorting of
proteins in endosomes or the trans-Golgi network and is able to lead to intracellular
retention (BOS et al. 1993; AROETI & MOSTOV 1994; MALLABIABARRENA et al.
1995; ROHRER et al. 1996). Additionally, tyrosine signals are involved in lysosomal
targeting and are responsible for internalization/endocytosis of receptors and other
cellular proteins as well as for basolateral sorting (LETOURNEUR & KLAUSNER
1992; THOMAS & ROTH 1994; BOGE et al. 1998; LI et al. 2000). However, for
TGEV and IBV S proteins the tyrosine-based motif results in intracellular retention at
the ERGIC and Golgi compartment (SCHWEGMANN-WESSELS et al. 2004;
110
Manuscript III
WINTER et al. 2008; PAUL et al. 2014). Thus, in single expressing cells S is
expressed intracellularly and rarely at the cell surface (LONTOK et al. 2004;
SCHWEGMANN-WESSELS et al. 2004; SHIRATO et al. 2011). Regarding
Betacoronaviruses, like Middle-East respiratory syndrome coronavirus (MERS-CoV)
and severe acute respiratory syndrome coronavirus (SARS-CoV), S sequences
contain a potential ER retrieval signal whereas mouse hepatitis virus (MHV) as well
as bovine coronavirus (BCoV) do not show such signals within their S sequence
(LONTOK et al. 2004; MCBRIDE et al. 2007). S proteins of SARS-CoV and MHV are
detected on the cell surface when independently expressed but are intracellularly
retained when co-expressed with the corresponding M protein (OPSTELTEN et al.
1995; MCBRIDE et al. 2007). During virus assembly interaction of S with M protein is
required for S incorporation into virus particles resulting in infectious virions or viruslike particles (VLPs) (OPSTELTEN et al. 1995; VENNEMA et al. 1996; NGUYEN &
HOGUE 1997). The S cytoplasmic domain seems to be essential during S-M
interaction whereas its ectodomain as well as its transmembrane domain are
dispensable as shown by the help of MHV- and FIPV-S chimeras (GODEKE et al.
2000; BOSCH et al. 2005). In case of MHV S, the charge-rich region plays a major
role during assembly, accomplished by S-M interaction while its cysteine-rich region
is more involved in cell-to-cell fusion (YE et al. 2004). Concerning SARS-CoV S
proteins its charge-rich region especially its dibasic motif plays a role in S-M
interaction as well (MCBRIDE et al. 2007). Due to several differences in S sequences
and expression pattern along and within the CoV genera, the role of the charge-rich
region and the tyrosine-based motif of the Alphacoronavirus S protein during S-M
interaction was examined in more detail. Therefore, the TGEV S sequence was used
as backbone while its charge-rich region was exchanged by analogue human- or batderived Alpha- and Betacoronavirus S charge-rich regions either containing a
complete, an incomplete or no tyrosine-based signal.
CoV M proteins are important during virus assembly due to their ability to interact
with viral structural proteins (LANSER & HOWARD 1980; STURMAN et al. 1980;
OPSTELTEN et al. 1995; NARAYANAN et al. 2000; LIM & LIU 2001). M proteins
contain a short ectodomain, three transmembrane domains, and a long cytoplasmic
tail (HOGUE & MACHAMER 2008). Concerning, the SARS M cytoplasmic domain a
tyrosine at position 195 seem to be important for S retention and interaction while for
MHV M a tyrosine at position 211 of the cytoplasmic tail may be involved (DE HAAN
111
Manuscript III
et al. 1999; MCBRIDE & MACHAMER 2010). Therefore, three TGEV M Y/A mutants,
where the tyrosine at position 235, 236 and 243 were exchanged by an alanine, were
tested for potential S retention in S and M protein co-expressing cells.
4.2 Material and Methods
4.2.1 Cell lines and virus strains
Baby hamster kidney cells (BHK-21) were grown in Dulbecco’s modified Eagle
medium supplemented with 5 % fetal calf serum.
4.2.2 Plasmids
TGEV Swt in pCG1 was used as backbone cDNA. The nucleotide sequence
encoding for the charge-rich region was exchanged by human-derived CoV S
sequences of SARS, MERS, NL63, and 229E as well as by bat-derived CoV S
sequences of F52 and F56 (Tab. 6). Due to the fact that amino acid sequences of
229E and F56 S charge-rich regions are identical, figures in the results were
declared just as 229E, but they show the same results as for F56. The amino acid
sequence of the SARS S charge-rich region is identical to the corresponding one of
the bat SARS-like WIV1 virus.
The TGEV S Y/A mutant harbors a destroyed
tyrosine-retention signal and is known to be expressed at the cell surface
(SCHWEGMANN-WESSELS et al. 2004; GELHAUS et al. 2014). Regarding the S
Y/A mutant, the tyrosine of the retention signal (position 1440) was replaced by an
alanine (Tab. 1). TGEV Mwt and TGEV M-HA cDNA were used for S-M interaction
studies. Regarding M-HA, wildtype M was tagged by HA (GELHAUS et al. 2014).
TGEV M Y/A mutants were cloned using standard hybridization PRC technique and
two of them were HA tagged (Tab. 7).
112
Manuscript III
Tab. 6 Amino acid sequences of coronavirus (CoV) spike (S) charge-rich regions.
TGEV S wildtype sequence was used as backbone, while its charge-rich region was exchanged by the
represented corresponding human- and bat-derived CoV S sequences. Transmissible gastroenteritis
virus (TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory
syndrome coronavirus (MERS-CoV), human CoV 229E and NL63, bat CoV F52 and F56. Complete
and incomplete tyrosine-based sorting signals are in red and bold letters. S amino acid sequence of
SARS-CoV is the same as for bat SARS-like WIV1 virus. Sequences of 229E and F56 are identical.
Virus
Group
Species
Sequence
TGEV
α-CoV
pig
RQFENYEPIEKVHVH
TGEV (S Y/A mutant)
α-CoV
pig
RQFENAEPIEKVHVH
SARS-CoV
β-CoV b
human
DDSEPVLKGVKLHYT
MERS-CoV
β-CoV c
human
DRYEEYDLEPHKVHVH
229E
α-CoV
human
ESTKLPYYDVEKIHIQGGDYKDDDDK
NL63
α-CoV
human
GSTKLPYYEFEKVHVQ
F52
α-CoV
bat
DTFEEEYEPLVSEEEEPPVKVHYA
F56
α-CoV
bat
ESTKLPYYDVEKIHIQGGDYKDDDDK
Tab. 7 Amino acid sequence of TGEV M Y/A mutants.
Tyrosines of TGEV M cytoplasmic domain were mutated to alanine (red and bold letters).
Declaration
Y/A position
227 – Sequence - 244
TGEV M Y/A-1
235
SSATGWAAYVKSKAGDYS
TGEV M Y/A-HA
236
SSATGWAYAVKSKAGDYS
TGEV M Y/A+8-HA
243
SSATGWAYYVKSKAGDAS
4.2.3 Transfection
BHK-21 cells were transfected by Lipofectamine® 2000 (Life Technologies) following
the manufacturer’s instructions.
4.2.4 Immunoflurescence analysis of single S or M protein expression
Cells were seeded in 24-well plates. One day later, the cells were transfected with
certain S constructs by the help of Lipofectamine®. After 16 h cells were fixed with
3 % paraformaldehyde. To observe protein expression on the cell surface cells were
113
Manuscript III
not permeabilized, whereas for the detection of protein overall expression cells were
permeabilized by the help of 0.2 % Triton-X-100. Afterwards, cells were treated with
monoclonal antibodies (mAb) against TGEV S (6A.C3, 1:200, kindly provided by Luis
Enjuanes) followed by FITC-conjugated anti-mouse antibody (1:100, Sigma). Nuclei
were stained with DAPI (4',6-diamidino-2-phenylindole). The Nikon Eclipse Ti
microscope was used for fluorescence analysis.
Regarding M Y/A mutants, overall expression was detected 16 h after transfection
with Lipofectamine®. Therefore, cells were fixed by the help of 3 % paraformaldehyde
followed by permeabilization using 0.2 % Triton-X-100. Afterwards, cells were treated
with mAb against TGEV M (9D.B4, 1:200, kindly provided by Luis Enjuanes) followed
by Cy3-conjugated anti-mouse antibody (1:500, Sigma). Nuclei were stained with
DAPI (4',6-diamidino-2-phenylindole). The Nikon Eclipse Ti microscope was used for
fluorescence analysis.
4.2.5 Immunofluorescence analysis of S and M-HA co-expression
Cells were seeded in 24-well plates. The next day cells were transfected with certain
S constructs and TGEV M-HA at a ratio 1:4 by the help of Lipofectamine ®. After 16 h
cells were fixed with 3 % paraformaldehyde and S protein surface expression as well
as S protein overall expression (by the help of 0.2 % Triton-X-100) were examined.
Afterwards, cells were treated with mAb against TGEV S (6A.C3, 1:200) followed by
FITC-conjugated anti-mouse antibody (1:100, Sigma). To detect M-HA, cells were
treated with anti-HA antibody (1:200, Sigma) followed by Cy3-conjugated anti-rabbit
antibody (1:500, Sigma). Nuclei were stained with DAPI (4',6-diamidino-2phenylindole). The Leica TCS SP5 confocal microscope was used for fluorescence
analysis.
4.2.6 Immunofluorescence analysis of S and Mwt co-expression
Cells were seeded in 24-well plates. The following day, cells were transfected with
cDNA encoding for the different S constructs and TGEV Mwt at a ratio 1:4 by the
help of Lipofectamine®. After 16 h and in the case of TGEV SSARS-S-Tail and TGEV
SMERS-S-Tail
constructs
additionally
after
18 h,
cells
were
fixed
with
3%
paraformaldehyde. To visualize S surface expression cells were treated with mAb
against TGEV S (6A.C3, 1:200) followed by FITC-conjugated anti-mouse antibody
114
Manuscript III
(1:100, Sigma), before permeabilization. Afterwards, cells were permeabilized with
0.2 % Triton-X-100 and treated with mAb against TGEV M protein (9D.B4, 1:200).
Fluorescence-labeling of the M protein was done by the help of Cy3-conjugated antimouse antibody (1:500, Sigma). Nuclei were stained with DAPI (4',6-diamidino-2phenylindole). The Leica TCS SP5 confocal microscope was used for fluorescence
analysis.
4.2.7 Immunofluorescence analysis of S Y/A and M Y/A mutant co-expression
Cells were seeded in 24-well plates. The next day cells were transfected with S Y/A
constructs and certain TGEV M Y/A cDNAs at a ratio 1:4 by the help of
Lipofectamine®. After 16 h cells were fixed with 3 % paraformaldehyde and S Y/A as
well as M Y/A protein overall expression (by the help of 0.2 % Triton-X-100) were
examined. Therefore, cells were treated with mAb against TGEV S (6A.C3, 1:200)
followed by FITC-conjugated anti-mouse antibody (1:100, Sigma). To detect M Y/A,
cells were treated with anti-HA antibody (1:200, Sigma) followed by Cy3-conjugated
anti-rabbit antibody (1:500, Sigma). Nuclei were stained with DAPI (4',6-diamidino-2phenylindole). The Nikon Eclipse Ti microscope was used for fluorescence analysis.
Additionally, S Y/A surface expression was observed 16 hours post transfection (hpt).
Cells were treated with mAb against TGEV S (6A.C3, 1:200) followed by FITCconjugated anti-mouse antibody (1:100, Sigma), before permeabilization. Afterwards,
cells were permeabilized with 0.2 % Triton-X-100. Here, intracellular expressed
TGEV M Y/A was detected using mAb against TGEV M (9D.B4, 1:200) followed by
Cy3-conjugated secondary anti-mouse antibody (1:500, Sigma). Nuclei were stained
with DAPI (4',6-diamidino-2-phenylindole). The Nikon Eclipse Ti microscope was
used for fluorescence analysis.
4.2.8 Co-immunoprecipitation, surface biotinylation, and cell lysis
BHK-21 cells were seeded on 6-well plates. One day later they were transfected with
S constructs plus empty pCG1 or co-transfected with S constructs and TGEV Mwt
cDNA by the help of Lipofectamine®. The next day cells were put on ice and washed
three times with ice-cold PBS. Afterwards, cells were treated with 1ml/ well of
0.5 mg/ml EZ-Link® Sulfo-NHS-LC-Biotin/PBS (Thermo Scientific) for 30 min at 4 °C
on a shaker. Then, cells were washed three times with 0.1 M glycine/ PBS and
115
Manuscript III
subsequently incubated with 0.1 M glycine/ PBS for 15 min at 4 °C. In the following
step, cells were washed with PBS and were scratched from the plate within 1 ml
PBS. After centrifugation at 3,800 x g at 4 °C supernatants were discarded and
pellets were resuspended in 100 µl NP-40 lysis buffer plus cOmpleteTM (protease
inhibitor cocktail, Roche) for 30 min at 4 °C. After incubation, the suspension was
centrifuged at 17,900 x g at 4 °C. Supernatants were transferred to a new tube. For
co-immunoprecipitation, 80 µl of the lysates were mixed with 50 µl Protein-ASepharose® beads (Sigma), 2 µl of mAb against TGEV S (6A.C3) and with 368 µl
NP-40 lysis buffer plus cOmpleteTM (Roche). Samples were incubated overnight at
4 °C under rotation. The next day, beads were washed 3 times with ice cold NP-40
lysis buffer followed by centrifugation at 17,900 x g at 4 °C to remove unspecific
binding; supernatants were discarded. Protein-antibody complexes were eluted from
the beads by heating them in 50 µl 6 x SDS sample buffer at 96 °C for 10 min. Beads
were pelleted by centrifugation at 17,900 x g and the supernatants were transferred
to a new tube and subsequently run on SDS-PAGE. The remaining 20 µl lysates
were mixed with 2 × SDS sample puffer and subjected to SDS-PAGE. By a semi-dry
technique (KYHSE-ANDERSEN 1984) SDS gels were transferred to nitrocellulose
membranes (GE Healthcare) which were subsequently blocked with 1 % blocking
reagent (Roche) in blocking buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5)
overnight at 4 °C. Membranes were washed 3 times with phosphate buffered saline +
0.1 % Tween (PBS+T) and once with PBS. The nitrocellulose membranes containing
lysates were treated with antibodies against the TGEV S protein (mAb 6A.C3, 1:200)
or the TGEV M protein (mAb 9D.B4, 1:200) overnight. The blot containing coimmunoprecipitated samples was treated with antibodies against TGEV M protein
(mAb 9D.B4, 1:200). The next day, blots were washed 3 times with PBS+T and PBS
followed by secondary antibody treatment. Lysates and co-immunoprecipitated
samples were treated with anti-mouse peroxidase-conjugated antibody (Dako;
1:1000) for 1 h at 4 °C. The membrane for the detection of surface biotinylated S was
treated
with
Streptavidin-biotinylated
horseradish
peroxidase
(1:1000,
GE
Healthcare). Chemiluminescent peroxidase substrate (Thermo Scientific) and the
Chemi Doc system (Biorad) were used for chemiluminescence signal detection.
Additionally, surface biotinylation and cell lysis was done with cells co-expressing
TGEV S Y/A and certain TGEV M Y/A mutant proteins. The experimental approach
was the same as described above for the tested S constructs.
116
Manuscript III
4.3 Results
4.3.1 S expression pattern alters when co-expressed with M proteins resulting
in intracellular S accumulation
All examined S proteins were expressed in BHK-21 cells and could be detected by
the used mAb against TGEV S (Fig. 4-1 & Fig. 4-2). In single expressing cells TGEV
S Y/A, TGEV SSARS-S-Tail, TGEV SMERS-S-Tail, and TGEV S229E-S-Tail were detected at the
cell surface as well as widely and diffuse distributed within the cytoplasm, whereas
TGEV Swt, TGEV SF52-S-Tail, and TGEV SNL63-S-Tail were only expressed intracellular at
the perinuclear region (Fig. 4-1a & Fig. 4-2a). Nevertheless, in the case of TGEV
S229E-S-Tail, only in a few cells S was noticed at the surface. Furthermore, TGEV SF52-STail
was distributed in vesicle-like structures as it is described for the TGEV S K/M
mutant, where the 1445 lysine is exchanged by methionine (PAUL et al. 2014).
After co-expression of S and M, S proteins were strongly accumulated near the
nucleus and close to the TGEV M-HA protein (Fig. 4-1b & Fig. 4-2b). S expression
pattern differed completely from single expressed S proteins, except from TGEV
SNL63-S-Tail. An intracellular retention of the S constructs was observed. Only regarding
TGEV SSARS-S-Tail a surface expression on a few cells was detectable but less
compared to single expression.
Additionally, TGEV M-HA single expressing cells were treated either with anti-HA
antibody or mAb against TGEV M. By detecting HA, M-HA proteins seem to be more
distributed probably due to antibody binding to single HA peptides (Fig. 4-1c).
117
Manuscript III
Fig. 4-1 Surface and total protein expression of S constructs in single expressing or M-HA coexpressing cells (part 1).
Transfection of BHK-21 cells with indicated S construct inserted in pCG1 expression plasmid.
Detection of S proteins (green) on cell surfaces or intracellularly (total), when treated with Triton-X-100
(a). Co-localization analysis of parental TGEV S or TGEV S mutant proteins with TGEV M-HA (HA
tagged). Detection of S surface expression and co-localization of S and M-HA proteins within the cells.
S protein (green), M-HA protein (red), co-localized proteins (yellow), (b). PFA fixation 16 hpt. Nuclei
stained with DAPI.
118
Manuscript III
Fig. 4-2 Surface and total protein expression of S constructs in single expressing or M-HA coexpressing cells (part 2).
Transfection of BHK-21 cells with indicated S construct inserted in pCG1 expression plasmid.
Detection of S proteins (green) on cell surfaces or intracellularly (total), when treated with Triton-X-100
(a). Co-localization analysis of parental TGEV S or TGEV S mutant proteins with TGEV M-HA (HA
tagged). Detection of S surface expression and co-localization of S and M-HA proteins within the cells.
S protein (green), M-HA protein (red), co-localized proteins (yellow), (b). PFA fixation 16 hpt. Nuclei
stained with DAPI.
119
Manuscript III
Due to the fact that in S and M-HA cDNA co-transfected cells TGEV SSARS-S-Tail was
detected in a reduced amount at the cell surface, we wanted to find out if those cells
are expressing both proteins. Therefore, cells co-transfected with S and TGEV Mwt
cDNA were first treated with antibodies against S, and then permeabilized followed
by treatment with antibodies against TGEV Mwt. For examination of a timedependent manner this experiment was done with cells either fixed 16 or 18 hpt (Fig.
4-3). 16 hpt only TGEV SSARS-S-Tail was less retained intracellularly although Mwt was
detected in the same cell. 18 hpt a higher amount of cells with TGEV SSARS-S-Tail
expressed at their cell surface was observed. Also here, Mwt was detectable within S
positive cells. Rare surface expression of TGEV SMERS-S-Tail was detected 18 hpt even
though Mwt was expressed within the same cells (Fig. 4-4; compare to single
expression 18 hpt, in Fig. 4-5). Nevertheless, Mwt was less accumulated near the
nucleus 18 hpt compared to its expression pattern 16 hpt.
i
g
.
4
3
T
G
E
V
S
S
A
R
S
S
T
a
i
l
120
a
n
d
T
G
Manuscript III
i
g
.
4
4
T
G
E
V
S
M
E
R
S
S
T
a
i
l
a
n
d
T
G
E
V
M
Fig. 4-5 TGEV SMERS-S-Tail single expression
Overview of TGEV SMERS-S-Tail at cell surfaces in single expressing BHK-21 cells 18 w
hpt.
i
l the M proteins
All S proteins have been detected in BHK-21 cell lysates as well as
d
when co-expressed. Surface biotinylation experiments showed similar
results as
t
y
noticed by immunofluorescence analysis (Fig. 4-6). In single transfected
cells TGEV
p
S Y/A, TGEV SSARS-S-Tail, TGEV SMERS-S-Tail, and TGEV S229E-S-Tail were
e expressed at
121
p
r
o
t
Manuscript III
the cell surface but not TGEV Swt, TGEV S F52-S-Tail, and TGEV SNL63-S-Tail. When cells
were co-transfected with Mwt a reduced amount of S was detectable at the cell
surface. Concerning the cell lysates, in TGEV S Y/A, TGEV S SARS-S-Tail, TGEV SMERSS-Tail,
TGEV S229E-S-Tail, and TGEV SNL63-S-Tail single expressing cells two bands have
been noticed. In the case of TGEV SSARS-S-Tail and TGEV SMERS-S-Tail the upper band
was more prominent then the lower one. Regarding TGEV S Y/A, TGEV S 229E-S-Tail,
and TGEV SNL63-S-Tail the lower band was predominant compared to the upper one.
Most likely, the upper band represents complex glycosylated S proteins, whereas the
lower
band
signifies
high-mannose
S
proteins.
Regarding
the
co-
immunoprecipitation, M proteins have been detected in every sample when TGEV M
cDNA was transfected, also slightly in M single expressing cells. Thus, M protein
unspecific binding to the beads is likely. This nonspecific binding of M proteins was
confirmed in cells co-expressing vesicular stomatitis virus glycoprotein and TGEV M
with pCG1 plasmid with inserted TGEV S wt or S mutant constructs. TGEV S
The charge-rich region was exchanged by different human- and bat-derived
he case of TGEV S Y/A the tyrosine of the tyrosine-based retention signal was
r 16 hpt surface biotinylation was performed. One part of lysates was analyzed for
tern blot. The other part of the lysates was used for surface expression and coof S proteins at cell surfaces and of S and M proteins in cell lysates. For coe treated with protein-A-sepharose beads and antibodies against TGEV S, while on
TGEV M were used. Dashed lines indicate changed order of the bands or point at
parental TGEV S or TGEV S mutant proteins and total protein expression of S and
proteins (data not shown).
4.3.2 TGEV M Y/A protein mutants still lead to S protein retention
All tested TGEV M Y/A proteins were detected by using a mAb against TGEV M.
These mutants were expressed in the perinuclear region similar to TGEV Mwt (Fig.
4-7a). To examine if M Y/A mutants are able to retain TGEV S proteins, cells were
co-transfected with TGEV M Y/A and TGEV S Y/A (surface expression due to
122
Manuscript III
destroyed tyrosine-based retention signal) cDNAs followed by immunofluorescence
analysis. In co-expressing cells, S Y/A proteins have been noticed accumulating
intracellularly near the nucleus and near M Y/A mutants (Fig. 4-7b). Nevertheless, S
Y/A protein was still observed at cell surfaces but in lower amounts compared to S
Y/A single expressing cells (Fig. 4-7c). These results were confirmed by S Y/A
surface biotinylation (Fig. 4-8). Results of co-immunoprecipitation using mAb against
TGEV S for precipitation are not shown due to failed M protein detection on the
western blot. All in all we could show that the three tested TGEV M Y/A mutants were
able to retain the TGEV S Y/A protein, although it was no complete retention as it
was observed for TGEV Mwt and TGEV S Y/A co-expressing cells (see results
above).
123
Manuscript III
Fig. 4-7 Immunofluorescence analysis of TGEV M Y/A protein mutants single-expressed or coexpressed with TGEV S Y/A protein in BHK-21 cells.
Total protein expression of TGEV M Y/A mutants intracellularly in single expressing cells (a); Coexpression of TGEV M Y/A mutants and TGEV S Y/A protein, both detected intracellular. TGEV M Y/A
mutant proteins were detected by HA-antibody (b); TGEV M Y/A mutant protein expression
intracellularly and TGEV S Y/A protein detection on cell surfaces (c); S protein (green), M protein
(red), nuclei (DAPI). PFA fixation 16 hpt.
124
Manuscript III
Fig. 4-8 Surface biotinylation and cell lysates of BHK-21 cells single transfected with TGEV M
Y/A mutant constructs or co-transfected with S Y/A cDNA.
After 16 hpt surface biotinylation was performed. One part of lysates was analyzed for total protein
expression on western blot. The other part of the lysates was analyzed for surface expression.
Detection of S Y/A proteins on cell surfaces and of S Y/A and M Y/A proteins in cell lysates. Dashed
lines indicate changed order of the bands or point at different blots.
4.4 Discussion
The CoV S protein is the determinant for host specificity as well as cell tropism. Its
main function includes receptor recognition and binding to host cells as well as
induction of fusion of the viral envelope with the endosomal membrane or from cellto-cell resulting in syncytia formation (YOO et al. 1991; SUZUKI & TAGUCHI 1996;
GALLAGHER & BUCHMEIER 2001). Assembly of infectious virus particles requires
S and M interaction resulting in S incorporation. Although S-M interaction has been
shown for IBV, BCoV, MHV, FIPV, and SARS-CoV, the specific regions responsible
for this association are mainly unknown (NGUYEN & HOGUE 1997; GODEKE et al.
2000; YOUN et al. 2005; HSIEH et al. 2008). In the case of SARS and MHV S
proteins their cytoplasmic domains, especially the charge-rich region appear to be
critical during the interaction with M (YE et al. 2004; MCBRIDE et al. 2007). By taking
a look at the Alphacoronavirus TGEV the cysteine-rich region in the S protein
cytoplasmic domain as well as the palmitoylation of these cysteines are not sufficient
for S-M interaction although S incorporation into virus particles is palmitoylation
dependent (GELHAUS et al. 2014). To examine if the tyrosine-based motif in the
charge-rich region of TGEV S is important for S-M interaction, different S constructs
125
Manuscript III
were cloned. Here, the charge-rich region of TGEV S was exchanged by the one of
human- and bat-derived S sequences of Alpha- and Betacoronaviruses. Those
constructs either contain a complete retention signal within their cytoplasmic tail, an
incomplete one or no retention motif. By immunofluorescence analysis S single
expression was investigated. It was noticed, that S constructs harboring a complete
tyrosine-dependent localization signal (YXXI; YXXL or YXXF) were expressed
intracellularly and not at the cell surface, except from constructs containing a YXXV
motif. S constructs with a YXXV signal, an incomplete or no tyrosine-based motif
were localized intracellularly as well as at the plasma membrane. The TGEV S
protein is intracellularly retained due to its tyrosine-based retention signal
(SCHWEGMANN-WESSELS et al. 2004). In the case of TGEV SF52-S-Tail and TGEV
SNL63-S-Tail the complete tyrosine motif may function as a retention signal as well.
Nevertheless, the well-known tyrosine-dependent localization signal YXXΦ: (Y =
tyrosine; X = any amino acid; Φ = bulky hydrophobic amino acid like phenylalanine
(F), isoleucine (I), leucine (L), methionine (M) or valine (V)) may lead to
internalization/
endocytosis
from
cell
surfaces
as
well
(BONIFACINO
&
DELL'ANGELICA 1999; KELLY et al. 2008). Additionally, the YXXΦ motif affects
membrane protein trafficking inside cells (BONIFACINO & TRAUB 2003; PANDEY
2009; ILINSKAYA et al. 2010). Determination of protein sorting and trafficking is due
to an interaction of tyrosine-based motifs with cellular proteins (BONIFACINO &
DELL'ANGELICA 1999). Regarding human T-lymphotropic virus type 1 (HTLV-1), its
envelope (Env) protein is synthesized at the ER, known to form trimers and contains
a receptor-binding domain in its surface subunit and a YXXI motif within its
cytoplasmic domain (DELAMARRE et al. 1996; DELAMARRE et al. 1999). The
tyrosine-based signal of HTLV-1 Env protein interacts with µ subunits of various
adaptor protein complexes whereas the interaction with each adaptor complex
results in different routes of transport (NORRIS et al. 1995; TRAUB et al. 1995;
DELL'ANGELICA et al. 1998; DELL'ANGELICA et al. 1999; BONIFACINO & TRAUB
2003; NAKATSU & OHNO 2003; JANVIER & BONIFACINO 2005). In the case of
TGEV S proteins, a potential cellular interaction partner of its tyrosine-based
retention signal is conjectural but not known yet (SCHWEGMANN-WESSELS et al.
2004). The tyrosine-based signal of TGEV S229E-S-Tail with valine as hydrophobic
amino acid does not lead to complete S retention. Additionally, TGEV S 229E-S-Tail
harbors a di-acidic signal (DXE), as it is present in SARS-CoV and MERS-CoV S
126
Manuscript III
proteins. The di-acidid signal may lead to protein transport from the trans-Golgi
network towards the plasma membrane, as it is reported for vesicular stomatitis virus
glycoprotein (NISHIMURA et al. 2002). Thus, the DXE motif may be the reason for
the surface expression of TGEV S229E-S-Tail, as well as of TGEV SF56-S-Tail.
Furthermore, TGEV S229E-S-Tail, TGEV SSARS-S-Tail, TGEV SMERS-S-Tail as well as TGEV
SNL63-S-Tail showed a double band profile on western blot when single expressed,
while for TGEV Swt only the lower band was observed. Retained S proteins are not
fully glycosylated and contain high mannose oligosaccharides, whereas S proteins
on cell surfaces have been transported through the Golgi complex towards the
plasma membrane and contain complex N-glycans (SCHWEGMANN-WESSELS et
al. 2004; PAUL et al. 2014). Fully glycosylated S proteins have a higher molecular
weight leading to the additional upper band on the western blot. Although, TGEV
SNL63-S-Tail is characterized by a complex glycosylation profile, it is not expressed at
the cell surface. Hence, this chimeric protein may be transported to the Golgi
compartment where it obtains N-glycans but is not moved further towards the plasma
membrane. However, TGEV SF52-S-Tail was characterized as vesicle-like structures
when expressed alone by immunofluorescence. Similar observations are known for
the TGEV S K/M mutant. Here, the lysine-methionine exchange leads to protein
transport to the plasma membrane subsequently followed by endocytosis (PAUL et
al. 2014). Although, TGEV SF52-S-Tail contains a complete ER retrieval signal (KXHXX)
as well as a tyrosine-based sorting signal like TGEV Swt, it has additional 9 amino
acids between these two motifs unlike TGEV Swt, resulting in a perinuclear, vesiclelike expression pattern.
In the next step, cells were co-transfected with S constructs of interest and TGEV MHA (M tagged to HA) cDNA to investigate if they still interact with each other. All
tested S constructs were strongly accumulated near the nucleus and closely
expressed to the TGEV M-HA protein 16 hpt. Only for TGEV SSARS-S-Tail a minor
expression at the cell surface was still detectable but less compared to single
transfected cells. These results demonstrate M-induced S retention probably due to
S-M interaction for all tested S constructs. As control double positive cells expressing
TGEV SSARS-S-Tail at the surface while M is expressed in the same cells are noticed.
Additionally, S expression was examined 18 hpt. Here, rare surface expression of
TGEV SMERS-S-Tail was detected while TGEV M was co-expressed in the same cell.
Regarding TGEV SSARS-S-Tail even higher amounts of S were expressed on cell
127
Manuscript III
surfaces after 18 h compared to 16 h. This is accounted by overexpression resulting
in S transport to the plasma membrane. If cells are fixed 18 h or later than 18 hpt
slightly S surface expression is notable for the other tested S constructs as well due
to overexpression, even though TGEV M proteins are expressed in the same cell
(data not shown).
By surface biotinylation of single and co-transfected cells similar results regarding S
surface expression were detected as observed by immunofluorescence. Also here,
less or no surface expression of S was noticed in co-transfected cells although S
protein was located at the plasma membrane in single transfected ones. For direct
examination
of
S-M
protein
interaction
co-immunoprecipitation
was
done.
Unfortunately, M proteins have been precipitated using mAb against TGEV S and
protein-A sepharose beads even in M single expressing cells. CoV M proteins are
able to form homomultimeric protein complexes, which may be the reason for
unspecific binding to the beads (LOCKER et al. 1992).
Due to the fact that the SARS-CoV M protein harbors a tyrosine in its cytoplasmic tail
at position 195 and the MHV M protein at position 211, respectively, both involved in
S protein retention, three TGEV M tyrosine mutants were cloned and validated (DE
HAAN et al. 1999; MCBRIDE & MACHAMER 2010). The TGEV M protein contains
tyrosines at position 235, 236, and 243 which were exchanged by alanine. All three
mutants were able to mainly retain the TGEV S Y/A protein, although this S mutant is
expressed at the cell surface in single expressing cells. Nevertheless, higher
amounts of TGEV S Y/A proteins were observed on cell surfaces in M Y/A protein coexpressing cells compared to TGEV S Y/A and Mwt co-expressing cells. Thus, two of
the tested tyrosines of the TGEV M cytoplasmic domain are enough for S-interaction
and retention. However, for a complete retention of the S protein a cooperation of all
three tyrosines seems to be necessary. Additionally, co-immunoprecipitation studies
were performed but did not produce results regarding potential S-M interaction (data
not shown). Again, we were not able to demonstrate S-M protein complexes via coimmunoprecipitaion as it is shown for MHV (DE HAAN et al. 1999). Therefore,
methods like bimolecular fluorescence complementation or fluorescence resonance
energy transfer seem to be adequate to examine direct S-M association (FORSTER
1959; HU et al. 2002).
128
Manuscript III
4.5 Conclusion
All in all we were able to show that TGEV S-M interaction was not inhibited by the
exchange of the S charge-rich region but slightly affected by TGEV M tyrosine
mutants. The charge-rich region, mainly sorting signals like the tyrosine-based
retention motif, influenced S single expression but had no effect on S-M interaction.
Due to the fact that sequence changes within the TGEV S cysteine-rich region as
well as in the charge-rich region do not influence S-M interaction other parts of this
protein seem to be crucial (GELHAUS et al. 2014). Nevertheless, the S charge-rich
region may be important for S-M association as long as it contains certain amounts of
charged amino acids. Therefore, S mutants with less or no charged amino acids
within their sequence should be tested. Additionally, potential functions of the dibasic
signal during S-M interaction have to be examined as well. The TGEV S ectodomain
may be another potential key player. Investigating different S mutants where parts of
the ectodomain or the whole domain are exchanged may provide more information
regarding critical S sequence regions important for successful S-M interaction.
Concerning specific tyrosines of the TGEV M cytoplasmic tail involved in S
interaction, at least two of them are sufficient for S retention. For further
investigations a TGEV M mutant where all of these three tyrosines are exchanged
(for instance by alanine) have to be verified. Such a mutant would give a hint if other
parts of its sequence are involved in the association with S proteins as well.
Authors’ contributions
ATR and CSW conceived and designed the study. ATR performed the experiments.
ATR and CSW analyzed the data and drafted the manuscript. All authors read and
approved the final manuscript. Sandra Bauer cloned TGEV M Y/A mutant constructs.
Competing interests
The authors have declared that no competing interests exist.
Funding
Financial support was provided by a grant to CSW (SCHW 1408/1.1) from the
German Research Foundation (DFG). CSW is funded by the Emmy Noether
129
Manuscript III
Programme from the DFG. ATR is a recipient of a Georg Christoph Lichtenberg PhD
fellowship from the Ministry for Science and Culture of Lower Saxony.
Acknowledgements
Thanks to Sandra Bauer for technical assistance. This work was performed by ATR
in partial fulfillment of the requirements for a Dr. rer. nat. degree from the University
of Veterinary Medicine Hannover. We are grateful to L. Enjuanes for providing
antibodies.
4.6 References
AROETI, B. & K. E. MOSTOV (1994): Polarized sorting of the polymeric immunoglobulin
receptor in the exocytotic and endocytotic pathways is controlled by the same amino acids.
EMBO J 13, 2297-2304
BOGE, M., S. WYSS, J. S. BONIFACINO & M. THALI (1998): A membrane-proximal tyrosinebased signal mediates internalization of the HIV-1 envelope glycoprotein via interaction with
the AP-2 clathrin adaptor. J Biol Chem 273, 15773-15778
BONIFACINO, J. S. & E. C. DELL'ANGELICA (1999): Molecular bases for the recognition of
tyrosine-based sorting signals. The Journal of cell biology 145, 923-926
BONIFACINO, J. S. & L. M. TRAUB (2003): Signals for sorting of transmembrane proteins to
endosomes and lysosomes. Annual review of biochemistry 72, 395-447
BOS, K., C. WRAIGHT & K. K. STANLEY (1993): TGN38 is maintained in the trans-Golgi network
by a tyrosine-containing motif in the cytoplasmic domain. EMBO J 12, 2219-2228
BOSCH, B. J., C. A. DE HAAN, S. L. SMITS & P. J. ROTTIER (2005): Spike protein assembly into
the coronavirion: exploring the limits of its sequence requirements. Virology 334, 306-318
CORSE, E. & C. E. MACHAMER (2000): Infectious bronchitis virus E protein is targeted to the
Golgi complex and directs release of virus-like particles. J Virol 74, 4319-4326
DE HAAN, C. A., M. SMEETS, F. VERNOOIJ, H. VENNEMA & P. J. ROTTIER (1999): Mapping of
the coronavirus membrane protein domains involved in interaction with the spike protein. J
Virol 73, 7441-7452
DELAMARRE, L., C. PIQUE, A. R. ROSENBERG, V. BLOT, M. P. GRANGE, I. LE BLANC & M. C.
DOKHELAR (1999): The Y-S-L-I tyrosine-based motif in the cytoplasmic domain of the human Tcell leukemia virus type 1 envelope is essential for cell-to-cell transmission. Journal of virology
73, 9659-9663
DELAMARRE, L., A. R. ROSENBERG, C. PIQUE, D. PHAM, I. CALLEBAUT & M. C. DOKHELAR
(1996): The HTLV-I envelope glycoproteins: structure and functions. Journal of acquired immune
deficiency syndromes and human retrovirology : official publication of the International Retrovirology
Association 13 Suppl 1, S85-91
DELL'ANGELICA, E. C., J. KLUMPERMAN, W. STOORVOGEL & J. S. BONIFACINO (1998):
Association of the AP-3 adaptor complex with clathrin. Science 280, 431-434
130
Manuscript III
DELL'ANGELICA, E. C., C. MULLINS & J. S. BONIFACINO (1999): AP-4, a novel protein complex
related to clathrin adaptors. The Journal of biological chemistry 274, 7278-7285
FORSTER, T. (1959): 10th Spiers Memorial Lecture. Transfer mechanisms of electronic
excitation. Discussions of the Faraday Society 27, 7-17
GALLAGHER, T. M. & M. J. BUCHMEIER (2001): Coronavirus spike proteins in viral entry and
pathogenesis. Virology 279, 371-374
GELHAUS, S., B. THAA, K. ESCHKE, M. VEIT &
C. SCHWEGMANN-WESSELS (2014):
Palmitoylation of the Alphacoronavirus TGEV spike protein S is essential for incorporation into
virus-like particles but dispensable for S-M interaction. Virology 464-465, 397-405
GODEKE, G. J., C. A. DE HAAN, J. W. ROSSEN, H. VENNEMA & P. J. ROTTIER (2000): Assembly
of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike
protein. J Virol 74, 1566-1571
HOGUE, B. G. & C. E. MACHAMER (2008): Coronavirus Structural Proteins and Virus Assembly.
In: S. PERLMAN, T. GALLAGHER &E. J. SNIJDER "Nidoviruses" American Society of Microbiology,
Washington, DC, pp. 179-200
HSIEH, Y. C., H. C. LI, S. C. CHEN & S. Y. LO (2008): Interactions between M protein and other
structural proteins of severe, acute respiratory syndrome-associated coronavirus. Journal of
biomedical science 15, 707-717
HU, C. D., Y. CHINENOV & T. K. KERPPOLA (2002): Visualization of interactions among bZIP
and Rel family proteins in living cells using bimolecular fluorescence complementation.
Molecular cell 9, 789-798
ILINSKAYA, A., G. HEIDECKER & D. DERSE (2010): Opposing effects of a tyrosine-based
sorting motif and a PDZ-binding motif regulate human T-lymphotropic virus type 1 envelope
trafficking. Journal of virology 84, 6995-7004
JANVIER, K. & J. S. BONIFACINO (2005): Role of the endocytic machinery in the sorting of
lysosome-associated membrane proteins. Molecular biology of the cell 16, 4231-4242
KELLY, B. T., A. J. MCCOY, K. SPATE, S. E. MILLER, P. R. EVANS, S. HONING & D. J. OWEN
(2008): A structural explanation for the binding of endocytic dileucine motifs by the AP2
complex. Nature 456, 976-979
KLUMPERMAN, J., J. K. LOCKER, A. MEIJER, M. C. HORZINEK, H. J. GEUZE & P. J. ROTTIER
(1994): Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion
budding. J Virol 68, 6523-6534
KRIJNSE-LOCKER, J., M. ERICSSON, P. J. ROTTIER & G. GRIFFITHS (1994): Characterization of
the budding compartment of mouse hepatitis virus: evidence that transport from the RER to
the Golgi complex requires only one vesicular transport step. J Cell Biol 124, 55-70
KYHSE-ANDERSEN, J. (1984): Electroblotting of multiple gels: a simple apparatus without
buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. Journal of
biochemical and biophysical methods 10, 203-209
LANSER, J. A. & C. R. HOWARD (1980): The polypeptides of infectious bronchitis virus (IBV-41
strain). J Gen Virol 46, 349-361
LETOURNEUR, F. & R. D. KLAUSNER (1992): A novel di-leucine motif and a tyrosine-based
motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 69, 11431157
131
Manuscript III
LI, Y., M. P. MARZOLO, P. VAN KERKHOF, G. J. STROUS & G. BU (2000): The YXXL motif, but
not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein
receptor-related protein. J Biol Chem 275, 17187-17194
LIM, K. P. & D. X. LIU (2001): The missing link in coronavirus assembly. Retention of the avian
coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and
physical interaction between the envelope and membrane proteins. J Biol Chem 276, 1751517523
LOCKER, J. K., D. J. OPSTELTEN, M. ERICSSON, M. C. HORZINEK & P. J. ROTTIER (1995):
Oligomerization of a trans-Golgi/trans-Golgi network retained protein occurs in the Golgi
complex and may be part of its retention. J Biol Chem 270, 8815-8821
LOCKER, J. K., J. K. ROSE, M. C. HORZINEK & P. J. ROTTIER (1992): Membrane assembly of
the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred
orientation. J Biol Chem 267, 21911-21918
LONTOK, E., E. CORSE & C. E. MACHAMER (2004): Intracellular targeting signals contribute to
localization of coronavirus spike proteins near the virus assembly site. J Virol 78, 5913-5922
MACHAMER, C. E. & J. K. ROSE (1987): A specific transmembrane domain of a coronavirus E1
glycoprotein is required for its retention in the Golgi region. The Journal of cell biology 105, 12051214
MALLABIABARRENA, A., M. A. JIMENEZ, M. RICO & B. ALARCON (1995): A tyrosine-containing
motif mediates ER retention of CD3-epsilon and adopts a helix-turn structure. EMBO J 14, 22572268
MCBRIDE, C. E., J. LI & C. E. MACHAMER (2007): The cytoplasmic tail of the severe acute
respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum
retrieval signal that binds COPI and promotes interaction with membrane protein. J Virol 81,
2418-2428
MCBRIDE, C. E. & C. E. MACHAMER (2010): A single tyrosine in the severe acute respiratory
syndrome coronavirus membrane protein cytoplasmic tail is important for efficient interaction
with spike protein. Journal of virology 84, 1891-1901
NAKATSU, F. & H. OHNO (2003): Adaptor protein complexes as the key regulators of protein
sorting in the post-Golgi network. Cell structure and function 28, 419-429
NARAYANAN, K., A. MAEDA, J. MAEDA & S. MAKINO (2000): Characterization of the
coronavirus M protein and nucleocapsid interaction in infected cells. J Virol 74, 8127-8134
NGUYEN, V. P. & B. G. HOGUE (1997): Protein interactions during coronavirus assembly. J
Virol 71, 9278-9284
NISHIMURA, N., H. PLUTNER, K. HAHN & W. E. BALCH (2002): The delta subunit of AP-3 is
required for efficient transport of VSV-G from the trans-Golgi network to the cell surface. Proc
Natl Acad Sci U S A 99, 6755-6760
NORRIS, F. A., E. UNGEWICKELL & P. W. MAJERUS (1995): Inositol hexakisphosphate binds to
clathrin assembly protein 3 (AP-3/AP180) and inhibits clathrin cage assembly in vitro. The
Journal of biological chemistry 270, 214-217
OPSTELTEN, D. J., M. J. RAAMSMAN, K. WOLFS, M. C. HORZINEK & P. J. ROTTIER (1995):
Envelope glycoprotein interactions in coronavirus assembly. J Cell Biol 131, 339-349
PANDEY, K. N. (2009): Functional roles of short sequence motifs in the endocytosis of
membrane receptors. Frontiers in bioscience 14, 5339-5360
132
Manuscript III
PAUL, A., A. TRINCONE, S. SIEWERT, G. HERRLER & C. SCHWEGMANN-WESSELS (2014): A
lysine-methionine exchange in a coronavirus surface protein transforms a retention motif into
an endocytosis signal. Biological chemistry 395, 657-665
ROHRER, J., A. SCHWEIZER, D. RUSSELL & S. KORNFELD (1996): The targeting of Lamp1 to
lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to
the membrane. J Cell Biol 132, 565-576
ROTTIER, P. J. & J. K. ROSE (1987): Coronavirus E1 glycoprotein expressed from cloned cDNA
localizes in the Golgi region. J Virol 61, 2042-2045
SCHWEGMANN-WESSELS, C., M. AL-FALAH, D. ESCORS, Z. WANG, G. ZIMMER, H. DENG, L.
ENJUANES, H. Y. NAIM & G. HERRLER (2004): A novel sorting signal for intracellular
localization is present in the S protein of a porcine coronavirus but absent from severe acute
respiratory syndrome-associated coronavirus. J Biol Chem 279, 43661-43666
SHIRATO, K., M. MAEJIMA, S. MATSUYAMA, M. UJIKE, A. MIYAZAKI, N. TAKEYAMA, H. IKEDA &
F. TAGUCHI (2011): Mutation in the cytoplasmic retrieval signal of porcine epidemic diarrhea
virus spike (S) protein is responsible for enhanced fusion activity. Virus Res 161, 188-193
STURMAN, L. S., K. V. HOLMES & J. BEHNKE (1980): Isolation of coronavirus envelope
glycoproteins and interaction with the viral nucleocapsid. J Virol 33, 449-462
SUZUKI, H. & F. TAGUCHI (1996): Analysis of the receptor-binding site of murine coronavirus
spike protein. J Virol 70, 2632-2636
TEASDALE, R. D. & M. R. JACKSON (1996): Signal-mediated sorting of membrane proteins
between the endoplasmic reticulum and the golgi apparatus. Annual review of cell and
developmental biology 12, 27-54
THOMAS, D. C. & M. G. ROTH (1994): The basolateral targeting signal in the cytoplasmic
domain of glycoprotein G from vesicular stomatitis virus resembles a variety of intracellular
targeting motifs related by primary sequence but having diverse targeting activities. J Biol
Chem 269, 15732-15739
TOOZE, J., S. TOOZE & G. WARREN (1984): Replication of coronavirus MHV-A59 in sac- cells:
determination of the first site of budding of progeny virions. European journal of cell biology 33,
281-293
TRAUB, L. M., S. KORNFELD & E. UNGEWICKELL (1995): Different domains of the AP-1 adaptor
complex are required for Golgi membrane binding and clathrin recruitment. The Journal of
biological chemistry 270, 4933-4942
VENNEMA, H., G. J. GODEKE, J. W. ROSSEN, W. F. VOORHOUT, M. C. HORZINEK, D. J.
OPSTELTEN & P. J. ROTTIER (1996): Nucleocapsid-independent assembly of coronavirus-like
particles by co-expression of viral envelope protein genes. EMBO J 15, 2020-2028
WINTER, C., C. SCHWEGMANN-WESSELS, U. NEUMANN & G. HERRLER (2008): The spike
protein of infectious bronchitis virus is retained intracellularly by a tyrosine motif. J Virol 82,
2765-2771
YE, R., C. MONTALTO-MORRISON & P. S. MASTERS (2004): Genetic analysis of determinants
for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich
and cysteine-rich regions of the endodomain. J Virol 78, 9904-9917
YOO, D. W., M. D. PARKER & L. A. BABIUK (1991): The S2 subunit of the spike glycoprotein of
bovine coronavirus mediates membrane fusion in insect cells. Virology 180, 395-399
133
Manuscript III
YOUN, S., E. W. COLLISSON & C. E. MACHAMER (2005): Contribution of trafficking signals in
the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection. Journal of
virology 79, 13209-13217
134
Discussion
5 Discussion
5.1 Replication of the Alphacoronavirus TGEV within chiropteran
cell lines
CoVs are host specific and show a narrow host range as well as tissue tropism. For
successful infection CoV S protein interaction with a specific receptor is required
(SANCHEZ et al. 1999; KUO et al. 2000; HAIJEMA et al. 2003). Several receptors for
CoVs have been identified (DVEKSLER et al. 1991; BEAUCHEMIN et al. 1999; W. LI
et al. 2003; PYRC et al. 2007; RAJ et al. 2013). TGEV is using pAPN to enter the
cells (DELMAS et al. 1992). APN is a type II transmembrane glycoprotein. It is
expressed as homodimer on the apical membrane of epithelial cells in the respiratory
and gastrointestinal tract, on liver cells, on endothelial cells of the kidney, as well as
on cells of the immune system like monocytes, dendrites or granulocytes (DELMAS
et al. 1993; LENDECKEL et al. 2000). Beside TGEV also other Alphacoronavirus S
proteins bind to APN, as this is known for HCoV 229E, FIPV, feline CoV, canine CoV,
PRCoV, and PEDV (YEAGER et al. 1992; DELMAS et al. 1993; TRESNAN et al.
1996; B. X. LI et al. 2007). PEDV and TGEV recognize pAPN as receptor and sialic
acids as sugar co-receptor (SCHWEGMANN-WESSELS & HERRLER 2006; LIU et
al. 2015). Regarding TGEV, point mutations in the S protein may result in the lack of
sialic acid binding activity as well as in a strong reduction of its enteropathogenicity.
Additionally, sialylated macromolecules which bind to the TGEV particle surface may
stabilize the virus in the gastrointestinal tract (KREMPL et al. 1998). Thus, the usage
of sialic acids as co-receptor may explain the enteric tropism of TGEV and PEDV.
Nevertheless, PEDV and TGEV S sequences show relatively low similarities. Thus,
PEDV also recognizes APN orthologues like human APN. Additionally, PEDV is able
to infect monkey and bat cells (LIU et al. 2015). Therefore, host range and tropism is
depending on the interaction of S proteins with the cellular receptor (SANCHEZ et al.
1999; KUO et al. 2000; LIU et al. 2015). Genetic changes within the S sequence may
result not only in different receptor usage, but also in altered tissue tropism and
virulence as it is shown for TGEV and PRCoV (SANCHEZ et al. 1999; KUO et al.
2000; HAIJEMA et al. 2003). PRCoV, a S gene deletion mutant of TGEV, infects and
replicates mainly in the respiratory tract, whereas TGEV causes enteric diseases
(COX et al. 1990a; COX et al. 1990b). Deletions or alterations within the receptor
135
Discussion
binding site (RBS) of PRCoV S protein may lead to tissue-specific tropism
(SANCHEZ et al. 1992). Furthermore, neutralization-resistant mutants of TGEV
containing point mutations or small deletions within the S gene result in a strongly
reduced enteropathogenicity (BERNARD & LAUDE 1995). Thus, N-terminal
subregions of S genes may influence the stability and folding of the expressed S
protein, resulting in differentially affected (like less fusogenic or attachment capacity)
S wildtype, mutant or truncated proteins within the gastrointestinal tract (LAUDE et al.
1993; BERNARD & LAUDE 1995).
However, CoVs are known to possess a
relatively high frequency of recombination and mutation rates which allow these
viruses to adapt to new hosts.
A close link between bat and human CoVs is described and lead to the assumption
of a zoonotic origin of human CoVs from bats (GLOZA-RAUSCH et al. 2008;
PFEFFERLE et al. 2009). The HCoV NL63, an Alphacoronavirus, is using ACE2 as
receptor in contrast to most other Alphacoronaviruses which recognize APN.
However, HCoV NL63 is able to infect human, primate as well as bat cells (VAN DER
HOEK et al. 2004; DONALDSON et al. 2008; HUYNH et al. 2012). The HCoV 229E
is predicted to share its most recent common ancestor with a bat CoV found in a leafnosed bat in Ghana (PFEFFERLE et al. 2009). Furthermore, the bat SARS-like CoVWIV1 isolated from Chinese horseshoe bat faecal samples was able to enter and
replicate in HeLa cells exogenously expressing ACE2 receptor from humans, civets,
and bats. Thus, a direct bat-to-human infection by some bat SARS-like CoVs is
possible. Moreover, this study provides strong evidence for Chinese horseshoe bats
being the natural reservoir of the SARS-CoV (W. LI et al. 2005; GE et al. 2013).
Both genera, Alpha- as well as Betacoronaviruses have been detected in chiropteran
by PCR. However, different circulation and transmission dynamics in bats are
noticed. Regarding this, Alphacoronaviruses demonstrate a wider host range as well
as a greater genetic diversity compared to Betacoronaviruses (HE et al. 2014). A lot
of Alphacoronaviruses infecting diverse bat species are known but their isolation
wasn’t successful yet. Therefore, the use of a model virus, like TGEV, may help to
understand their replication within chiropteran cell lines. Swine testis cells
endogenously express pAPN and were used as positive control, whereas chiropteran
cells of interest do not. Thus, tested chiropteran cell lines were not susceptible for
TGEV infection. After transfection of the chiropteran cells with pAPN-GFP TGEV was
136
Discussion
able to enter these cells. The results confirm a pAPN-dependent entry of TGEV
(HOFFMANN et al. 2013).
However, viruses have to overcome much more hurdles than entering host cells. To
replicate successfully in foreign hosts the pathogen must be able to amplify by the
help of host machinery and host factors, has to evade inhibitory host products, must
be able to leave the cell and spread to neighbor cells and the virus must exit the
initial host and transmit to another one (WEBBY et al. 2004). In pAPN expressing
chiropteran cells TGEV was able to replicate and to egress infectious progeny.
Nevertheless, in the various chiropteran cells lines differences in the amount of
released viral particles which did not correlate with receptor expression levels on the
cell surface were observed. Furthermore, S and M proteins within infected
chiropteran cells displayed different expression pattern 24 hpi. This leads to the
assumption that beside the abundance of the species-specific receptor other cellular
as well as viral factors influence TGEV replication. Cellular regulatory elements
affecting transcription, translation, as well as RNA and protein processing, stability
and transport influence viral pathogenesis (LEVINE 1984). Therefore, host factors
interacting with TGEV S proteins were identified and their role during virus life cycle
was investigated.
5.2 Alphacoronavirus interaction with host cell microtubules
For murine CoVs several studies represent the importance of intact microtubules for
successful replication, viral protein trafficking and assembly, as well as neuronal
spread (PASICK et al. 1994; KALICHARRAN & DALES 1995, 1996; BISWAS & DAS
SARMA 2014). Regarding Alphacoronaviruses like TGEV, an alteration in the
expression pattern of alpha and beta tubulin (up-regulation) in infected ST cells is
known (ZHANG et al. 2013). Nevertheless, the precise functions of microtubules
during the TGEV infection cycle as well as specific viral components interacting with
these filaments haven’t been identified, so far. In this study Alphacoronavirus S
proteins of TGEV, HCoV NL63, and HCoV 229E were detected to directly associate
with different tubulin alpha and beta chains, especially their last 39 aa stretch of the S
protein cytoplasmic domain. Presence of dynamic microtubules is relevant for TGEV
replication and assembly. Due to microtubule depolymerization S and M proteins are
expressed different compared to TGEV infected cells with intact microtubules. Here,
137
Discussion
TGEV S and M proteins are scattered within the cytoplasm and no perinuclear
accumulation is detectable anymore. Furthermore, less TGEV S protein is
incorporated into virus particles as well as a reduced amount of infectious virus yield
is measured when microtubules are drug-induced depolymerized. Although an
interaction of TGEV with tubulin does not seem to be essential, significantly fewer
infectious progeny emerges in the absence of intact microtubules. This was also
shown for TGEV infected chiropteran cells. Due to the fact that alpha and beta
tubulins are endogenously expressed among all eukaryotic cells a highly conserved
coronavirus replication strategy utilizing microtubules is likely. Especially, for spillover events the use of cellular proteins which are conserved among several host
species may facilitate or promote virus transmission.
Beside the direct interaction of tested Alphacoronavirus S proteins with tubulin an
additional association to microtubules by the help of molecular motors is possible.
Co-immunoprecipitation studies or confocal microscopy analysis in absence or
presence of microinjected anti-dynein or anti-kinesin antibodies may be adjuvant for
further investigations (SUIKKANEN et al. 2003; THEISS et al. 2005). Many viruses
like herpes simplex virus, African swine fever virus, lyssavirus, rabies virus,
papillomavirus and Ebola virus are associated to dynein light chains for example for
their transport towards the nucleus (JACOB et al. 2000; RAUX et al. 2000; ALONSO
et al. 2001; MARTINEZ-MORENO et al. 2003; DOUGLAS et al. 2004; KUBOTA et al.
2009). Nevertheless, dynein light chains homodimerization may occur in the
cytoplasm
without
being
associated
to
microtubules.
Therefore,
co-
immunoprecipitation of viral components and dynein intermediate or heavy chains
would be necessary (MERINO-GRACIA et al. 2011). Regarding CoVs, only for
SARS-CoV envelope protein an interaction with dynein heavy chain isoform I has
been observed so far (ALVAREZ et al. 2010).
Although CoV replication takes place within the cytoplasm and not inside the nucleus
viral protein association with molecular motors and/or microtubules may play a
crucial role during their movement on secretory pathways. Here, protein trafficking is
explained by the cisternal maturation model combined with the stable ERGIC model
(BANNYKH & BALCH 1997; GLICK et al. 1997; MIRONOV et al. 1997; BENTEKAYA et al. 2005; APPENZELLER-HERZOG & HAURI 2006; UJIKE & TAGUCHI
2015), (Fig. 5-1a). The newly synthesized CoV proteins S, M, and E are packaged
into coat protein complex II (COPII) vesicles which bud from ER exit sites and be
138
Discussion
anterograde transported towards the ERGIC (GLICK & NAKANO 2009). At the
ERGIC CoV particle assembly and budding takes place. Budded COPII vesicles fuse
homotypically, resulting in new cis-cisternae of the Golgi. Cisternae move and mature
to medial- and then to trans-cisternae. Arrived at the trans-Golgi network cisternae
break down and transport carriers emerge. Proteins/ virions are transported to the
plasma membrane, either directly or indirectly by recycling endosomes (LUINI et al.
2008). COPI vesicles mediate retrograde transport of proteins towards younger
cisternae, the ERGIC or the ER, function as recycling process (GLICK & NAKANO
2009; GLICK & LUINI 2011; GEVA & SCHULDINER 2014). Microtubules facilitate
the protein traffic from the ER towards the Golgi complex and the plasma membrane
as well as their way back (COLE & LIPPINCOTT-SCHWARTZ 1995; KREIS et al.
1997). Molecular motors such as kinesin are involved and power the transport from
the Golgi towards the ER (LIPPINCOTT-SCHWARTZ et al. 1995), while the motor
complex of dynein/dynactin supports ER-to-Golgi transport (PRESLEY et al. 1997),
(Fig. 5-1b).
Golgi apparatus organization is microtubule-dependent and its compact cisternae
cluster at the microtubule organizing center (MTOC), where the microtubule minusend is located (THYBERG & MOSKALEWSKI 1985; HO et al. 1989). A drug-induced
depolymerization of microtubules leads to a breakdown of Golgi cisternae into many
ministacks, distributed within the cytoplasm, by keeping their normal activities and
functions (ROGALSKI & SINGER 1984; TURNER & TARTAKOFF 1989). Regarding
mitosis, cytoplasmic microtubules disappear during prophase, resulting in diffusely
spread Golgi cisternae throughout the cytosole. During telophase cytoplasmic
microtubules reappear and the Golgi apparatus shows its characteristic shape
(MOSKALEWSKI et al. 1977). A Golgi-to-ER recycling pathway may be an
explanation for Golgi scattering after microtubule depolymerization. Recycling of
Golgi glycosylation enzymes is relevant for structural and functional maintenance of
the Golgi apparatus (STORRIE et al. 1998). In infected ST cells, where microtubules
were drug-induced depolymerized, TGEV S and M proteins partly co-localized with
the scattered ERGIC and Golgi ministacks. Although the S and M protein synthesis
was unaffected by organelle disruption, virus yield of infectious progeny was
reduced. This supports the idea, that trafficking of TGEV components along
microtubules is involved in CoV assembly and budding processes. Similar
observations are found for Sendai virus (CHAMBERS & TAKIMOTO 2010).
139
Discussion
Nevertheless, virus assembly or budding are not basically microtubule-dependent, for
example Bursal disease virus life cycle is not critically affected when microtubule
dynamic is inhibited (DELGUI et al. 2013).
Fig. 5-1 Intracellular protein transport, modified (KREIS et al. 1997; GLICK & NAKANO 2009).
Cisternal maturation model combined with stable ER-Golgi intermediate compartment (ERGIC) model.
Coat protein complex (COP), trans-Golgi network (TGN), (a); vesicular transport from ER towards
Golgi and Golgi towards ER or plasma membrane via microtubules (MT) and molecular motors.
Microtubule organizing center (MTOC), COPI (blue), COPII (pink), kinesin (orange), dynein/dynactin
(green), (b).
5.3 Interaction of coronaviral S and M proteins
S and M proteins of MHV interact specifically with each other and form large
heterocomplexes post-translationally but with different kinetics. While unmodified M
proteins immediately associate with S proteins after synthesis, newly synthesized S
proteins are found in these complexes after 10-20 min. S protein folding is necessary
for its interaction with M proteins and is the rate-limiting step, whereas under
reducing conditions no M-S complexes are formed (OPSTELTEN et al. 1994).
Correct S protein folding involves formation of disulfide bonds, addition of N-linked
sugars, as well as its assembly into homo-oligomers, which occurs slowly
140
Discussion
(VENNEMA et al. 1990; OPSTELTEN et al. 1993; OPSTELTEN et al. 1995). In
contrast, the final conformation of M proteins is glycosylated but without inter- or
intramolecular disulfide bonds (OPSTELTEN et al. 1993). In MHV M and S protein
co-expressing cells, heteromultimeric complexes are transported beyond the ERGIC
and accumulate at the Golgi apparatus (OPSTELTEN et al. 1995). M proteins
themselves are able to form complexes by M-M interactions as well. They
accumulate at the Golgi complex in homomultimeric and detergent-insoluble
structures (LOCKER et al. 1995). During CoV particle assembly only M and E
proteins are required and VLPs are built, whereas S proteins are incorporated when
present (BOS et al. 1996; VENNEMA et al. 1996). Interestingly, N proteins are not
required for VLP assembly and were not always taken into VLPs when present
(VENNEMA et al. 1996). Such nucleocapsid-less particles show a typical CoV
morphology but a lower density compared to virions, as this is shown for IBV
(MACNAUGHTON & DAVIES 1980). Beside M-S complexes, M proteins are able to
interact with HE proteins and form M-HE complexes leading to HE incorporation into
virus particles. Thus, M proteins play a crucial role in virus assembly retaining viral
glycoproteins at internal membranes (NGUYEN & HOGUE 1997).
However, CoV S proteins show low sequence conservation. Approximately 30 %
sequence identity is found in the S2 region responsible for membrane fusion
(CAVANAGH 1995). The highly conserved sequence KWPW(Y/W)VWL is present at
the transition of the transmembrane and ectodomain, which is probably involved in
membrane fusion as well but is dispensable for S incorporation into virus particles
(BOSCH et al. 2005). Additionally, in the cytoplasmic domain conserved cysteines
(approximately 24 %) are present (BOSCH et al. 2005). Regarding MHV and SARSCoV S cytoplasmic tail, especially their charge-rich region seems to play a key role
during M interaction (YE et al. 2004; BOSCH et al. 2005; MCBRIDE et al. 2007). By
the help of a recombinant fMHV mutant, containing a chimeric S gene (ectodomain of
FIPV, transmembrane and endodomain of MHV), a host switching event to feline
cells was detected due to the FIPV ectodomain. Nonetheless, interaction with MHV
M protein was still possible, resulting in S chimera incorporation into virus particles
due to the S carboxy-terminus derived from MHV (KUO et al. 2000). In contrast, for
TGEV S-M interaction neither the S cytoplasmic domain cysteine-rich motif nor the
tyrosine-based retention signal are essential (GELHAUS et al. 2014). All tested
TGEV S chimera, where the charge-rich region was exchanged by corresponding
141
Discussion
human- and bat-derived S sequences, were intracellularly retained closely to M
proteins in co-transfected cells. Nevertheless, investigated TGEV S chimera showed
different expression pattern when expressed alone. However, all testes S constructs
still contained positively or negatively charged amino acids. For further investigations,
S mutants harboring only uncharged amino acids downstream their cysteine-rich
region as well as the dibasic signal should be examined regarding their importance
during S-M protein interaction. Results may give a hint if the charge-rich region and
the dibasic signal of the TGEV S protein are necessary at all.
S protein regions important for the interaction with M proteins may depend on the
CoV genera. For Betacoronaviruses the S cytoplasmic domain seems to be
necessary, whereas for Alphacoronaviruses the S ectodomain is probably involved in
M protein association. A reason therefore may lay in S protein modifications during
maturation. For example, concerning MHV and SARS-CoV, S protein ectodomains
are cleaved into S1 and S2 subunits to reach full functional activity, which is
regarding TGEV not tested yet.
5.4 Conclusion
CoVs underlay high mutation and recombination rates during their replication cycle,
although they show a narrow host range. Nonetheless, several host shifting events
are known for these enveloped RNA viruses including successful animal-to-human
transmission. Especially, bats represent high risk factors for zoonotic host shifting.
Therefore, better understanding of CoV life cycle is necessary. In this study, we
confirmed that the Alphacoronavirus S protein is the determinant for species
restriction due to its function of receptor binding. Virus attachment to the species
specific receptor is the first hurdle the virus has to overcome. However, during virus
replication, assembly and budding, further cellular factors seem to be involved and
play a crucial role in host restriction. We were able to identify tubulins as interaction
partners of the CoV S protein cytoplasmic domain. This association highly likely
supports the S protein transport during the virus assembly and budding process. Due
to the fact that tubulins are conserved among eukaryotic cells this host factor is
available also in potential new hosts and does not represent a species barrier.
Moreover, the TGEV S protein tyrosine-based retention signal is not essential for SM interaction. Here, its ectodomain, the charged amino acids or the dibasic motif
142
Discussion
within the charge-rich region of the cytoplasmic domain might play a key role.
Sequence differences in the S charge-rich region may not be a barrier for coronaviral
evolution, as long as it contains a certain amount of charged amino acids.
Recombination events which may not disturb S-M interaction and incorporation of S
proteins into virions might extend the host range of newly evolved CoVs.
Further insights into CoV life cycle, usage of host cell machinery as well as the
identification and characterization of additional involved cellular factors may promote
antiviral target investigations.
5.5 References
ALONSO, C., J. MISKIN, B. HERNAEZ, P. FERNANDEZ-ZAPATERO, L. SOTO, C. CANTO, I.
RODRIGUEZ-CRESPO, L. DIXON & J. M. ESCRIBANO (2001): African swine fever virus protein
p54 interacts with the microtubular motor complex through direct binding to light-chain dynein.
J Virol 75, 9819-9827
ALVAREZ, E., M. L. DEDIEGO, J. L. NIETO-TORRES, J. M. JIMENEZ-GUARDENO, L. MARCOSVILLAR & L. ENJUANES (2010): The envelope protein of severe acute respiratory syndrome
coronavirus interacts with the non-structural protein 3 and is ubiquitinated. Virology 402, 281291
APPENZELLER-HERZOG, C. & H. P. HAURI (2006): The ER-Golgi intermediate compartment
(ERGIC): in search of its identity and function. J Cell Sci 119, 2173-2183
BANNYKH, S. I. & W. E. BALCH (1997): Membrane dynamics at the endoplasmic reticulum-Golgi
interface. J Cell Biol 138, 1-4
BEAUCHEMIN, N., P. DRABER, G. DVEKSLER, P. GOLD, S. GRAY-OWEN, F. GRUNERT, S.
HAMMARSTROM, K. V. HOLMES, A. KARLSSON, M. KUROKI, S. H. LIN, L. LUCKA, S. M. NAJJAR,
M. NEUMAIER, B. OBRINK, J. E. SHIVELY, K. M. SKUBITZ, C. P. STANNERS, P. THOMAS, J. A.
THOMPSON, M. VIRJI, S. VON KLEIST, C. WAGENER, S. WATT & W. ZIMMERMANN (1999):
Redefined nomenclature for members of the carcinoembryonic antigen family. Exp Cell Res
252, 243-249
BEN-TEKAYA, H., K. MIURA, R. PEPPERKOK & H. P. HAURI (2005): Live imaging of bidirectional
traffic from the ERGIC. J Cell Sci 118, 357-367
BERNARD, S. & H. LAUDE (1995): Site-specific alteration of transmissible gastroenteritis virus
spike protein results in markedly reduced pathogenicity. J Gen Virol 76 ( Pt 9), 2235-2241
BISWAS, K. & J. DAS SARMA (2014): Effect of microtubule disruption on neuronal spread and
replication of demyelinating and nondemyelinating strains of mouse hepatitis virus in vitro. J
Virol 88, 3043-3047
BOS, E. C., W. LUYTJES, H. V. VAN DER MEULEN, H. K. KOERTEN & W. J. SPAAN (1996): The
production of recombinant infectious DI-particles of a murine coronavirus in the absence of
helper virus. Virology 218, 52-60
BOSCH, B. J., C. A. DE HAAN, S. L. SMITS & P. J. ROTTIER (2005): Spike protein assembly into
the coronavirion: exploring the limits of its sequence requirements. Virology 334, 306-318
CAVANAGH, D. (1995): The Coronavirus Surface Glycoprotein. In: S. SIDDELL "The
Coronaviridae" Plenum Press, New York, pp. 73-113
143
Discussion
CHAMBERS, R. & T. TAKIMOTO (2010): Trafficking of Sendai virus nucleocapsids is mediated
by intracellular vesicles. PLoS One 5, e10994
COLE, N. B. & J. LIPPINCOTT-SCHWARTZ (1995): Organization of organelles and membrane
traffic by microtubules. Current opinion in cell biology 7, 55-64
COX, E., J. HOOYBERGHS & M. B. PENSAERT (1990a): Sites of replication of a porcine
respiratory coronavirus related to transmissible gastroenteritis virus. Res Vet Sci 48, 165-169
COX, E., M. B. PENSAERT, P. CALLEBAUT & K. VAN DEUN (1990b): Intestinal replication of a
porcine respiratory coronavirus closely related antigenically to the enteric transmissible
gastroenteritis virus. Veterinary microbiology 23, 237-243
DELGUI, L. R., J. F. RODRIGUEZ & M. I. COLOMBO (2013): The endosomal pathway and the
Golgi complex are involved in the infectious bursal disease virus life cycle. J Virol 87, 89939007
DELMAS, B., J. GELFI, R. L'HARIDON, L. K. VOGEL, H. SJOSTROM, O. NOREN & H. LAUDE
(1992): Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV.
Nature 357, 417-420
DELMAS, B., J. GELFI, H. SJOSTROM, O. NOREN & H. LAUDE (1993): Further characterization of
aminopeptidase-N as a receptor for coronaviruses. Adv Exp Med Biol 342, 293-298
DONALDSON, E. F., B. YOUNT, A. C. SIMS, S. BURKETT, R. J. PICKLES & R. S. BARIC (2008):
Systematic assembly of a full-length infectious clone of human coronavirus NL63. J Virol 82,
11948-11957
DOUGLAS, M. W., R. J. DIEFENBACH, F. L. HOMA, M. MIRANDA-SAKSENA, F. J. RIXON, V.
VITTONE, K. BYTH & A. L. CUNNINGHAM (2004): Herpes simplex virus type 1 capsid protein
VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular
transport. J Biol Chem 279, 28522-28530
DVEKSLER, G. S., M. N. PENSIERO, C. B. CARDELLICHIO, R. K. WILLIAMS, G. S. JIANG, K. V.
HOLMES & C. W. DIEFFENBACH (1991): Cloning of the mouse hepatitis virus (MHV) receptor:
expression in human and hamster cell lines confers susceptibility to MHV. J Virol 65, 6881-6891
GE, X. Y., J. L. LI, X. L. YANG, A. A. CHMURA, G. ZHU, J. H. EPSTEIN, J. K. MAZET, B. HU, W.
ZHANG, C. PENG, Y. J. ZHANG, C. M. LUO, B. TAN, N. WANG, Y. ZHU, G. CRAMERI, S. Y.
ZHANG, L. F. WANG, P. DASZAK & Z. L. SHI (2013): Isolation and characterization of a bat
SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535-538
GELHAUS, S., B. THAA, K. ESCHKE, M. VEIT & C. SCHWEGMANN-WESSELS (2014):
Palmitoylation of the Alphacoronavirus TGEV spike protein S is essential for incorporation into
virus-like particles but dispensable for S-M interaction. Virology 464-465, 397-405
GEVA, Y. & M. SCHULDINER (2014): The back and forth of cargo exit from the endoplasmic
reticulum. Current biology : CB 24, R130-136
GLICK, B. S., T. ELSTON & G. OSTER (1997): A cisternal maturation mechanism can explain the
asymmetry of the Golgi stack. FEBS Lett 414, 177-181
GLICK, B. S. & A. LUINI (2011): Models for Golgi traffic: a critical assessment. Cold Spring Harbor
perspectives in biology 3, a005215
GLICK, B. S. & A. NAKANO (2009): Membrane traffic within the Golgi apparatus. Annual review of
cell and developmental biology 25, 113-132
144
Discussion
GLOZA-RAUSCH, F., A. IPSEN, A. SEEBENS, M. GOTTSCHE, M. PANNING, J. F. DREXLER, N.
PETERSEN, A. ANNAN, K. GRYWNA, M. MULLER, S. PFEFFERLE & C. DROSTEN (2008):
Detection and prevalence patterns of group I coronaviruses in bats, northern Germany. Emerg
Infect Dis 14, 626-631
HAIJEMA, B. J., H. VOLDERS & P. J. ROTTIER (2003): Switching species tropism: an effective
way to manipulate the feline coronavirus genome. Journal of virology 77, 4528-4538
HE, B., Y. ZHANG, L. XU, W. YANG, F. YANG, Y. FENG, L. XIA, J. ZHOU, W. ZHEN, Y. FENG, H.
GUO, H. ZHANG & C. TU (2014): Identification of diverse alphacoronaviruses and genomic
characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in
China. J Virol 88, 7070-7082
HO, W. C., V. J. ALLAN, G. VAN MEER, E. G. BERGER & T. E. KREIS (1989): Reclustering of
scattered Golgi elements occurs along microtubules. European journal of cell biology 48, 250-263
HOFFMANN, M., M. A. MULLER, J. F. DREXLER, J. GLENDE, M. ERDT, T. GUTZKOW, C.
LOSEMANN, T. BINGER, H. DENG, C. SCHWEGMANN-WESSELS, K. H. ESSER, C. DROSTEN &
G. HERRLER (2013): Differential sensitivity of bat cells to infection by enveloped RNA viruses:
coronaviruses, paramyxoviruses, filoviruses, and influenza viruses. PloS one 8, e72942
HUYNH, J., S. LI, B. YOUNT, A. SMITH, L. STURGES, J. C. OLSEN, J. NAGEL, J. B. JOHNSON, S.
AGNIHOTHRAM, J. E. GATES, M. B. FRIEMAN, R. S. BARIC & E. F. DONALDSON (2012):
Evidence supporting a zoonotic origin of human coronavirus strain NL63. J Virol 86, 1281612825
JACOB, Y., H. BADRANE, P. E. CECCALDI & N. TORDO (2000): Cytoplasmic dynein LC8
interacts with lyssavirus phosphoprotein. J Virol 74, 10217-10222
KALICHARRAN, K. & S. DALES (1995): Involvement of microtubules and the microtubuleassociated protein tau in trafficking of JHM virus and components within neurons. Adv Exp Med
Biol 380, 57-61
KALICHARRAN, K. & S. DALES (1996): The murine coronavirus as a model of trafficking and
assembly of viral proteins in neural tissue. Trends in microbiology 4, 264-269
KREIS, T. E., H. V. GOODSON, F. PEREZ & R. RÖNNHOLM (1997): Golgi apparatus-cytoskeleton
interactions. In: E. G. BERGER und J. ROTH "The Golgi Apparatus" Birkhäuser Verlag, Basel, pp.
179-193
KREMPL, C., H. LAUDE & G. HERRLER (1998): Is the sialic acid binding activity of the S protein
involved in the enteropathogenicity of transmissible gastroenteritis virus? Adv Exp Med Biol
440, 557-561
KUBOTA, T., M. MATSUOKA, T. H. CHANG, M. BRAY, S. JONES, M. TASHIRO, A. KATO & K.
OZATO (2009): Ebolavirus VP35 interacts with the cytoplasmic dynein light chain 8. J Virol 83,
6952-6956
KUO, L., G. J. GODEKE, M. J. RAAMSMAN, P. S. MASTERS & P. J. ROTTIER (2000): Retargeting
of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell
species barrier. Journal of virology 74, 1393-1406
LAUDE, H., K. VAN REETH & M. PENSAERT (1993): Porcine respiratory coronavirus: molecular
features and virus-host interactions. Vet Res 24, 125-150
LENDECKEL, U., T. KAHNE, D. RIEMANN, K. NEUBERT, M. ARNDT & D. REINHOLD (2000):
Review: the role of membrane peptidases in immune functions. Adv Exp Med Biol 477, 1-24
145
Discussion
LEVINE, A. (1984): Viruses and Differentiation: The Molecular Basis of Viral Tissue Tropisms. In:
A. NOTKINS und M. A. OLDSTONE "Concepts in Viral Pathogenesis" Springer New York, pp. 130134
LI, B. X., J. W. GE & Y. J. LI (2007): Porcine aminopeptidase N is a functional receptor for the
PEDV coronavirus. Virology 365, 166-172
LI, W., M. J. MOORE, N. VASILIEVA, J. SUI, S. K. WONG, M. A. BERNE, M. SOMASUNDARAN, J.
L. SULLIVAN, K. LUZURIAGA, T. C. GREENOUGH, H. CHOE & M. FARZAN (2003): Angiotensinconverting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450-454
LI, W., Z. SHI, M. YU, W. REN, C. SMITH, J. H. EPSTEIN, H. WANG, G. CRAMERI, Z. HU, H.
ZHANG, J. ZHANG, J. MCEACHERN, H. FIELD, P. DASZAK, B. T. EATON, S. ZHANG & L. F.
WANG (2005): Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676-679
LIPPINCOTT-SCHWARTZ, J., N. B. COLE, A. MAROTTA, P. A. CONRAD & G. S. BLOOM (1995):
Kinesin is the motor for microtubule-mediated Golgi-to-ER membrane traffic. J Cell Biol 128,
293-306
LIU, C., J. TANG, Y. MA, X. LIANG, Y. YANG, G. PENG, Q. QI, S. JIANG, J. LI, L. DU & F. LI (2015):
Receptor usage and cell entry of porcine epidemic diarrhea coronavirus. J Virol 89, 6121-6125
LOCKER, J. K., D. J. OPSTELTEN, M. ERICSSON, M. C. HORZINEK & P. J. ROTTIER (1995):
Oligomerization of a trans-Golgi/trans-Golgi network retained protein occurs in the Golgi
complex and may be part of its retention. J Biol Chem 270, 8815-8821
LUINI, A., A. A. MIRONOV, E. V. POLISHCHUK & R. S. POLISHCHUK (2008): Morphogenesis of
post-Golgi transport carriers. Histochemistry and cell biology 129, 153-161
MACNAUGHTON, M. R. & H. A. DAVIES (1980): Two particle types of avian infectious bronchitis
virus. J Gen Virol 47, 365-372
MARTINEZ-MORENO, M., I. NAVARRO-LERIDA, F. RONCAL, J. P. ALBAR, C. ALONSO, F.
GAVILANES & I. RODRIGUEZ-CRESPO (2003): Recognition of novel viral sequences that
associate with the dynein light chain LC8 identified through a pepscan technique. FEBS Lett
544, 262-267
MCBRIDE, C. E., J. LI & C. E. MACHAMER (2007): The cytoplasmic tail of the severe acute
respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum
retrieval signal that binds COPI and promotes interaction with membrane protein. J Virol 81,
2418-2428
MERINO-GRACIA, J., M. F. GARCIA-MAYORAL & I. RODRIGUEZ-CRESPO (2011): The
association of viral proteins with host cell dynein components during virus infection. The FEBS
journal 278, 2997-3011
MIRONOV, A. A., P. WEIDMAN & A. LUINI (1997): Variations on the intracellular transport theme:
maturing cisternae and trafficking tubules. J Cell Biol 138, 481-484
MOSKALEWSKI, S., J. THYBERG, A. HINEK & U. FRIBERG (1977): Fine structure of the golgi
complex during mitosis of cartilaginous cells in vitro. Tissue & cell 9, 185-196
NGUYEN, V. P. & B. G. HOGUE (1997): Protein interactions during coronavirus assembly. J Virol
71, 9278-9284
OPSTELTEN, D. J., P. DE GROOTE, M. C. HORZINEK & P. J. ROTTIER (1994): Folding of the
mouse hepatitis virus spike protein and its association with the membrane protein. Archives of
virology. Supplementum 9, 319-328
146
Discussion
OPSTELTEN, D. J., P. DE GROOTE, M. C. HORZINEK, H. VENNEMA & P. J. ROTTIER (1993):
Disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins. J Virol
67, 7394-7401
OPSTELTEN, D. J., M. J. RAAMSMAN, K. WOLFS, M. C. HORZINEK & P. J. ROTTIER (1995):
Envelope glycoprotein interactions in coronavirus assembly. The Journal of cell biology 131,
339-349
PASICK, J. M., K. KALICHARRAN & S. DALES (1994): Distribution and trafficking of JHM
coronavirus structural proteins and virions in primary neurons and the OBL-21 neuronal cell
line. J Virol 68, 2915-2928
PFEFFERLE, S., S. OPPONG, J. F. DREXLER, F. GLOZA-RAUSCH, A. IPSEN, A. SEEBENS, M. A.
MULLER, A. ANNAN, P. VALLO, Y. ADU-SARKODIE, T. F. KRUPPA & C. DROSTEN (2009): Distant
relatives of severe acute respiratory syndrome coronavirus and close relatives of human
coronavirus 229E in bats, Ghana. Emerg Infect Dis 15, 1377-1384
PRESLEY, J. F., N. B. COLE, T. A. SCHROER, K. HIRSCHBERG, K. J. ZAAL & J. LIPPINCOTTSCHWARTZ (1997): ER-to-Golgi transport visualized in living cells. Nature 389, 81-85
PYRC, K., B. BERKHOUT & L. VAN DER HOEK (2007): The novel human coronaviruses NL63 and
HKU1. J Virol 81, 3051-3057
RAJ, V. S., H. MOU, S. L. SMITS, D. H. DEKKERS, M. A. MULLER, R. DIJKMAN, D. MUTH, J. A.
DEMMERS, A. ZAKI, R. A. FOUCHIER, V. THIEL, C. DROSTEN, P. J. ROTTIER, A. D.
OSTERHAUS, B. J. BOSCH & B. L. HAAGMANS (2013): Dipeptidyl peptidase 4 is a functional
receptor for the emerging human coronavirus-EMC. Nature 495, 251-254
RAUX, H., A. FLAMAND & D. BLONDEL (2000): Interaction of the rabies virus P protein with the
LC8 dynein light chain. J Virol 74, 10212-10216
ROGALSKI, A. A. & S. J. SINGER (1984): Associations of elements of the Golgi apparatus with
microtubules. J Cell Biol 99, 1092-1100
SANCHEZ, C. M., F. GEBAUER, C. SUNE, A. MENDEZ, J. DOPAZO & L. ENJUANES (1992):
Genetic evolution and tropism of transmissible gastroenteritis coronaviruses. Virology 190, 92105
SANCHEZ, C. M., A. IZETA, J. M. SANCHEZ-MORGADO, S. ALONSO, I. SOLA, M. BALASCH, J.
PLANA-DURAN & L. ENJUANES (1999): Targeted recombination demonstrates that the spike
gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and
virulence. J Virol 73, 7607-7618
SCHWEGMANN-WESSELS, C. & G. HERRLER (2006): Sialic acids as receptor determinants for
coronaviruses. Glycoconjugate journal 23, 51-58
STORRIE, B., J. WHITE, S. ROTTGER, E. H. STELZER, T. SUGANUMA & T. NILSSON (1998):
Recycling of golgi-resident glycosyltransferases through the ER reveals a novel pathway and
provides an explanation for nocodazole-induced Golgi scattering. J Cell Biol 143, 1505-1521
SUIKKANEN, S., T. AALTONEN, M. NEVALAINEN, O. VALILEHTO, L. LINDHOLM, M. VUENTO & M.
VIHINEN-RANTA (2003): Exploitation of microtubule cytoskeleton and dynein during parvoviral
traffic toward the nucleus. J Virol 77, 10270-10279
THEISS, C., M. NAPIREI & K. MELLER (2005): Impairment of anterograde and retrograde
neurofilament transport after anti-kinesin and anti-dynein antibody microinjection in chicken
dorsal root ganglia. European journal of cell biology 84, 29-43
THYBERG, J. & S. MOSKALEWSKI (1985): Microtubules and the organization of the Golgi
complex. Exp Cell Res 159, 1-16
147
Discussion
TRESNAN, D. B., R. LEVIS & K. V. HOLMES (1996): Feline aminopeptidase N serves as a
receptor for feline, canine, porcine, and human coronaviruses in serogroup I. J Virol 70, 86698674
TURNER, J. R. & A. M. TARTAKOFF (1989): The response of the Golgi complex to microtubule
alterations: the roles of metabolic energy and membrane traffic in Golgi complex organization.
J Cell Biol 109, 2081-2088
UJIKE, M. & F. TAGUCHI (2015): Incorporation of spike and membrane glycoproteins into
coronavirus virions. Viruses 7, 1700-1725
VAN DER HOEK, L., K. PYRC, M. F. JEBBINK, W. VERMEULEN-OOST, R. J. BERKHOUT, K. C.
WOLTHERS, P. M. WERTHEIM-VAN DILLEN, J. KAANDORP, J. SPAARGAREN & B. BERKHOUT
(2004): Identification of a new human coronavirus. Nat Med 10, 368-373
VENNEMA, H., G. J. GODEKE, J. W. ROSSEN, W. F. VOORHOUT, M. C. HORZINEK, D. J.
OPSTELTEN & P. J. ROTTIER (1996): Nucleocapsid-independent assembly of coronavirus-like
particles by co-expression of viral envelope protein genes. EMBO J 15, 2020-2028
VENNEMA, H., P. J. ROTTIER, L. HEIJNEN, G. J. GODEKE, M. C. HORZINEK & W. J. SPAAN
(1990): Biosynthesis and function of the coronavirus spike protein. Adv Exp Med Biol 276, 9-19
WEBBY, R., E. HOFFMANN & R. WEBSTER (2004): Molecular constraints to interspecies
transmission of viral pathogens. Nat Med 10, S77-81
YE, R., C. MONTALTO-MORRISON & P. S. MASTERS (2004): Genetic analysis of determinants
for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich
and cysteine-rich regions of the endodomain. J Virol 78, 9904-9917
YEAGER, C. L., R. A. ASHMUN, R. K. WILLIAMS, C. B. CARDELLICHIO, L. H. SHAPIRO, A. T.
LOOK & K. V. HOLMES (1992): Human aminopeptidase N is a receptor for human coronavirus
229E. Nature 357, 420-422
ZHANG, X., H. Y. SHI, J. F. CHEN, D. SHI, H. W. LANG, Z. T. WANG & L. FENG (2013):
Identification of cellular proteome using two-dimensional difference gel electrophoresis in ST
cells infected with transmissible gastroenteritis coronavirus. Proteome science 11, 31
148
Appendix
Appendix
Nucleotide and amino acid sequences
TGEV (PUR-46 MAD) S protein
Nucleotide sequence (4344 bp)
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
1351
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
2101
atgaaaaaac
caattttcct
atctcattga
tcagatgtgg
ttgcattcgc
aagcattgta
caacggttaa
aacaacccgc
agggctcacc
ggtagtgagt
agaggcaaat
aggttgttgc
caatggtctg
agtcgctggc
tcagttatta
tgcactgatc
aggaggtttt
ctaatagctc
ttagttaatt
attttgtttt
ataatactgt
caatcaggta
cactcttgaa
acggtgaaat
cactataatg
caaggagatt
atttctttag
ggtgatagtg
attagtacaa
gtcacgttaa
ggattttatc
tgtgttacta
gtcttggtat
agtaacatca
tcgttctgac
tatgggacaa
gctgttataa
ttacttaact
attgtaagtt
agaagtttgt
gtctgataat
gcacagatta
actaacagga
tatttgtggt
tgttctaaat
aaccttcctt
tgttaggtga
aatgatagta
ttgggattat
acgtagtcgt
aattttaatt
acctactacc
gcaggttaaa
tgtggtaata
ttatttacat
gcactgtcac
acgcttgtag
tagggttaat
aatgtgctag
ataccatcag
cacgttggtt
gcttatggcc
gagggtgctg
agacgtcatt
agggtgccac
atttcatgtt
tccgttcggc
gcacagctct
gctattagta
cacatttcct
acgttttctg
gttgaaaaca
taacattaaa
ctgtttcttc
cctagctttt
gaagcgtagt
cactaccaat
caattttcag
tatttttaag
aaactggtac
tttaacaagt
tgatgtagct
atgtaatata
agtggtgtgc
caatatatat
cgctacttag
tttggtcgta
tgactaatag
ctaaactata
ttattttcct
atgaccttta
gctacagaaa
taatggatac
ctgctgaagg
accacagaat
ccataagttc
tgctgtatgg
ggtgctagtt
atttggtgat
acctttggtg
aataaaaatg
ttatgtggct
attttagttt
agtggtaaat
agtccctagc
gctttgatca
aggttcaacc
agtgttttca
atacagtgag
gtaactgatg
taagtattta
agtggggcca
attgattgta
gacaatagct
cagctattac
tgctctcaaa
aagtgaagtt
acacacatac
ggttatggtc
gcaggatcac
tttatgttca
cgaaactgca
ttgtcctttc
tctgtttgtc
gcccgtacaa
tgaagaagga
acgatttgtc
ggtagaactg
tggcttatat
atgccattga
aactataggc
gtagtaggtt
actgtacaac
tgttacactg
atatcacttg
ccatactcca
tgctattata
ctagtttgac
cctatatgtc
cctacaatgg
accgtattag
atgcgtgcga
gtttaatcct
gtactaccgt
aatgttttta
taataattgg
tagttaccaa
tttgaagaag
atgtaatggt
ttaattttac
ttgaacacaa
tgactcgagc
gaccacggta
ggaacattac
tttttatatt
tatcttttaa
tacacatcgt
aaaggtgacg
ttactgctaa
ggtcttgtca
cattgttaac
aacccatagc
aacaccgatg
ttctacttgc
cggacgtttt
tcatttgata
gttgagtcct
gaaccaatga
gacaacatag
agtgctacac
gtgttggtat
tacacatcac
tttatggaga
aaccagtgga
accacctaat
cttggtttaa
gaaaatctta
gaatcacaga
tcacagttac
tgcatttgta
ttgcaattgg
cttctaattc
tttgcagatg
ttttgaaaat
caacattaga
gtttatgatg
agtttccaat
ctacacagcc
ttccttctaa
acagccgtta
cagcttctac
gctgttttaa
tacaaatgta
cgggtggtgt
tttttcagtt
ctgttacgta
cacctagtgt
aatggttaca
tttgaccact
acactgaagc
tattgtaata
tttgaataat
ataagagtgt
ataactattg
ctcaacatta
tgtactgtat
aaaagtgctt
agatgccaca
aattgaacaa
gttggtgcta
gcaggttgtt
tgggtgtacc
ctagattcct
tattagacaa
tatcaggtga
Appendix
2151
2201
2251
2301
2351
2401
2451
2501
2551
2601
2651
2701
2751
2801
2851
2901
2951
3001
3051
3101
3151
3201
3251
3301
3351
3401
3451
3501
3551
3601
3651
3701
3751
3801
3851
3901
3951
4001
4051
4101
4151
4201
4251
4301
tttgttaggt
catgtgatgt
gctatcactt
aacacctaat
ctcgtggcac
acctattcta
cgtcacacat
cgatacctac
tacactacac
ccctaggtgt
ttgagcaagc
tccatgttgt
attcaatagt
taggtggttc
agcaaacgta
tgtaacatct
gtggttatga
atggtgctac
atcccttgca
ctataccttt
caaactgatg
tcaagctatt
tacatcaaac
gtgcaagatg
acaattgcaa
ataataggct
acaggaagac
acaagcggag
aatgcgttag
catttgtttt
cacagtgcta
tttgtgcttc
cagttgactt
aactatgtat
aagggtgcga
attatacctg
aaattttaga
acgcaaccta
tcagaaaagc
cattaacaat
atgtaaaatg
ttttgcatac
atgcataggt
aatttgaaaa
tttaaaaatg
aagcgcacaa
ccattaacag
ttttattact
tgcaattgac
acataggtgt
tctgatggag
aaactttacc
cagtgtcaat
aacaaattgt
acttgcaatg
ttgtttctga
tcagaaactt
ttggctagaa
agtatcgttc
ggtttaggta
catagctgac
ctggtgtggc
ggtggtataa
tgcagtagca
tattgaacaa
ggtaacatta
atcacgaggt
ttgtcaacat
aataatttcc
tgacgaattg
ttacagcact
gttagggcta
gtctcagtct
cactcgcaaa
ttaccaacgg
agatggtgat
tgtttcgtaa
cagcctagag
tgtgctgttt
attatattga
ccaaattgga
tttaaacctg
tacataacac
acattagtca
gccttggtat
cattactgct
tgtttaggaa
ttacgaacca
ttagtgatgg
gcagctgtta
tgaactgtta
actctatata
agtaatgatg
ttgtaaaaat
acgtgcaacc
atatccgtgc
agactgttca
taacacaata
ggtgccagac
aaatgccctt
tagaccctat
ggtctaaaat
agctatagag
cagttgatga
ttagtatgtg
taatgctgac
cattaggtgc
gttcaggcta
aaaccagcag
cacagtcatt
cttgctactg
acaagggcaa
aagccattag
agtgctgatg
taatgcattt
gtagacaact
cagagattcg
tgcagcacca
cttatgaaac
cgcacttttg
tctagatgac
ttgcaactag
gttaatgcaa
tattaatcag
ctgtacctga
actggtgaaa
cactgtagaa
atcttgaatg
gtgtggctac
attttgctgt
gttgttgtca
attgaaaaag
150
tgtcatctac
ttgatggtac
ggtctaacac
taattacaca
ttgattgtga
ggtgcttttg
aattagcact
aagtcgaata
agatatgttt
cgtttctgca
ttgaaaacat
aaattggcat
ttacaaagaa
acatacttcc
gacttgcttt
agattataaa
ctcaatacta
aaaatgacta
acttggtgga
gacttaatta
attctggcta
tggtaaggtt
ttgctaaagc
gctttaagcc
tagttctatt
cacaagttga
gtgtctcaga
tgccaaagac
gattctgtgg
aatggcatga
tgtgactgct
gacttgtcgt
aagttctatt
ttctgacttt
ctgtaagtga
actgttcaag
gttgacattt
ttgatgactt
cttgccattc
gctcaataga
taataggctt
tgtagtacag
ctctatatgt
tgcacgtcca
tctgtaacgc
catagttggg
attggacaac
aatgatagga
acctgtcata
tttttattaa
ggtaatgtca
tattcaggtt
gtaatggtaa
tgtcaaacta
ggaggttgat
ctgttgaagc
tggcctaata
gtcccataat
ttgataaggt
cgttgtacag
taatggcatc
tgtacacagc
ggcgccgtgg
tgttgctcta
gtgctttcaa
aatgatgcta
attggcaaaa
acctaacagt
agtgacattt
caggctgatc
ctctaaccag
aaggttaatg
taatggtaca
ttttctttca
tggccaggta
taaagatgtc
tgacccccag
gttcaaattg
tttgcctagt
acatattaga
gacattttta
agaatttagg
tcattgacaa
attgaaacct
agtagtaata
gttgctgtgg
agtagaagac
ttaa
Appendix
Amino acid sequence (1447 aa)
accession no. CAB91145
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
1351
1401
MKKLFVVLVV
SDVVLGDYFP
QRLNVVVNGY
GSECRLNHKF
QWSGTVTFGD
CTDQCASYVA
LVNCLWPVPS
QSGKGATVFS
HYNGTALKYL
GDSDVFWTIA
GFYPVSSSEV
SNITLPMQDH
AVIKTGTCPF
RSLYVIYEEG
TNRTLLSGLY
AITSINSELL
TYSNIGVCKN
YTTPVSIDCS
SMLFVSENAL
SKRKYRSAIE
MVLPGVANAD
QTDVLNKNQQ
VQDVVNIQGQ
TGRLTALNAF
HLFSLANAAP
QLTLFRNLDD
IIPDYIDINQ
SEKLHNTTVE
FCIPLLLFCC
MPLIYGDNFP
TVQPWFNCIR
PYSITVTTTR
PICPSNSEAN
MRATTLEVAG
NVFTTQPGGF
FEEAASTFCF
LNTTGGVTLE
GTLPPSVKEI
YTSYTEALVQ
GLVNKSVVLL
NTDVYCIRSD
SFDKLNNYLT
DNIVGVPSDN
YTSLSGDLLG
GLTHWTTTPN
GAFVFINVTH
RYVCNGNPRC
KLASVEAFNS
DLLFDKVVTS
KMTMYTASLA
ILASAFNQAI
ALSHLTVQLQ
VSQTLTRQAE
NGMIFFHTVL
KFYLTPRTMY
TVQDILENFR
LAILIDNINN
CSTGCCGCIG
CSKLTNRTIG
NDSNDLYVTL
NFNSAEGAII
CGNMLYGLQW
TLVDLWWFNP
IPSDFSFNNW
EGAGFDQCNG
ISCYTVSDSS
AISKWGHFYI
VENTAITKVT
PSFYTHTIVN
QFSVYVHSTC
FNKFCLSLSP
SGVHDLSVLH
FKNVSDGVIY
FYYYSIYNYT
SDGDVQPIST
NKLLTQYVSA
SETLDPIYKE
GLGTVDEDYK
GGITLGALGG
GNITQSFGKV
NNFQAISSSI
VRASRQLAKD
LPTAYETVTA
QPRVATSSDF
PNWTVPELTF
TLVNLEWLNR
CLGSCCHSIC
NQWNLIETFL
ENLKALYWDY
CICKGSPPTT
FADEVVAYLH
VYDVSYYRVN
FLLTNSSTLV
AVLNNTVDVI
FFSYGEIPFG
NGYNFFSTFP
YCNSHVNNIK
ITIGLGMKRS
KSALWDNIFK
VGANCKFDVA
LDSCTDYNIY
SVTPCDVSAQ
NDRTRGTAID
GNVTIPTNFT
CQTIEQALAM
WPNIGGSWLE
RCTGGYDIAD
GAVAIPFAVA
NDAIHQTSRG
SDIYNRLDEL
KVNECVRSQS
WPGICASDGD
VQIEGCDVLF
DIFNATYLNL
IETYVKWPWY
SRRQFENYEP
LNYSSRLPPN
ATENITWNHR
TTESSLTCNW
GASYRISFEN
NKNGTTVVSN
SGKLVTKQPL
RFNLNFTTNV
VTDGPRYCYV
IDCISFNLTT
CSQITANLNN
GYGQPIASTL
RNCTDVLDAT
ARTRTNEQVV
GRTGVGIIRQ
AAVIDGTIVG
SNDVDCEPVI
ISVQVEYIQV
GARLENMEVD
GLKYILPSHN
LVCAQYYNGI
VQARLNYVAL
LATVAKALAK
SADAQVDRLI
QRFGFCGNGT
RTFGLVVKDV
VNATVSDLPS
TGEIDDLEFR
VWLLIGLVVI
IEKVHVH
agcgtgtgtg
ccgatacaga
tgcttcaacg
ctggtctata
ctcaattcag
ttatggcccg
ccaagtgtcc
ttacatttgt
agaaggacta
ttgcgttagt
caactggtgt
ttcaaaattg
attgcatgcg
tttgtcatgt
gaggcgatct
atattgatcg
ctggttcgtg
ttgttttggc
agatatgtaa
actctggatt
agtcttggtg
gcattaggaa
cactctaact
caggtggtat
catgtggaga
cgcaatagta
tatttggcat
tttttataac
tatggcatta
tcttacgatt
tgttcggctt
atgtattttg
gtctttcaac
gaagctatgt
ttgctttcag
gaacatcgac
TGEV (PUR-46 MAD) M protein
Nucleotide sequence (789 bp)
1
51
101
151
201
251
301
351
401
451
501
551
atgaagattt
acgctattgt
cagcgtctga
cttgcaaact
tgtgctacaa
aaatgcttat
tttaatgcat
tagtattgca
taagatccat
cctgaaacta
gcttcctctc
ggaatttgta
tgttaatatt
gctatgaaat
ttgtgagtca
ggaacttcag
tatggaagac
aatgtggcta
actcggaata
ggtgcaattg
tcagttgtac
aagcaattct
gaaggtgtgc
cgctgaaggg
151
Appendix
601
651
701
751
aatttaccaa
cacacttgtt
actatgtaaa
aatttgagtg
aatacgtaat
ggcaagaagt
atctaaagct
agcaagaaaa
ggttgcatta
tgaaagcaag
ggtgattact
attattacat
cctagcagga ctattgtcta
tagtgcgact ggatgggctt
caacagaggc aagaactgat
atggtataa
AMKSDTDLSC
YGRPQFSWFV
GAIVTFVLWI
EGVPTGVTLT
GKKLKASSAT
RNSTASDCES
YGIKMLIMWL
MYFVRSIQLY
LLSGNLYAEG
GWAYYVKSKA
CFNGGDLIWH
LWPVVLALTI
RRTKSWWSFN
FKIAGGMNID
GDYSTEARTD
ctgggcatcc
cgctctgtct
tcccccaagc
ttggaccaga
gcctgattcc
atggcctgta
gagtccaccg
ccaggggcac
agatcgacag
ctcaagggct
ccagggggaa
tggagggcaa
gatgcccgga
gttcaacatc
tgccgcccaa
gacactgagt
catcgtgagc
tgatccggat
tgggcatcct
gtggtgtatg
ccccacgtcg
gcaagccgtg
tacttcgtga
catcttcaag
atgtcatcat
atggtggtcc
gactgagctg
cgctgcagcc
ctcgccgacg
cgtcaaaaag
aatccttccc
actctcatcc
aggttccagc
tcgaaaccac
gagtcccaga
ctgggctcgg
Amino acid sequence (262 aa)
accession no. CAB91149
1
51
101
151
201
251
MKILLILACV
LANWNFSWSI
FNAYSEYQVS
PETKAILCVS
NLPKYVMVAL
NLSEQEKLLH
IACACGERYC
ILIVFITVLQ
RYVMFGFSIA
ALGRSYVLPL
PSRTIVYTLV
MV
HA peptide
Nucleotide sequence (30 bp)
1
tacccatacg atgttccaga ttacgct
Amino acid sequence (10 aa)
1
YPYDVPDYA
pAPN
Nucleotide sequence (2892 bp)
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
atggccaagg
cctcggcgtg
cccaggagaa
cccaccatca
gaaccggtac
cgctgagacc
ggcaaaagca
catccatagc
tgcggggcgt
gtagagctca
cggccacatg
acctggcagg
gtgctggcca
atgctttgac
accctaacaa
accccacttg
acctgtgatg
gcgtgaatga
gattctacat
gcggccgtgg
gaacaagaat
ccaccacagc
cgcctaccca
ctacctcact
tcgtccgctt
aagaagctca
gggggactcc
ctgagtacct
tacgagatgg
cttctaccgc
cgacacagat
gagccagcca
cctcacggcc
cagaagaccc
tccacgtacc
aacggcccaa
ttccaaggcc
ccaccatcat
gccgagcatg
cgccatcacc
caacgctgtt
cccaacgcgg
actctgccag
actacaccac
caggtcccag
ggtggtccac
agagtgaatt
agcgagtaca
gcagtctaca
tgaaggccac
ctgtccaata
caactggtct
ttctggccta
aatggcgtcc
152
Appendix
901
951
1001
1051
1101
1151
1201
1251
1301
1351
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
2101
2151
2201
2251
2301
2351
2401
2451
2501
2551
2601
2651
2701
2751
2801
2851
cctaatgcaa
tcccatccta
ccaaatccga
aactgggggc
gtcctcctcc
aactggccca
gacctgtggc
tgaccacgca
acgtgtaccg
accacccctg
tgactccatc
acttcctgac
gcctttgcct
gaaggctgtg
ccatcatgga
gacaccaaga
atccaacgtc
tctcatctat
gtttcccaag
cttgctgaac
acaactggag
cctgtcatca
tgcccacatg
acggagagaa
tacttcagcc
atacctcagg
ctaaaaactg
attaatgcca
tctggccaag
cgatccaccc
ggcggccagg
gctggtaaat
aggtctggct
attcggaagc
catcgggcag
tctttcagga
ggtgtgaccc
gttcaagaag
tggagcaagc
aacaaggagg
ttgcagaggg
aacttctttg
ccagattgcc
tggtgaccta
atcagcaaca
ccagtggttt
tgaatgaggg
gagcccacct
agtgatggct
ctgaggaggt
tcctacagca
tgaggacctg
atcagaacac
gatgctcaga
tcgatggacc
caggaaacat
acccgctcct
taaaaatggt
cccagaatga
gtcaacgtga
gatgattcag
atcgggctca
gtccctgtca
agagtacatg
tcatgttcga
aagcaggtcg
gaccgagcgc
tcagcactgc
acccttttcg
caacctgcgg
accagtggga
gaggccgaca
cctgaacagg
aagacgccac
cctctggcct
ctatggcggt
gaagattctc
aacaacatgg
cctggagaag
tggtgttgaa
ccatggcatg
ccaatcatta
ttgcccgact
ccgggagaac
aagagcgagt
ggcaacctgg
ctttgcctcc
ggaatctgaa
gtggatgctc
caacacacct
agggagcctc
ttcaaggagg
cacctacctg
cgtccatcag
ctgcagatgg
ctcacagaag
cagcgttcga
gtgatgcagg
tttgttcaaa
caggctattt
catcagctgc
ggtcatctac
ccctggctct
ccctggcagg
ccgctccgag
aacccctctt
ccagaaaatc
ctgctccaat
accagtggat
tccaccatct
ctttgcctgg
aactccgctc
tacctggatt
ctccactatt
gggattttgt
ggttccttct
ctctgagttt
atgtgggctt
accaaggcca
ttggttcata
153
tatgccctga
taatacatcc
tcaatgccgg
gcgctgctgt
tgtcactgtg
tgaccctggc
tatgtggagt
agacctcatc
tggcttcctc
gcccagatca
ggttatcagg
gcctggcgtc
gacctgtggg
gctgccagac
gcttccccgt
cacttcctcc
ctacctctgg
atcactactg
accgcatcgg
ccaggtgaac
agacaaacct
gacagcttca
ggacaacacc
ccgccctgag
gtctatggcc
ccaacatttc
tgatggacca
ggattgcctc
gagcgaccca
actgcaatgc
gggcagttac
agcgctggcc
acaccctgaa
aacagcattg
ccagagcaac
ccttctccaa
gagctgcagc
cggctccggc
acatcaagtg
gagcacagct
atgtgacagg
tacccactcc
tgccatggag
ttgacccaca
attgctcacg
ctggtggaat
acctgggtgc
gtgccaggcg
ccacctgctg
gcgagatgtt
atgctctcca
ctacttgcat
agcacctgca
actgtgagag
catcaccgtg
tcgactccga
attgttccca
gctgcgggat
acgattgggt
tacgacgagg
gtcggtcatc
acctggccac
ctcttcctga
cagcctgagc
ccatgaagaa
gaaactctca
gtacagtgag
aatgtgagaa
gaaaataacc
catagcccag
aacaagccca
tgcagcaacg
cccggacctc
ccagcaatgt
tggaagaagc
cctcattcag
agctggagca
acccgggctc
ggtgaaggag
aa
Appendix
Amino acid sequence (963 aa)
accession no. NP_999442
1
MAKGFYISKA LGILGILLGV AAVATIIALS VVYAQEKNKN AEHVPQAPTS
51
PTITTTAAIT LDQSKPWNRY RLPTTLLPDS YFVTLRPYLT PNADGLYIFK
101
GKSIVRLLCQ ESTDVIIIHS KKLNYTTQGH MVVLRGVGDS QVPEIDRTEL
151
VELTEYLVVH LKGSLQPGHM YEMESEFQGE LADDLAGFYR SEYMEGNVKK
201
VLATTQMQST DARKSFPCFD EPAMKATFNI TLIHPNNLTA LSNMPPKGSS
251
TPLAEDPNWS DTEFETTPVM STYLLAYIVS ESQSVNETAQ NGVLIRIWAR
301
PNAIAEGHGM YALNVTGPIL NFFANHYNTS YPLPKSDQIA LPDFNAGAME
351
NWGLVTYREN ALLFDPQSSS ISNKERVVTV IAHELAHQWF GNLVTLAWWN
401
DLWLNEGFAS YVEYLGADHA EPTWNLKDLI VPGDVYRVMA VDALASSHLL
451
TTPAEEVNTP AQISEMFDSI SYSKGASVIR MLSNFLTEDL FKEGLASYLH
501
AFAYQNTTYL DLWEHLQKAV DAQTSIRLPD TVRAIMDRWT LQMGFPVITV
551
DTKTGNISQK HFLLDSESNV TRSSAFDYLW IVPISSIKNG VMQDHYWLRD
601
VSQAQNDLFK TASDDWVLLN VNVTGYFQVN YDEDNWRMIQ HQLQTNLSVI
651
PVINRAQVIY DSFNLATAHM VPVTLALDNT LFLNGEKEYM PWQAALSSLS
701
YFSLMFDRSE VYGPMKKYLR KQVEPLFQHF ETLTKNWTER PENLMDQYSE
751
INAISTACSN GLPQCENLAK TLFDQWMSDP ENNPIHPNLR STIYCNAIAQ
801
GGQDQWDFAW GQLQQAQLVN EADKLRSALA CSNEVWLLNR YLDYTLNPDL
851
IRKQDATSTI NSIASNVIGQ PLAWDFVQSN WKKLFQDYGG GSFSFSNLIQ
901
GVTRRFSSEF ELQQLEQFKK NNMDVGFGSG TRALEQALEK TKANIKWVKE
951
NKEVVLNWFI EHS
154
Affidavit
Affidavit
I herewith declare that I autonomously carried out the dissertation entitled “Analysis
of viral and host factors influencing Alphacoronavirus life cycle in chiropteran and
porcine cell lines”.
No third party assistance has been used.
I did not receive any assistance in return for payment by consulting agencies or any
other person. No one received any kind of payment for direct or indirect assistance in
correlation to the content of the submitted thesis.
I conducted the project at the following institute: Institute of Virology, University of
Veterinary Medicine Hannover.
The thesis has not been submitted elsewhere for an exam, as thesis or for evaluation
in a similar context.
I hereby affirm the above statements to be complete and true to the best of my
knowledge.
____________________________
Acknowledgement
Acknowledgement
Firstly, I would like to express my deepest gratitude to my supervisor PD, Dr.
Christel Schwegmann-Weßels, for providing me the opportunity to work on this
fascinating topic as well as for her continuous guidance, support, patience,
motivation, and knowledge.
I would also like to thank Prof. Dr. Andreas Beinecke and PD, Dr. Eike Steinmann,
for valuable discussions, their insightful comments and encouragement.
Furthermore, I am grateful to our collaboration partners Dr. Albrecht von Brunn and
Peter Mayrhofer for good cooperation and for teaching me how to perform GFP
Trap® pull down assay during my stay in Munich.
I am thankful to my colleagues and former employees from the Institute of Virology.
Special thanks go to Dr. Markus Hoffmann and Dr. Sandra Gelhaus for their
helpfulness and support.
Furthermore, I’d like to thank my roommates Dr. Sandra Gelhaus and Tanja
Krimmling for stimulating discussions but also for all the fun we’ve had.
My thanks go to the HGNI for getting the Georg-Christoph-Lichtenberg scholarship
and to the Deutsche Forschungsgesellschaft, especially the Emmy-NoetherProgramm for funding this project.
I would like to thank my beloved family and friends, particularly my mum and sister,
Knobi, Marie and Conny, for being by my side, for their support and continuous
encouragement and for letting me withstand the strain during my work in the lab and
writing the thesis.
My heartfelt thanks go to my loving ‘American parents’ Sue & Larry for helping me
improving my English during my stays, for their mental support and for always feeling
close despite the distance.