YEB
30/2015 Anna Knuuttila
Diagnostics and Epidemiology of Aleutian Mink Disease Virus
31/2015 Jose Martin Ramos Diaz
Use of Amaranth, Quinoa, Kañiwa and Lupine for the Development of Gluten-Free Extruded
Snacks
32/2015 Aleksandar Klimeski
Characterization of Solid Phosphorus-Retaining Materials – from Laboratory to Large-Scale
Treatment of Agricultural Runoff
33/2015 Niina Idänheimo
The Role of Cysteine-rich Receptor-like Protein Kinases in ROS signaling in Arabidopsis thaliana
34/2015 Susanna Keriö
Terpene Analysis and Transcript Profiling of the Conifer Response to Heterobasidion annosum
s.l. Infection and Hylobius abietis Feeding
35/2015 Ann-Katrin Llarena
Population Genetics and Molecular Epidemiology of Campylobacter jejuni
1/2016 Hanna Help-Rinta-Rahko
The Interaction Of Auxin and Cytokinin Signalling Regulates Primary Root Procambial
Patterning, Xylem Cell Fate and Differentiation in Arabidopsis thaliana
2/2016 Abbot O. Oghenekaro
Molecular Analysis of the Interaction between White Rot Pathogen (Rigidoporus microporus)
and Rubber Tree (Hevea brasiliensis)
3/2016 Stiina Rasimus-Sahari
Effects of Microbial Mitochondriotoxins from Food and Indoor Air on Mammalian Cells
4/2016 Hany S.M. EL Sayed Bashandy
Flavonoid Metabolomics in Gerbera hybrida and Elucidation of Complexity in the Flavonoid
Biosynthetic Pathway
5/2016 Erja Koivunen
Home-Grown Grain Legumes in Poultry Diets
6/2016 Paul Mathijssen
Holocene Carbon Dynamics and Atmospheric Radiative Forcing of Different Types of Peatlands
in Finland
7/2016 Seyed Abdollah Mousavi
Revised Taxonomy of the Family Rhizobiaceae, and Phylogeny of Mesorhizobia Nodulating
Glycyrrhiza spp.
8/2016 Sedeer El-Showk
Auxin and Cytokinin Interactions Regulate Primary Vascular Patterning During Root
Development in Arabidopsis thaliana
9/2016 Satu Olkkola
Antimicrobial Resistance and Its Mechanisms among Campylobacter coli and Campylobacter
upsaliensis with a Special Focus on Streptomycin
10/2016 Windi Indra Muziasari
Impact of Fish Farming on Antibiotic Resistome and Mobile Elements in
Baltic Sea Sediment
11/2016 Kari Kylä-Nikkilä
Genetic Engineering of Lactic Acid Bacteria to Produce Optically Pure
Lactic Acid and to Develop a Novel Cell Immobilization Method Suitable
for Industrial Fermentations
ISSN 2342-5423
ISBN 978-951-51-2239-1
12/2016
Helsinki 2016
JANE E. BESONG-NDIKA Molecular Insights into a Putative Potyvirus RNA Encapsidation Pathway and Potyvirus Particles as Enzyme Nano-Carriers
Recent Publications in this Series
dissertationes schola doctoralis scientiae circumiectalis,
alimentariae, biologicae. universitatis helsinkiensis
JANE E. BESONG-NDIKA
Molecular Insights into a Putative Potyvirus RNA
Encapsidation Pathway and Potyvirus Particles as
Enzyme Nano-Carriers
DIVISION OF MICROBIOLOGY AND BIOTECHNOLOGY
DEPARTMENT OF FOOD AND ENVIRONMENTAL SCIENCES
FACULTY OF AGRICULTURE AND FORESTRY
DOCTORAL PROGRAMME IN MICROBIOLOGY AND BIOTECHNOLOGY
UNIVERSITY OF HELSINKI
12/2016
MOLECULAR INSIGHTS INTO A PUTATIVE POTYVIRUS
RNA ENCAPSIDATION PATHWAY AND POTYVIRUS
PARTICLES AS ENZYME NANO-CARRIERS
Jane E. Besong-Ndika
Division of Microbiology and Biotechnology
Department of Food and Environmental Sciences
Faculty of Agriculture and Forestry
University of Helsinki
Doctoral School in Environmental, Food and Biological Sciences (YEB)
Doctoral Programme in Microbiology and Biotechnology (MBDP)
University of Helsinki
Ecole Doctoral de Science de le Vie
Université de Bordeaux
ACADEMIC DISSERTATION
To be presented with permission of the Faculty of Agriculture and Forestry of the University of
Helsinki for public examination in auditorium 1041 of Biocenter 2 (Viikinkaari 5) on the 14th of
June 2016 at 12 noon.
Helsinki
Supervisors
Docent Kristiina Mäkinen
Department of Food and Environmental Sciences
University of Helsinki, Finland
Dr. Thierry Michon
Equipe de Virologie
UMR 1332 Biologie du Fruit et Pathologie
INRA et Université de Bordeaux, France
Follow-up Group
Professor Teemu Teeri
Department of Agricultural Sciences
University of Helsinki, Finland
Dr. Harry Boer
VTT Technical Research Center – Biotechnology
Espoo, Finland
Pre-examiners
Professor Michel Taliansky
The James Hutton Institute
University of Dundee
Scotland, United Kingdom
Docent Vesa Hytönen
BMT / Protein Dynamics
University of Tampere, Finland
Opponent
Professor Christina Wege
Institute of Biomaterials and biomolecular systems
University of Stuttgart, Germany
Cover image by Jane Besong-Ndika: Represents native and enzyme-coated potyvirus particles.
Cover layout by Anita Tienhaara
ISBN 978-951-51-2239-1 (paperback)
ISBN 978-951-51-2240-7 (PDF)
ISSN 2342-5431 (Online)
ISSN 2342-5423 (print)
Hansaprint
Helsinki 2016
II
‘I can do all things through Christ who strengthens me’
-Philippians 4:1
‘If you're walking down the right path and you're willing to keep walking, eventually you'll make
progress.’
-Barack Obama
This PhD dissertation is dedicated to my late dad, Dr. Moses Besong Besong who inspired me to
embark on this journey, my mother, my husband and kids
III
TABLE TO CONTENTS
LIST OF PUBLICATIONS AND AUTHOR’S CONTRIBUTIONS
ABBREVIATIONS
VI
VII
ABSTRACT
IX
FRENCH SUMMARY
X
INTRODUCTION
11
Section 1: Viruses as Nanoparticles
11
1. Definition of viral nanoparticles (VNPs)
2. Why plant viruses?
3. Architecture of VNPs
3.1. Icosahedral VNPs
3.2. Rod-shaped VNPs
3.3. RNA-driven viral architectures
4. Production of VNPs
4.1. Native VPs
4.2. VLPs
5. Functionalization of VNPs
5.1. Covalent attachment by bioconjugation
5.2. Genetic engineering of the capsid protein
5.3. Non-covalent strategies
5.3.1. Bio-conjugation via streptavidin-biotin interaction
5.3.2. Utilization of coiled coils for functionalization
5.4. Other functionalization strategies
6. Application of VNPs
6.1. Nanomedicine
6.2. Nano-electronics
6.3. Modern enzymology
Section 2: Potyviruses as nano-platforms
11
11
12
12
13
14
14
14
16
16
16
17
18
18
18
18
19
19
20
20
20
7. Potyviruses
8. Structure of Potyvirus CP and virions
9. Potyvirus assembly
10. Potyvirus VLP production
11. Application of potyviruses
11.1. Biotechnology
11.2. Nanotechnology
20
21
22
23
23
23
23
Section 3: Multi-enzyme systems and Immobilization
IV
24
AIMS OF THE STUDY
25
SUMMARY OF MATERIALS AND METHODS
26
SUMMARY OF RESULTS AND DISCUSSION
29
Section 1: Insights into the assembly process of PVA particles (II)
29
1.
2.
3.
4.
Dose-dependent inhibition of translation
Translation inhibition occurs via the CP mRNA
Co-translational CP-CP interactions inhibit viral RNA translation
Possible regulation of the shift from viral RNA translation to encapsidation by CP
Section 2: Building functionalized potyvirus-based nano-carriers
5. Via IgG binding peptide, z33 (I,II)
5.1. Expression of z33-tagged enzymes
5.2. z33-IgG parameters
5.3. Macromolecular assembly in solution and on carbon coated grids
5.4. Potyvirus particles as multi-enzyme nano-carriers
6. Leucine zipper strategy (unpublished results)
7. PVA-binding peptide strategy (unpublished results)
29
29
30
31
32
33
33
33
34
34
35
37
CONCLUDING REMARKS AND PERSPECTIVES
39
ACKNOWLEDGEMENTS
41
REFERENCES
43
REPRINTS OF ORIGINAL PUBLICATIONS
58
V
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications, referred to by their roman numerals
throughout the text.
I.
Pille J, Cardinale D, Carette N, Di Primo C, Besong-Ndika J, Walter J, Lecoq H, van
Eldijk MB, Smits FC, Schoffelen S, van Hest JC, Mäkinen K, Michon T. General
strategy for ordered noncovalent protein assembly on well-defined nanoscaffolds.
Biomacromolecules. 2013 Dec 9;14 (12):4351-9. doi: 10.1021/bm401291u
II.
Besong-Ndika J, Ivanov KI, Hafrèn A, Michon T, Mäkinen K. Cotranslational coat
protein-mediated inhibition of potyviral RNA translation. J Virol. 2015 Apr;89
(8):4237-48. doi: 10.1128/JVI.02915-14
III.
Besong-Ndika, J., Wahlsten, M., Cardinale, D., Pille, J., Walter, J., Michon, T., and
Mäkinen, K. Toward the Reconstitution of a Two-Enzyme Cascade for Resveratrol
Synthesis
on
Potyvirus
Particles.
2016.
Front.
Plant
Sci.
DOI:10.3389/FPLS.2016.00089
THE AUTHOR’S CONTRIBUTION
I.
Jane Besong-Ndika participated in the research work, analysis and data interpretation.
II.
Jane Besong-Ndika designed and executed most of the experimental work with the
exception of RNA gel shift experiments. She analysed the results and interpreted the
results with co-authors. JBN wrote the first draft of the manuscript.
III.
Jane Besong-Ndika designed and executed most of the experiments except for the MS
analysis. She interpreted the results together with co-authors and wrote the first draft
of the manuscript.
Unpublished data is also presented which showcase the work JBN carried out to identify a
successful strategy to graft functional biological molecules on potyvirus scaffolds.
VI
ABBREVIATIONS
4CL2
AFM
Arg
Asp
ATP
BSA
CI
CoA
CP
CPIP
CVP
DAS-ELISA
DNA
DTT
E. coli
EM
EMSA
ENC
fluc
GFP
Gln
Glu
Gly
GOx
GUS
HC-Pro
HRP
icDNA
IPTG
LC-MS
Lys
MQ
MRI
MS
MS/MS
Mt
MWCO
NIb
OD600
PCR
PEG
PMSF
polyA
PVLP
4-coumarate:coenzyme A ligase
atomic force microscopy
arginine
asparagine
adenosine triphosphate
bovine serum albumin
cylindrical inclusion protein
coenzyme A
coat protein
coat protein interacting protein
chimeric or hybrid virus particles
double-antibody sandwich enzyme-linked immunosorbent assay
deoxyribonucleic acid
dithiothreitol
Escherichia coli
electron microscopy
electrophoretic mobility shift assay
enzyme nano-carriers
firefly luciferase
green fluorescent protein
glutamine
glutamic acid
glycine
glucose oxidase
β-glucuronidase
helper component proteinase
horseradish peroxidase
infectious complementary DNA
isopropyl-β-D-thiogalactopyranoside
liquid chromatography mass spectrometry
lysine
milliQ water
magnetic resonance imaging
mass spectrometry
tandem mass spectrometry
molecule touching
molecular weight cut off
nuclear inclusion protein B
optical density measured at 600
polymerase chain reaction
polyethylene glycol
phenylmethanesulfonyl fluoride
polyadenylic acid
potyvirus-like particles
VII
RES
RLUC
RNA
RT
RT-PCR
SDS-PAGE
SECM
Ser
STS
Tat
TEM
Tyr
UTR
VLP
VNP
VP
VPg
WT
YFP
resveratrol
Renilla reniformis luciferase
ribonucleic acid
room temperature
real-time PCR
sodium dodecylsulphate-polyacrylamide gel electrophoresis
scanning electrochemical microscopy
serine
stilbene synthase
trans-acting transcriptional activator
transmission electron microscopy
tyrosine
untranslated region
virus-like particles
viral nano-particles
native virus particles
genome-linked viral protein
wild type
yellow fluorescence protein
Viruses
BMV
CCMV
CPMV
FMDV
HCV
HIV
JGMV
LMV
M13
MS2
PVA
PVBV
PVX
RCNWV
RuV
TMV
TuMV
TVCM
TYMV
ZYMV
Brome mosaic virus
Cowpea chlorotic mottle virus
Cowpea mosaic virus
Foot-and-mouth disease virus
Hepatitis C virus
Human immunodeficiency virus
Johnsongrass moasic virus
Lettuce mosaic virus
Bacteriophage M13
Bacteriophage MS2
Potato virus A
Pepper vein banding virus
Potato virus X
Red clover necrotic mottle virus
Rubella virus
Tobacco mosaic virus
Turnip mosaic virus
Turnip vein clearing virus
Turnip yellow mosaic virus
Zucchini yellow mosaic virus
VIII
ABSTRACT
The present study intended to identify new
strategies for the selective presentation of
biocatalysts on the surface of viral
nanoparticles with potential application in
biosensor technology or protein chips.
Potyviruses were chosen as model nanoscaffolds for biocatalysts. Potyviruses are the
largest genus in the family Potyviridae and
cause significant plant damage. They form
flexible rod-shaped capsids surrounding a
single stranded positive sense RNA
molecule. The molecular events leading to
the specific selection and encapsidation of
potyviral RNA are unknown. To better
exploit the potential of these viruses as nanocarriers, the first step in this study was to look
into their in vivo RNA encapsidation process.
Earlier studies showed that Potato virus A
(PVA) coat protein (CP) interferes with viral
RNA translation when provided in excess in
trans and it was suggested this could occur to
initiate viral RNA encapsidation. In this
follow up study, we used the agroinfiltration
approach for the transient expression of full
length, truncated or mutated viral RNAs with
wild type CP (CPwt) and showed that this
inhibition is mediated by co-translational CPCP interactions occurring between two CP
populations, produced in trans and in cis.
Because CP inhibited translation of the entire
viral genome and virus particles were formed
later than during normal infection, it was
assumed that the CP acted during this
inhibition process to specifically recruit viral
RNA for encapsidation. In line with
previously published in vitro assembly
studies, we propose a mechanism through
which viral RNA encapsidation is initiated
through co-translational CP-CP interactions.
The second part of this work entailed the
investigation of novel approaches for
organizing biocatalysts on virus platforms.
The aim was to control the display of
enzymes on virus surfaces while maximizing
channelling of reaction intermediates. Three
strategies were tested but only one involving
an antibody binding peptide, the z33 peptide
from Staphylococcus aureus was successful.
An 87 % occupancy of accessible sites on the
potyvirus particles by the enzyme was
achieved. The same strategy was used to graft
potyvirus particles with two enzymes: 4coumarate:coenzyme A ligase (4CL2) and
stilbene
synthase
(STS),
catalysing
consecutive steps in resveratrol synthetic
pathway or a protein chimera, generated by
the genetic fusion of both enzymes. This was
achieved by trapping either the monoenzymes or the protein chimera from
clarified soluble E. coli cell lysates on to the
surface of potyvirus particles preimmobilized in a polypropylene tube.
Resveratrol was synthesized from both
mono-enzymes and the protein chimera in
solution and on potyvirus particles. This
strategy brings together a bottom-up and top
down approach for designing virus based
nano-materials and offers a cost effective and
efficient way to co-immobilize and purify
enzymes.
IX
FRENCH ABSTRACT
La présente étude avait pour but d'identifier
de nouvelles stratégies pour la présentation
sélective d'enzymes à la surface de
nanoparticules virales dans le but d’une
application potentielle dans la technologie
des biocapteurs ou des puces à protéines. Les
potyvirus ont été choisis comme nanosupports modèles. Les Potyvirus, le genre le
plus large de la famille des Potyviridae, la
seconde plus grande famille de virus de
plante, sont responsables de très graves pertes
dans les cultures. Ils forment des capsides
flexibles en forme de bâtonnet entourant une
seule molécule d'ARN positif simple brin.
Les événements moléculaires conduisant à la
sélection et à l'encapsidation spécifiques de
l'ARN potyviral sont inconnus. Afin de
mieux exploiter le potentiel de ces virus
comme nano-supports, la première étape de
ce travail a porté sur l’étude, in vivo, du
processus d'encapsidation de l'ARN de
particules de potyvirus. Des études
précédentes ont montré que la protéine
d'enveloppe (CP) du virus de la pomme de
terre A (PVA) interfère avec la traduction de
l'ARN viral lorsqu'elle est fournie en excès en
trans suggérant que cela pourrait se produire
pour initier l’encapsidation de l'ARN viral.
Dans cette étude, nous avons montré que
cette inhibition est médiée par des
interactions CP-CP co-traductionnelles se
produisant entre deux populations de CP,
produites en trans et en cis et permettant très
probablement le recrutement spécifique de
l'ARN potyviral pour son encapsidation. En
accord avec les études d'assemblage in vitro
publiées précédemment nous proposons un
mécanisme selon lequel l’encapsidation de
l'ARN viral est initiée par des interactions
CP-CP co-traductionnelles.
Dans la deuxième partie de ce travail,
différentes approches ont été testées afin
d’organiser des enzymes sur les platesformes virales dans le but d’optimiser la
canalisation des intermédiaires réactionnels.
Parmi les trois stratégies testées seule celle
utilisant un peptide qui se liant aux anticorps,
le peptide z33 de la protéine A de
Staphylococcus aureus a été couronnée de
succès. Une couverture de 87 % des sites sur
les particules de potyvirus avec l'enzyme a
été obtenue. Cette stratégie a été utilisée pour
piéger deux enzymes, la 4-coumarate:
coenzyme A ligase (4Cl2) et stilbène
synthase (STS), catalysant des étapes
consécutives dans la voie de synthèse de
resvératrol à partir de lysats cellulaires
solubles d’E. coli clarifiés, à la surface de
particules de potyvirus immobilisées sur les
parois d'un tube en polypropylène. Cette
stratégie rassemble les approches ascendante
et descendante pour construire des
nanomatériaux à base de virus et offre un
moyen efficace et économique pour coimmobiliser et purifier des enzymes.
X
Introduction
INTRODUCTION
Section 1: Viruses as Nanoparticles
1. Definition
(VNPs)
of
viral
nanoparticles
2. Why plant viruses?
Viruses have emerged as interesting biotemplates for the construction of functional
devices with a broad range of applications.
The rationale behind the use of plant viruses
instead of artificial nanoparticles such as
carbon nanotubes (Baughman, Zakhidov &
de Heer 2002), graphene oxide (Du et al.
2011), polymersomes (Wang et al. 2013) or
dendrimer-grafted cellulose nanocrystals
(Chen et al. 2015) to name a few, stems from
the following features:
The size, shape and highly ordered backbone
structure of virus particles have made them
attractive building blocks for nanotechnology
applications. Notably, these natural nanoobjects have received a lot of interest over the
years as attractive bio-templates and
platforms for the construction of smart nanomaterials with potential applications in nanomedicine, biotechnology industries and in
material sciences.
Viruses are nano-sized pathogens ranging in
size from about 10 nm – 2 μm. The basic
structure of a virus consist of a genomic
nucleic acid molecule embedded in a
protective protein shell known as a capsid.
This protected viral genome only replicate in
the host cell. Viruses infect all kind of living
organisms, including plants, animals,
bacteria, fungi and humans. The viral genetic
material of viruses can be DNA or RNA
which can be single or double stranded.
Based on their genetic content, they are
classified as RNA or DNA viruses. The
interest in viruses for nanotechnology
applications originates from a plethora of
naturally existing features in them. The term
viral nanoparticles emerged as a generic term
to
describe
functional
virus-derived
nanomaterials. There are three forms in
which these virus particles are exploited: as a
native infectious virus particles (VP) known
as virions containing the genetic material, as
virus-like particles (VLP) which are noninfectious protein cages either devoid of
genetic material or encapsulating foreign
material and as chimeric or hybrid virus
particles (CVP) which is a genetically
modified form of the virion.
11
x
Their nano-size structures with a variety
of shapes and a broad range of sizes for
tailored applications and functions.
x
Their ability to form stable and
symmetrical protein cages with distinct
properties providing a uniform platform
(protein backbone) for dense presentation
of molecules with applications for
example in biosensor technology.
x
The ability of the virus protein cages to
assemble and disassemble under specific
conditions and for several of them, the
possible reconstitution of empty cages
devoid of genetic material, a useful
property for example in the controlled
packaging and delivery of biomolecules.
x
The potential for chemical modification
of accessible amino acid side chains on
the coat protein (CP) and genetic
engineering of the CP-encoding sequence
used for example for controlled
functionalization.
x
Their inability to infect and replicate in
humans which is a plus for biosafe
handling and medical applications.
Introduction
x
The ease of production of plant virus
particles either in planta or via
heterologous expression of the CP.
x
Their biodegradability important for
green/sustainable development.
x
Their ability to form a range of structures
with different architectures which are
stable over a broad range of pH,
temperature and ionic condition.
x
Their mono-dispersity in terms of
composition, size and shape.
2008). These data have been crucial in
designing
potyvirus-based
nanobiotemplates. For example, redox electron
cascades have been reconstituted on potyviral
particles and a viral protein present as a single
copy on the potyvirus particle surface was
conjugated to redox-antibodies and imaged
by immuno-redox molecule touching /
electrochemical atomic force microscopy
(Mt/AFM-SECM) (Nault et al. 2015). This
text will focus mainly on examples from nonenveloped plant virus particles utilized in
nanotechnology applications.
3.1. Icosahedral VNPs
These manifold properties have enabled the
exploitation of their exterior, interior and
interface surfaces in the design of a broad
range of nanomaterials (Young et al. 2008,
Steinmetz, Evans 2007, Fischlechner, Donath
2007, Cardinale, Carette & Michon 2012).
The simplest icosahedral virus consist of 60
CP subunits arranged in a 5:3:2 symmetry
(CASPAR, KLUG 1962). Their structures
are described in terms of T (triangulation) or
P (pseudo T) numbers (Speir, Johnson 2008).
Nonetheless, most viruses utilized as VNPs
have a quasi-equivalent or pseudo-equivalent
symmetry. The diameter of these particles
ranges from 10 nm to about 800 nm. A
combination of protein-protein and proteinnucleic acid interactions stabilize virus
particles but the contribution of each type of
interaction varies amongst virus genera. For
example comoviruses are mostly stabilized
by protein-protein interactions and form
virus-like particles in the absence of RNA
(Kaper 1973) whereas alfamoviruses and
bromoviruses are predominantly stabilized
by protein-RNA interactions (Kaper 1973,
Rao 2006). The most common spherical
VNPs are formed from the bromovirus
Cowpea chlorotic mottle virus (CCMV) and
Brome mosaic virus (BMW), the comovirus
Cowpea mosaic virus (CPMV), the
dianthovirus Red clover necrotic mottle virus
(RCNMV) and the tymovirus Turnip yellow
mosaic virus (TYMV). CCMV and CPMV
are further described below.
3. Architecture of VNPs
VNPs occur in various shapes. They can be
spherical with icosahedral symmetry or rodshaped with helical symmetry, as well as
enveloped or non-enveloped. More complex
virus structures also exist. Non-enveloped
icosahedral and rod-shaped plant viruses are
the most exploited as nano-templates (Culver
et al. 2015). Many of these viruses have been
extensively
studied
with
available
information on their tertiary and quaternary
structures. For many of these viruses,
information on their in vivo and in vitro
assembly and disassembly processes are
available and their encapsidation signals have
been identified. Notwithstanding, some
viruses have been used as nano-templates
despite limited knowledge of their assembly
processes. For example, the assembly process
of potyviruses is not well understood but the
secondary structure of its constituting CP has
been modelled (Baratova et al. 2001) its
ultrastructure studied (Torrance et al. 2006,
Gabrenaite-Verkhovskaya et al. 2008) and
the particle structure solved (Kendall et al.
CCMV is an icosahedral RNA plant virus. Its
particles are formed from 180 identical coat
12
Introduction
protein subunits with a T = 3 symmetry and a
diameter of 26 nm. The structure of CCMV
has been determined at a resolution of 3.2 Å
(Speir et al. 1995). This virus belongs to the
picornavirus-like major capsid fold group
which has a canonical β-barrel topology
(Rossmann, Johnson 1989). It can be
produced in its natural host (about 1-2 g per
kg of infected leaf material) and in
heterologous expression systems (Brumfield
S et al., 2004). Its assembly process is well
understood and one peculiar feature of this
virus is its ability to undergo reversible
conformational changes which are dependent
on pH, ionic strength and the presence of
specific metal ions (Bancroft, Hills &
Markham 1967, Hiebert, Bancroft & Bracker
1968, Tama, Brooks III 2002). At pH ˃ 6.5,
it forms a swollen structure with 60 2 nmsized pores which allow diffusion of
molecules into and out of the particle while at
pH ˂ 6.5, these pores are closed and no
exchange of material occurs. CCMV
particles are mainly stabilized by electrostatic
interactions between the enclosed RNA
molecule and the protective protein coat. The
interior cavity of CCMV particles is
positively charged with about nine basic
residues (Van der Graaf, Van Mierlo &
Hemminga 1991) while its exterior surface is
not highly charged (Speir et al. 1995).
Because of the in depth understanding of
CCMV VLP formation, it was possible to
compare
covalent
and non-covalent
approaches for loading CCMV particles with
a fluorescent protein (Rurup et al. 2014).
from 1 kg of infected leaf material from their
natural host (Johnson, Lin & Lomonossoff
1997).
3.2. Rod-shaped VNPs
Rod-shaped VNPs constitute several coat
protein subunits arranged in a helical manner
around a central axis. These VNPs can have
flexible rod-shaped structures for example
Potato virus X (PVX) (Kendall et al. 2008) or
rigid rod-shaped structures for example,
Tobacco mosaic virus, TMV, (Klug 1999).
The structure of rod-shaped VNPs is
described in terms of length, width, the
number of helical turns, the number of
protein monomers per turn and the pitch of
the helix. These particles range from about
300 nm to 2000 nm in size and from 10 nm to
80 nm in width. Their small central canal
limits their usage in encapsulation of
molecules hence their external surfaces are
the most exploited. The most common rodshaped VNPs are formed from the plant
tobamovirus TMV (Wen et al. 2012,
Borovsky et al. 2006, Love et al. 2014, Koch
et al. 2016), the plant potexvirus PVX
(Carette et al. 2007, Lico, Benvenuto &
Baschieri 2015) and bacteriophage M13
(Blaik et al. 2016, Park et al. 2014, Beech et
al. 2015). The structures of TMV and PVX
are briefly described.
TMV particles contain about 2130 identical
protein subunits surrounding a singlestranded positive sense RNA genome. Its
particles are 300 nm long, have a diameter of
18 nm with a pitch of ca. 2.3 nm and 16 1/3
protein subunits per turn (Namba, Pattanayek
& Stubbs 1989). Its crystal structure has been
determined at a resolution of 2.9 Å (Namba,
Pattanayek & Stubbs 1989). The interior
surface of TMV is negatively charged under
physiological conditions containing several
Asp and Glu residues while its exterior
surface is positively charged containing
several Lys and Arg residues (Namba, Stubbs
CPMV virus particles are formed from 60
copies of one domain of the small (S) proteins
and two domains of the large (L) protein with
a pseudo T = 3 symmetry (Rossmann,
Johnson 1989, Lin et al. 1999). Its structure
has been determined at a resolution of 2.8 Å
(Lin et al. 1999) and it has a diameter of 30
nm. Its constitutive subunits also form the
jellyroll β-barrel fold (Lin et al. 1999). About
1-2 g of CPMV particles can be produced
13
Introduction
1986). They are produced in high yields in
plants (about 2 g per kg leaf material). The Nand C-terminal of CP are exposed on the
external surface of the virus. In vitro
conditions for assembly and disassembly of
TMV VNPs are well established (Durham,
Klug 1971, Mandelkow, Stubbs & Warren
1981, Culver 2002).
packing signal sequence on an RNA
template, TMV-like rods of controlled length
and geometry can be produced (Geiger et al.
2013). In another study, TMV OA was also
used to control the assembly of TMV CP into
distinct architectures with applications in
material sciences (Eber et al. 2015). These
examples highlight the opportunities
embedded in these miniature nano-templates
and their potential when their assembly
process is well understood.
PVX particles contain 1270 identical coat
protein subunits assembled around a positive
sense RNA genome. It is 515 nm long and has
a diameter of 13 nm (Kendall et al. 2008,
Kendall et al. 2013). It can be produced in
gram quantities from infected leaf material.
Protocols have been established for in vitro
assembly and disassembly of its constitutive
subunits (Goodman et al. 1976, McDonald,
Beveridge & Bancroft 1976).
3.3. RNA-driven assembly
architectures
of
In addition, the size or length of the
encapsulated molecule can also modulate the
structure of the VNP. Longer and shorter
particles have been obtained this way. An
example of the former is shown with Potato
virus A (PVA) in which insertion of three
foreign genes into the viral genome resulted
in chimeric particles proportional in length to
the size of the engineered viral genome
(Kelloniemi, Makinen & Valkonen 2008).
Furthermore, the size of an encapsulated
molecule can alter the symmetry of a VNP.
For example, BMV VLPs of T = 1, T =2 and
T =3 symmetry were obtained when PEGcoated gold cores of sizes 6, 9 and 12 were
respectively encapsulated into these particles
(Sun et al. 2007).
viral
Structural diversity and the ability to mimic
different structural conformations is a pivotal
feature in virus usage as bio-templates. Viral
RNA can act as a nucleating agent directing
its encapsidation by CP subunits and the
specific RNA sequence involved in the initial
interaction between both molecules is called
an origin of assembly (OA) or encapsidation
signal (ES) or packing signal (PS). The
encapsidation signal of many plant viruses
has been identified as summarized by
Besong-Ndika and co-authors (BesongNdika, Walter & Mäkinen, 2015). Some have
more than one OA for example the Ross river
virus (RRV) whereas, for other viruses e.g.
Sindbis virus (SINV), conformational
changes are induced when the CP interacts
with RNA elements. The OA has become a
useful tool for the construction of predictable
supramolecular structures. The OA of TMV
is known and has been used to create several
RNA-guided functionalities and biostructures. It was reported that, by tuning the
positioning and number of copies of TMV
4. Production of VNPs
4.1. Native VPs
Native VPs can be produced in large scale in
their natural host post infection. Infection can
either be initiated by mechanical inoculation
or
by
Agrobacterium
mediated
transformation of the plant cell.
Mechanical inoculation is achieved by
rubbing homogenized infected plant tissue or
purified virions onto healthy plants. Viral
entry into the cell is often facilitated by mild
application of silicon carbide (carborundum)
to the surface of the leaves prior to
inoculation (Hull 2009).
14
Introduction
Agroinfiltration employs a plant bacterium
Agrobacterium tumefaciens to transfer genes
of interest into plant cells for transient
expression. This bacteria carries a tumorinducing plasmid called the Ti-plasmid. After
infiltration, a portion of the Ti-plasmid is
transferred into the plant cell after which
transient expression of the desired gene is
initiated. The transferred fragment called the
transfer
DNA
(T-DNA),
contains
homologous double stranded 25-bp sequence
borders flanking its right and left sides which
are targets of VirD1/VirD2 endonuclease
responsible for excising the T-DNA from the
Ti plasmid (Gelvin 2003). Acetosyringone, a
phenolic compound released from lacerated
plants and also available commercially,
induces expression of these virulence genes
and is sometimes included when preparing
Agrobacterium suspensions prior to
infiltration (Du, Rietman & Vleeshouwers
2014, Johansen, Carrington 2001, Eskelin et
al. 2011). Replacement of the T-DNA genes
with infectious cDNA is a strategy utilized to
initiate viral infection. Once in the plant cell,
the icDNA is transported to the nucleus for
transcription into viral RNA. This viral RNA
is then transported into the cytoplasm for
viral RNA translation and subsequent
initiation of viral infection. Several weeks
later, native VPs can be purified from
infected leaf material (Figure 1). For
example, the agroinfiltration method was
used to infect Nicotiana benthamiana with
TYMV, weeks later VPs accumulated in the
plants (Cho, Dreher 2006). This method is
also employed for high level expression of
viral components in planta (Leuzinger et al.
2013) to study virus assembly (Chaturvedi et
al. 2012) and to produce CVPs (Petukhova et
al. 2014).
Figure 1. Schematic representation of agroinfiltration procedure.
15
Introduction
Ez) formation minimized. Moreover,
depending on the application positional and
spatial control might also be required
(Cardinale, Carette & Michon 2015).
4.2. VLPs
VLPs are formed from a supramolecular
assembly of monomeric viral structural
proteins either empty or encapsulating
foreign materials. These cages can be
assembled in vivo by heterologous expression
of the CP (Phelps et al. 2007) or in vitro from
purified CP subunits under specific pH
conditions and ionic strengths (Anindya,
Savithri 2003). These CPs can be expressed
in a variety of host systems including:
bacteria (E. coli) (Diaz-Valle et al. 2015,
Brown et al. 2013, Kalnciema et al. 2012),
yeast (Brumfield et al. 2004), plant-based
systems (Sainsbury, Lomonossoff 2008,
Chen, Lai 2013), insect-based systems (Wu
et al. 2010, Thompson, Aucoin & Kamen
2016) and cell free systems (Bundy,
Franciszkowicz & Swartz 2008). Each of
these systems have their advantages and
disadvantages and their selection is virus and
application dependent (Schneemann, Young
2003, Steinmetz, Manchester 2011). CVPs
can also be produced using these systems.
For example PVX CVPs have been produced
in plant-based systems (Dickmeis et al.
2015).
5.1. Covalent
attachment
bioconjugation
by
The protein backbone of viruses makes them
suitable
candidates
for
chemical
modifications. Proteins are made up of amino
acids linked together by covalent peptide
bonds. The amino acid side chains do not take
part in the formation of the peptide bond and
the side chains of some amino acids are
available for chemical modifications. The
functionalization strategy is dependent on the
application (Stephanopoulos, Francis 2011).
Bioconjugation of VNPs has mostly been
achieved through the amine group of lysines,
the carboxylate groups of aspartic acids and
glutamic acids, the thiol group of cysteines
and the hydorxyl group of tyrosines. In rare
cases, methionine, arginine, histidine and
tryptophane reactive side groups have also
been utilized. The choice of the amino acid
depends on its spatial arrangement on the
VNP and the behavior of the target functional
group in the microenvironment. These amino
acids can occur naturally in the protein or can
be inserted by genetic modifications when
additional labelling sites are required. For
instance, a Cys residue was incorporated into
the CP of TMV for the attachment of
fluorescent chromophores (Miller, Presley &
Francis 2007) enabling some degree of
positional control. The spatial control and
orientation of foreign moieties was also
achieved for example on CPMV mutants by
attaching nano-gold particles to Lys 38 of the
capsid S subunit or to Lys 99 of the L protein
(Chatterji et al. 2004). The major drawbacks
with this conjugation method is that these
processes can be time consuming and require
the use of excess reagents and/or component
(Chatterji et al. 2004, Wang et al. 2002).
Another setback is aggregation for example
5. Functionalization of the VNPs
Several strategies have been put forward
which have permitted the chemical and
genetic modification of VNPs. The
availability of information on the structural
and functional properties of monomeric CPs
and virus particles have paved the way for
such modifications. Each strategy has its pros
and cons. The general considerations during
the choice of a specific strategy are: unaltered
assembly properties, relative stability of the
capsid (Mateu 2011), unaltered function of
the attached biomolecule and when
functionalization proceeds through the
attachment of a foreign biomolecule
(enzyme, fluorophores etc) to the CP,
heteropolymer (CP-Ez) formation needs to be
favored and homopolymers (CP-CP or Ez-
16
Introduction
by the formation of disulphide bonds which
have been encountered with CPMV particles
(Steinmetz, Evans & Lomonossoff 2007).
Click chemistry using Cu (I)-catalyzed azidealkyne, a bio-orthogonal reaction, emerged as
a more cost effective and efficient
conjugation method and has been applied to
several VNPs (Strable et al. 2008, Bruckman
et al. 2008, Sen Gupta et al. 2005, Steinmetz
et al. 2010).
of the 27 kDa GFP to PVX CP impaired
assembly (Cruz et al. 1996) however,
assembly was restored in the presence of
wild-type CP. There is no rule and steric
hindrance is definitely virus specific because
even a short insertion can impair the
assembly of some virus particles. This was
reported when a 12- amino acid long peptide
was fused to TMV CP (Hamamoto et al.
1993). Assembly occurred only when a six
base
sequence
(5’-TAGCAATTA-3’),
enabling stop codon read-through, (Skuzeski
et al. 1991) was introduced between the CP
and the foreign gene. The ratio of fusion CP
to wild-type CP in the particles was about 1
to 200. Later on, the same read-through,
leaky stop codon was used to produce
chimeric TMV particles displaying a
mosquito decapeptide hormone containing a
20:1 ratio of CP to fusion CP (Borovsky et al.
2006). A fusion protein lacking this leaky
stop codon did not assemble into virus like
particles.
Recently a new functionalization strategy
was reported. It consist of a spontaneous
isopeptide linkage between the SpyCatcher
from Streptococcus pyogenes and its binding
partner, the SpyTag. It was utilized to
decorate bacteriophage AP205 VLPs with a
malaria antigen (Brune et al. 2016). It is
thought to be an easier, faster and more stable
way to functionalized VLPs and to obtain a
homogenous population of these particles.
5.2. Genetic engineering of the capsid
protein
Another strategy to overcome steric
hindrance whilst enhancing flexibility of the
displayed molecule is by using short peptide
linkers. These linkers are often inserted
between the CP and the foreign molecule.
When Turnip vein-clearing virus (TVCN)
was decorated with a 133-amino acid
segment from protein A using a glycine rich
linker, (GGGGS)3 and a helical linker,
(EAAAK)5 full coverage of the particle was
observed (Werner et al. 2006). The Cterminal cleavage site of the 2A region of
Foot-and-mouth disease virus (FMDV)
inserted in frame within a protein encoding
sequence is also used to generate an alternate
translation of full length and truncated
polypeptides. (Ryan, King & Thomas 1991,
Donnelly et al. 2001). When inserted
between a foreign protein and PVX CP,
chimeric PVX particles consisting of a 1:3
ratio of a fusion CP and the native CP were
produced (Carette et al. 2007). A direct
fusion of an R9 peptide from hepatitis C to
The N- and C-terminal regions of the CP of
most rod-shaped viruses are often the target
for modifications due to their exposure on the
surface of the capsid for example in
potyviruses (Shukla et al. 1988) and
potexviruses (Baratova et al. 1992). At first
sight, a direct genetic fusion of the foreign
protein to the CP’s N- or C- terminal should
bypass any problem linked to chemical
coupling strategies. However, the constraints
associated with genetic modifications is
steric hindrance which in some cases hamper
particle assembly. This restricts the size of
the biomolecule that can be engineered to the
surface of some viruses. Short peptides such
as epitopes have been fused to the CP without
compromising its assembly properties (Hema
et al. 2008, Lim et al. 2002) whilst the fusion
of larger peptides can impair assembly due to
steric repulsion.
Some strategies have been implemented to
overcome this problem. For example, fusion
17
Introduction
PVX CP impaired systemic movement in
infected plants, but it was restored when the
2A peptide was engineered between both
open reading frames (Uhde-Holzem et al.
2010).
Glucose oxidase and horseradish peroxidase
streptavidin conjugates were coupled to the
virus with a 50 % coverage efficiency (Koch
et al. 2015)
5.3.2. Utilization of coiled
functionalization
5.3. Non-covalent strategies
5.3.1. Bio-conjugation via streptavidinbiotin interaction
coil
for
Coiled coils are naturally occurring helical,
oligomerization domains of varying lengths
and stability consisting of heptad amino acid
repeats, (abcdefg)n (Litowski, Hodges 2001,
Litowski, Hodges 2002). They contain
hydrophobic residues located at positions a
and d whilst charged residues involved in
electrostatic interactions are often located at
positions e and g. For functionalization,
genetic engineering is often used to fuse one
domain to the CP and the complementary
domain to the target molecule. Various
coiled-coils have been derived which form
either homodimeric or heterodimeric
associations (Muller, Arndt & Alber 2000).
The latter is generally preferred to avoid selfdimerization (Domeradzka et al. 2015). The
choice of the coiled-coil is often dependent
on the surface charge of the CP. A
heterodimeric coiled coil was utilized to
incorporate an enhanced green fluorescence
protein (EGFP) into CCMV particles (Minten
et al. 2009). About 15 EGFPs were
encapsulated per particle.
Streptavidin is a tetrameric protein capable of
interacting with four biotin molecules with
very high affinity, about Kd = 10-15 M and
with very high stability (Wilchek, Bayer &
Livnah 2006, Kay, Thai & Volgina 2009).
Increased knowledge and advances in this
conjugation method have encouraged its
usage in the functionalization of virus
particles. Biotinylation of the target
molecules, in this case the viral structural
proteins, can be achieved in two ways.
Chemical biotinylation is a less selective but
straight forward approach which targets εamines,
sulfhydryls
or
carboxylates
(Diamandis, Christopoulos 1991). Its
drawback is the simultaneous modification of
other non-target molecules. Enzymatic
biotinylation on the other hand is a more
selective approach that employs a biotin
acceptor peptide and an E. coli biotin
holoenzyme synthetase (BirA) (ChapmanSmith, Cronan 1999). BirA utilizes an E. coli
biotin carboxylate carrier protein, BCCP, to
insert biotin into the amino group of modified
lysine residues (Smith et al. 1998). The
chemical approached was applied in the
biotinylation of a reactive lysine inserted at
the N-terminus of the TMV CP (Smith et al.
2006). A fusion protein, GFP-Streptavidin,
was conjugated to this biotinylated CP and a
26 % loading between the tetrameric
streptavidin molecule and the CP was
achieved. A cysteine was also introduced by
site directed mutagenesis on an exposed part
of the TMV CP and was used to chemically
couple a biotin moiety to the TMV surface.
5.3.3. Other functionalization strategies
Phage display is an in vitro technique used for
the screening and selection of short peptide
binders (Blokhina et al. 2013). Simply
described, a foreign gene is expressed as a
fusion to the coat protein of a phage. This
produces phage particles displaying this
foreign peptide on their surfaces. Phage
libraries are created via a mixture of phages
displaying different peptides. After several
rounds of panning, amplification and
subsequent sequencing, potential binding
partners of the target can be identified from
the displayed peptides (Sidhu et al. 2000).
18
Introduction
Many other functionalization strategies are
not covered here. They are discussed
elsewhere (Steinmetz, Manchester 2011).
For bioimaging VNPs have potential as
platforms for in vivo targeting and imaging.
For example, CPMV particles conjugated to
fluorescent dyes have permitted high
resolution imaging of vascular endothelium
for up to 72 hours (Lewis et al. 2006) without
fluorescence quenching. In another example,
CCMV particles carrying Gadolinium (Gd3+)
showed great potential as high-performance
contrast agent for magnetic resonance
imaging (MRI) (Liepold et al. 2007).
6. Application of VNPs
6.1. Nanomedicine
For the efficient use of nanoparticles in
nanomedicine,
factors
such
as
biocompatibility and biodegradability need
to be considered and in vivo properties such
as immunogenicity, pharmacokinetics, tissue
distribution and toxicity of these
nanoparticles towards the specific target need
to be well understood and characterized.
Plant derived VNPs are of specific interest
compared to animal viruses such as
Adenoviruses because, they do not infect nor
replicate in mammalian systems and they are
less toxic and are more rapidly eliminated
from the body (Singh et al. 2007). VNPs
provide a large surface area for multivalent
display of epitopes or targeting peptides and
an interior cavity for encapsulation of for
example drug molecule, imaging molecules.
Some applications make use of only the
exterior or interior surfaces while others
exploit both the interior and exterior surfaces.
Below are some examples showcasing how
VNPs are being exploited as platforms in
biomedical research:
For targeted drug delivery VNPs have been
utilized to specifically target and deliver
therapeutic molecules to diseased cells. This
has been achieved by modifying the exterior
surface of the VNP with specific proteins that
bind receptors at the surface of diseased cells
and the interior with therapeutic molecules.
For example, modification of the exterior
surface of the Bacteriophage MS2 with a
Jurkat-specific aptamer and the interior with
porphyrins enabled selective targeting and
killing of about 76 % of Jurkat leukemia T
cells (Stephanopoulos, Francis 2011).
For gene therapy The natural ability of
viruses to deliver genetic material is being
exploited to either complement or inhibit
gene
expression.
Adenoviruses
and
lentiviruses have extensively been exploited
for this purpose but because these are
mammalian viruses, they are considered
unsafe (Tenenbaum, Lehtonen & Monahan
2003). Plant viruses and bacteriophages are
being exploited as safer transfer agents. For
examples bacteriophage MS2 VLPs modified
with the HIV-1 tat peptide and encapsulating
an antisense RNA were shown to inhibit
Hepatitis C virus (HCV) translation in vitro
(Wei et al. 2009).
For vaccine development Virus-based
vaccines are mostly developed from
attenuated viruses, inactivated viruses, VLPs
or chimeric viruses. VLPs are non-infectious
hence, regarded as the safest option for
vaccine production (Ludwig, Wagner 2007,
Bárcena, Blanco 2013). Displaying epitopes
on the highly ordered and repetitive protein
backbone of viruses, increases their
immunogenicity. For example, mink was
protected against Mink enteritis virus after
immunization with CPMV particles
displaying Mink enteritis virus VP2 epitope
(Dalsgaard et al. 1997). Other examples have
been reviewed (Plummer, Manchester 2011).
6.2. Nano-electronics
The distinct and tuneable surface properties
of VNPs make them suitable scaffolds for
bottom up approaches such as nucleation and
deposition of inorganic materials, known as
19
Introduction
biotemplating, during the fabrication of
molecular electronics. For example the
positively charged interior cavity of CCMV
has been used for size-constrained synthesis
of inorganic materials via electrostatic
interactions (Douglas, Young 1998). More
so, knowledge of the surface properties of
TMV has also paved the way for
electrostatically driven mineralization by
inorganic materials (Dujardin et al. 2003).
For example, platinum nanoparticles have
been coupled to the positively charged
exterior surface of TMV particles and these
particles inserted into a polyvinyl alcohol
matrix and sandwiched between two
aluminium electrodes were used to fabricate
a novel non-volatile electronic storage device
based on conductance switching (Tseng et al.
2006).
6.3. Modern enzymology
Virus particles have also been used as model
nano-reactors or enzyme nano-carriers
(Cardinale, Carette & Michon 2012, Koch et
al. 2016) to study enzyme kinetics because
their protein backbones can easily be
modified to achieve positional control and
confinement. The aim is to mimic the natural
organization of enzymes and substrates in the
cell and by this enhance catalytic efficiency.
For example, immobilization of two
enzymes, GOx and HRP onto TMV particles
resulted in a 45-fold increase in catalytic
activity, increased enzyme stability and
reusability (Koch et al. 2015). It is worth
noting here that immobilizing the enzymes
on virus scaffolds does not always enhance
the catalytic efficiency.
Section 2: Potyviruses as nano-platforms
proteolytically cleaved into ten mature
proteins (Figure 2). An 11th protein, P3NPIPO, was identified within the P3-encoding
region which is expressed as a consequence
of a transcriptional slippage at a conserved
GA6 motif (Chung et al. 2008, Olspert et al.
2015). Latest findings on potyviruses have
been reviewed (Ivanov et al. 2014, Revers,
Garcia 2015).
7. Potyviruses
Potyviruses are the largest of eight genera of
the Potyviridae family. A genome-linked
protein, VPg, is covalently linked to the 5’end and a polyadenylated tail is located at the
3’-end. These viruses belong to the picornalike supergroup of viruses. They form
flexible rod-shaped particles consisting of
about 2000 coat protein subunits surrounding
a single-stranded positive sense RNA
molecule of about 10 kb. They are mostly
transmitted by aphids. Potyviruses are
translated into a large polyprotein which is
Figure 2: Schematic representation of the Potyvirus genome
20
Introduction
8. Structure of Potyvirus CP and Virions
proteins are biologically active proteins
which lack a stable three dimensional
structure under physiological conditions and
occur as dynamic ensembles without any
stably folded structure (Habchi et al. 2014,
Xue et al. 2014). Charged residues are
abundant in these proteins and they are
involved in many interactions. In viruses
these regions are especially important for
example for interactions between viral
proteins and host components necessary for a
successful infection. Disordered prediction
analysis have shown that although CP
sequences display high amino acid
polymorphism within the potyvirus genius,
intrinsic disorder is conserved, suggesting its
functional relevance (Charon et al. 2016). An
empirical study of CP properties within the
PVA virion was recently published. It was
observed that the CP possess several
intrinsically disordered segments (Charon et
al. 2016, Ksenofontov et al. 2013) and it was
suggested that the CP’s thermostability at up
to 55 oC could be caused by these disordered
regions. Follow-up studies by the same
authors showed that upon heating to
temperatures between 60 to 70 oC the CP’s
secondary
structure
undergoes
conformational transitions while maintaining
some level of intrinsically disordered
structures. The alpha helices on its secondary
structure are changed to beta and cross beta
helices (Ksenofontov et al. 2016). The same
behaviour was observed with monomeric CP
molecules and this transition was attributed
to biological functions. These studies also
revealed some regions in the core area of the
CP are prone to aggregation highlighting the
role of this region in virus assembly.
Potyvirus particles contain VPg protruding as
a tip structure at the 5'-end of the viral RNA.
In addition, gold labelling experiments
revealed a subpopulation (about 1%) of
purified particles display the viral proteins
HC-Pro and CI associated to this tip structure
Potyviral CP is a structural protein and the
major component of the potyviral particle. It
comprises 269 to 350 amino acid residues. It
contains three structural regions: the Nterminus, the core region and the C-terminus.
A model of the CP structure was proposed,
based on secondary structure prediction and
tritium bombardment of the accessible parts
of the CP on the virus (Baratova et al. 2001).
In this model, the N-terminal region (amino
acids 1-60) consists of an unstructured and
disordered region immediately followed by
four β-strands. The core region (amino acids
61 to 180) constitutes five α-helices whereas
the C-terminal region comprising amino
acids 181 to 269 folds into two α-helices and
three β-strands. A Thr-242 situated after the
β7 strand, according to the CP secondary
structure put forward by Baratova and coworkers, is a phosphorylation site for CK2
(Ivanov et al. 2003). Phosphorylation at this
site prevents binding of PVA CP to viral
RNA.
Potyvirus virions have a flexible rod-shaped
structure with helical symmetry. The
structural coat protein constitutes 95 % of the
virions and the viral genome 5 %. These
particles are about 680 – 900 nm long and
have a diameter of about 11 -13 nm. The
potyvirus Soybean mosaic virus (SMV) was
shown to have 8.9 CP subunits per helical
turn (Kendall et al. 2008). Potyviruses have a
helical pitch of about 3.4 nm (Tollin, Wilson
1988). Potyvirus virions comprise the same
CP structural regions as described above. The
CPs N- and C-terminal ends are exposed on
the surface of the virus particle (Shukla et al.
1988). Most of the N-terminal is accessible
on the exterior of the virus (Baratova et al.
2001) and contains virus-specific epitopes
(Shukla et al. 1988). The core region is highly
conserved and unaffected by trypsin
treatment. Potyvirus CPs exhibit some
disordered structures. Intrinsically disordered
21
Introduction
(Torrance et al. 2006,
Verkhovskaya et al. 2008).
Gabrenaite-
ring-like intermediates can assemble into
VLPs or stacked rings in the presence or
absence of RNA (McDonald, Bancroft 1977).
Two conserved charged amino acid residues,
Arg194 and Asp238 situated in the conserved
AFDF motif in the C-terminal region of
potyviral CPs, from Johnsongrass mosaic
virus (JGMV) and its counterparts Arg154
and Asp198 from Tobacco etch virus (TEV)
are essential for assembly and/or
virus stability (Jagadish et al. 1991, Jagadish
et al. 1993, Dolja et al. 1994, Jagadish, Huang
& Ward 1993) but not through formation of a
salt bridge (Voloudakis AE et al., 2004).
Other conserved amino acids critical for
assembly are Gln195, Try130 and Tyr131.
Assembly defects were observed with Plum
pox virus (PPV) CP mutants with substitution
in the conserved Asp and Arg residues and
was attributed to the low expression levels of
these
mutants
(Varrelmann,
Maiss
2000). The above studies highlight the
importance of CP-CP interactions during the
assembly process but there is still insufficient
evidence on the involvement of an
encapsidation signal.
9. Potyvirus assembly
Although the CP is involved in several
processes during viral infection (Ivanov,
Makinen 2012), its major function is to
protect and transport viral RNA locally and
systemically in the plant. A few studies have
suggested a mechanism for potyviral CP
assembly in vitro but there is limited
information on how it occurs in vivo. An
earlier study predicted that the assembly
process is initiated from the 5'-end of newly
synthesized RNA molecules (Wu, Shaw
1998) but there is still no evidence to support
this study. A decade later, it was proposed
that the origin of assembly could be located
within the CP cistron (Hema et al. 2008). This
conclusion was drawn when heterologous
expression of a potyvirus CP resulted in the
formation of VLPs containing CP’s mRNA.
The assembly of potyvirus CPs into
filamentous flexible rods proceeds via a 16S
octameric ring-like intermediate (McDonald,
Beveridge & Bancroft 1976, McDonald,
Bancroft 1977, Anindya, Savithri 2003).
Early on, the core region of CP was thought
to contain all the component needed for
assembly (Jagadish, Huang & Ward 1993)
and it was suggested that the surface exposed
N-terminal part was dispensable for assembly
(Dolja et al. 1994). Contrary to this, it was
later shown that the N- and C-terminal
regions are indispensable and involved in a
head to tail electrostatic interaction crucial
for the formation of an octameric ring by CP
monomers (Anindya, Savithri 2003).
Nonetheless, trypsin treated particles were
readily disassembled into the ring-like
intermediates and reassembled into flexible
rods suggesting that these terminal regions
are solely needed for the formation of the
octameric ring. The N-terminal exposed
residues are not needed for protein-RNA
interactions (Voloudakis et al. 2004). The
10. Potyvirus VLP production
Potyviral VLPs have been produced in
different host systems by heterologous
expression of the CP. In E. coli, potyviral
VLPs have been produced from TEV CP
(Voloudakis et al. 2004); an Indian isolate of
Sugarcane streak mosaic virus- Andhra
Pradesh (AP) CP (Hema et al. 2008); in insect
and mammalian systems from JGMV CP
(Hammond et al. 1998) and in yeast systems
from JGMV CP (Jagadish et al. 1991). PVA
VLPs have also been produced in Nicotiana
benthamiana (Cerovska et al. 2008). In vitro
conditions for assembly and disassembly of
some potyvirus particles are known
(Anindya, Savithri 2003). PVBV VLPs have
been disassembled in 10 mM CAPS buffer,
pH 10 containing 50 mM NaCl at 10 °C and
CP subunits reassembled via dialysis against
22
Introduction
10 mM Tris-HCL, pH 8 containing NaCl at
the same temperature.
11.2.
Nanotechnology
In addition to the above biotechnological
applications, potyviruses have also been
exploited in nanotechnology as nano-carriers
mostly for medical applications. Jadagish and
co-workers have reviewed the properties of
potyvirus CPs and particles and some
examples of how chimeric potyviruses can be
produced (Jagadish et al. 1996). The N- and
C-terminal parts of potyvirus CP are exposed
on the surface of the particle making it
possible to adapt them for the display of
foreign biomolecules. As mentioned above,
coupling foreign molecules to the CP can
sometimes affect its assembly properties.
However, there are few examples in which
potyvirus CP’s encoding sequence has been
modified without assembly defects. For
example, VLPs were formed in E. coli when
36 amino acid residues were inserted at the
N-terminus of SCSMV (Hema et al. 2008). In
another example, assembly and systemic
infection were unaltered when Zucchini
yellow mosaic virus (ZYMV) CP's Nterminus was replaced with a human c-Myc
peptide (Arazi, Shiboleth & Gal-On 2001).
For possible application in nanomedicine,
JGMV hybrid particles displaying a
functional 26 kD Sj26-glutathione Stransferase from Schistosoma japonicum
with immunogenic properties have been
produced (Jagadish et al. 1993). Potyviral
VLPs with N- and C-terminal modifications
have been produced for use as multivalent
carriers. For example, the N- and C-terminus
of PVA CP has been modified to carry two
epitopes, L2 and E7 from Human
papillomavirus type 16 and these VLP
particles produced in E. coli associated into
bundles (Cerovska et al. 2008). These
examples show that knowledge of the
potyvirus CP and particle structure allows a
better design of VNPs for different
applications.
11. Application of Potyviruses
11.1. Biotechnology
Potyvirus-based expression vectors have
been used to express foreign proteins in
plants both for biotechnological and medical
purposes. Foreign genes have been
introduced into different locations in these
vectors. This has sometimes led to a
reduction in virus accumulation without
altering virus infectivity. In potyvirus-based
vectors, there are three prominent insertion
sites: the 5'-end of the P1 cistron (Rajamaki
et al. 2005), between P1 and HC-Pro cistrons
(Dolja, McBride & Carrington 1992) and
between the NIb and CP cistrons
(Varrelmann, Maiss 2000). For example, a
reporter enzyme has been expressed as an Nterminal fusion of HC-Pro from a TEV-based
vector (Dolja et al. 1997). In another
example, beet yellow closterovirus p20
protein was expressed from a TEV vector and
isolated from plants (Dolja et al. 1998). PVAbased vectors have also been used to express
Renilla luciferase inserted between NIb and
CP (Eskelin et al. 2010), and active soluble
catechol-O-methyltransferse (S-COMT) and
GFP (Kelloniemi, Makinen & Valkonen
2006). Simultaneous expressions are possible
with these vectors showing their robustness.
For instance, GFP and GUS have been
simultaneously expressed from Turnip
mosaic virus (TuMV) - based vector
(Beauchemin, Bougie & Laliberte 2005)
whilst
up
to
three
proteins
were simultaneously expressed from a PVAbased vector (Kelloniemi, Makinen &
Valkonen 2008). In both vectors, infectivity
was not compromised even with a 25 – 40 %
increase in the size of the viral genome. Other
examples of how potyviruses are exploited
for biotechnological applications are
discussed elsewhere (Majer, Navarro &
Daros 2015, Kamencayova et al. 2014).
23
Introduction
Section 3: Multi-enzyme systems and immobilization
In their natural environment, the cell,
enzymes are often organized into complexes
that act cooperatively, the product of one
reaction being the substrate of the next. The
active sites of these enzymes are usually in
close proximity with special spatial
arrangements that allow channelling of the
intermediate between active sites while
minimizing its diffusion into the bulk phase.
Metabolic channelling describes the
restricted
transfer
of
intermediates
(substrates and products) within a multienzyme system (Welch, Easterby 1994,
Winkel 2004). A plethora of advantages
accompany these systems: enhanced reaction
rates, reduced toxicity due to the
concentration of the intermediates in a
specific environment, reduced substrate
competition, modulation of metabolic fluxes
etc. (Zhang 2011). Metabolic channelling in
the cell is ensured by two main strategies: by
confining the enzymes in a cellular
compartment such as the mitochondria or by
anchoring the enzymes in close proximity in
a matrix such as a cellular membrane.
Inspired by these processes, scientists have
been able to build artificial multi-enzyme
systems on scaffolds such as virus scaffold
(Koch et al. 2015, Koch et al. 2016), DNA
scaffolds (Fu et al. 2014), porous and nonporous material (Jia, Narasimhan &
Mallapragada 2014) and nano-reactors (Van
Oers, Rutjes & Van Hest 2014). Viruses
because of their naturally occurring features
have become interesting scaffolds for
enzymes. The large surface area of rodshaped viruses provide a homogenous
surface for displays bringing enzymes in
close proximity whereas the interior of
icosahedral viruses can be used as cellular
compartments for enzyme confinement.
Strategies permitting the attachment of
enzymes to these scaffolds have extensively
been reviewed (Garcia-Galan et al. 2011,
Barbosa et al. 2015, Schoffelen, van Hest
2013).
24
Aims of the study
AIMS OF THE STUDY
The ability of viruses to self-assemble into highly ordered, symmetric nano-objects with protein
backbones have motivated the interest in their use in bio-nanotechnology applications. The
objectives of this study were:
1. To further understand the molecular mechanism inherent to CP-mediated inhibition of PVA
viral RNA translation and its link to viral RNA encapsidation (II).
2. To develop strategies for grafting functional biological molecules like enzymes on virus
scaffolds (I).
3. To exploit potyvirus particles as enzyme nano-carriers (ENCs) using the strategy developed
above and to use these ENCs in single and multi-enzyme catalysed processes (I & III).
25
Summary of materials and methods
SUMMARY OF MATERIALS AND METHODS
A list of methods, plasmids, proteins and peptides utilized in this study are presented in the tables
below. Details of the methods are found in the original publications.
Table 1. Methods used in this study
Methods
Molecular Biology/Biochemistry techniques
Affinity chromatography
Antibody purification
DNA cloning
DNA sequencing
Immunocapture RT-PCR
In vitro transcription
Macromolecular assembly
PCR amplification
Quantitative RT-PCR
Recombinant protein expression
RT PCR
SDS-PAGE analysis
Site directed mutagenesis
Size exclusion chromatography
Western Blot analysis
Yeast recombination
Described in
I, III
III
I, II, III
I, II, III
II
II
I, III
I, II, III
II
I, II, III
II
I, II, III
II
I, III
I, II, III
I, III
Assays
Affinity assays
Antibody/protein titration
DAS-ELISA
electrophoretic mobility shift assay (EMSA)
Enzyme assays
Luciferase assays
I, III
I, III
III
II
I, III
II
Microbiology
Agrobacterium-mediated plant transformation
Agrobacterium infiltration
Virus purification
II
II
I, III
Spectroscopy
Liquid chromatography-mass spectrometry (LC-MS)
Tandem mass spectrometry (MS/MS)
I, III
III
Imaging
Correlative microscopy
Immunosorbent electron microscopy (EM)
Transmission electron microscopy (TEM)
I
II
I, III
26
Summary of materials and methods
Software
IMAGEJ
CorelDraw
GraphPad Prism
Constructs
PVAwt
PVAGDD
PVAΔCP
PVASTOPCP
PVAΔGDDΔ5UTR
PVAΔGDDΔ5UTR
Constructs
CPwt
CPmut
FLuc
5’UTR-Rluc
RLuc-3UTR
RLuc-CP-3UTR
RLucSTOP-CP-3UTR
I
II, III
II
Table 2. Viral constructs used in this study
Gene cassette
35S-PVAwt::rlucint-nos, pRD400
35S-PVAGDD ::rlucint-nos, pRD400
35S-PVAΔCP::rlucint-nos, pRD400
35S-PVASTOPCP::rlucint-nos, pRD400
35S- PVAΔGDDΔ5UTR::rlucint-nos, pRD400
35S- PVAΔGDDΔ5UTR::rlucint-nos, pRD400
Table 3. Non-viral constructs use in this study
Gene cassette
35S-CPwt-nos, pRD400
35S-CPmut-nos, pRD400
35S-fluc-nos, pRD400
35S-5UTR-rluc-nos, pEAQ-HT
35S- RLuc:3’UTR:rluc-nos, pEAQ-HT
35S-RLuc:CP:3’UTR-nos, pEAQ-HT
35S:RLucSTOP:CP:3’UTR-nos, pEAQ-HT
Table 4. z33-containing constructs used in this study
Constructs
Gene cassette
T7-z33:Linker1:YFP:His-taa, pET21a
mYFPz
z
T7-z33:Linker1:STS:His-taa, pET21a
STSHis
z
T7-z33:Linker1:4CL2:His-taa, pET21a
4CL2His
z
T7:z33:Linker1:4CL2:Linker2:STS:His-taa, pET21a
4CL2::STSHis
Linker1: GGGGS; Linker 2: GSG
Publication
II
II
II
II
II
II
Publication
II
II
II
II
II
II
II
Publication
I
III
I, III
III
Table 5. Leucine zipper- and P12-containing constructs used in this study (unpublished)
Constructs Gene cassette
Host
LZEg
CP
T5-His:LZEg:CP-taa, pQE30
E. coli M15 (pREP4)
LZEgCP
Tac-GST:LZEg:CP-taa, pGEX-6P-1 E. coli BL21
LZGlu
CP
T5-His:LZGlu:CP-taa, pQE30
E. coli M15 (pREP4)
LZGlu
CP
Tac-GST:LZGlu:CP-taa, pGEX-6P-1 E. coli BL21
LZGlu
CP
35S-His:LZGlu:CP-nos, pEAQ-HT
Agrobacterium tumefaciens strain C58C1
P12
YFPHis
T7-P12:YFP:His-taa, PET21a
E. coli BL21
27
Summary of materials and methods
Table 6. Peptide sequences
Leucine zipper
LZGlu
LZArg
LZEg
LZKg
Xpress epitope
Z33 peptide
P12 peptide
Protein
4CL2
STS
mYFP
LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK
LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK
AQLEKELQALEKKLAQLEWENQALEKELA
AQLKKKLQANKKELAQLKWKLQALKKKLA
DLYDDDDK
FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD
FTTMAWRGGG
Table 7. Protein accession numbers
Host
GeneBank accession number
Nicotiana tabacum
U50846
Vitis Vinifera
EU156062
Aequorea victoria
(Zacharias et al. 2002)
Table 8. Viruses
Virus
PVA
Strain
B11
NCBI Reference Sequence
NC_004039.1
28
Summary of results and discussion
SUMMARY OF RESULTS AND DISCUSSION
Section 1: Insights into the assembly process of PVA particles (II)
Understanding the virus encapsidation
mechanism is important for nanotechnology
applications. Knowledge of the surface
exposed C- and N-terminal parts of the
CP on the surface of the virus particle
have paved the way for their usage as
carrier molecules. Potyvirus VLPs can be
assembled in vitro and in vivo from
recombinantly expressed CPs and evidence
for the in vitro mechanism is available but
little is known about the in vivo process.
How the CP preferentially selects viral
RNA in the host cell and how assembly
of the latter is initiated. Earlier studies
from our group showed that potyviral CP
inhibits viral RNA translation and because this occurred at high CP
concentrations in the cell, it was proposed
that virion assembly could prevent viral RNA
from being translated (Hafren et al. 2010). It
was speculated that the CP specifically
interacts with viral RNA to induce this
inhibition and in turn initiate viral RNA
encapsidation but the mechanism through
which it inhibits RNA translation was not
investigated. In most positive sense RNA
viruses, interaction between the CP and a
specific RNA signal known as the
encapsidation signal, occurs to initiate
encapsidation (Rao 2006). This study aimed
to look into the mechanism governing this
inhibition, identify the specific RNA
sequence involved and also examine its role
in encapsidation. A Renilla luciferase-based
system developed in our laboratory was used
to monitor viral gene expressions in
Nicotiana benthamiana leaves (Eskelin et al.
2010). A reduction in reporter gene
expression was interpreted as an interaction
occurring between the specific viral RNA
and the CP.
1. Dose-dependent
translation
inhibition
of
The CP modulates several events during
virus infection and even plays a role in the
transition from one stage during the virus
infectious cycle to another. Its functions are
usually concentration dependent (Boni et al.
2005, Yi et al. 2009). PVA CP was found to
inhibit viral RNA translation when present in
high titres. In this study, by co-expressing
different ascending concentrations of
Agrobacterium containing CPwt with
Agrobacterium containing PVAwt, we
showed
that
the
higher
the concentration of CPwt, the greater the inhibition of Renilla luciferase reporter gene expression in PVAwt (Figure 2, II). Notwithstanding, the extent of inhibition might also
have been enhanced by reduction in replication because the
CP-inhibitory
effect
also affects replication.
2. Translation inhibition occurs via the
CP mRNA
It was earlier speculated that potyvirus RNA
encapsidation is initiated from the 5’UTR
(Wu, Shaw 1998). However, we showed that
CPwt has no preference for this region nor the
3’UTR as co-expression of replication deficient mutants lacking the 5’UTR,
or
the
3’UTR,
PVAΔGDDΔ5UTR,
PVAΔGDDΔ3UTR, with CPwt still down
regulated viral RNA translation (Figure 3
A&B, II). Further evidence was shown when
CPwt had no effect on non-viral constructs,
5UTR-RLuc and RLuc-3UTR, containing
these untranslated regions and the renilla
reporter gene (Figure 3. C&D, II). Similar
experiments conducted with the CP cistron
showed the unequivocal involvement of this
region in the inhibition. A 14-fold decrease in
29
Summary of results and discussion
expressing PVASTOPCP / CPwt and plants
coexpressing PVASTOPCP / GUS control (Figure 5 C, II). Potyviral CP is not required for
virus replication but needs to be translated to
some extent probably to ensure that intact
viral RNAs are replicated (Mahajan,
Dolja & Carrington 1996). This could
explain the absence of infection in the
PVASTOPCP mutant. Also, the extent of
reduction in reporter gene activity was
evinced in the RNA levels in plants expressing PVAwt / GUS compared to those
expressing PVAwt / CP (Figure 5 B, II).
Disappearance of the inhibition after insertion of the stop codon indicated the inhibition by exogenous CP necessitated CP
translation from RLuc-CP- 3UTR and
PVAwt constructs even though the role of
specific structures induced during its translation cannot be completely excluded. Additionally, because CPwt severely inhibited translation when expressed with a
movement and assembly deficient mutant
virus, PVACPmut, (Hafren et al. 2010) but not
when a mutant CP, CPmut, with the same
mutation was expressed with PVAwt (Figure
3E, II) indicated that, exogenous CP needs
to be fully functional to induce this inhibition while this is not a requirement for
CP translated from the viral construct. The
CP is known for functioning via CP-CP
and CP- RNA interactions. It either coordinates the switch between the different
stages during infection or functions in the
encapsidation and movement of viral RNA.
For example, a mutant CP, T24W, with
enhanced CP-CP coupling abilities significantly minimized TMV infection by hampering the formation of replication complexes and this mutant was able to form
non-infectious
virus
particles
(Bendahmane
et
al.
1997,
Bendahmane et al. 2007). CP-CP have been
identified in potyviruses (Guo, Merits &
Saarma 1999, Guo et al. 2001) and in vitro
experiments show that this interaction is
important for the formation of an assembly
intermediate (Anindya, Savithri 2003). This
reporter gene expression was observed when
CPwt was co-expressed with RLuc-CP-3UTR
(Figure 3E, II) when compared to the control
and this inhibition was less than 2-fold, when
the CP cistron was deleted from PVAwt viral
RNA (Figure 3F, II). Potyvirus-like particles
have been formed in E. coli by
heterologous expression of a potyvirus
CP (Anindya, Savithri 2003). Because
these particles contained RNA, it was
thought that the origin of assembly could be
located within the CP mRNA. Unexpectedly, a comparative EMSA experiment to
further confirm CP’s interaction with
its mRNA showed that this interaction is
not sequence specific (Figure 4, II). The CP
had equal affinities for synthetic transcripts
obtained from the CP-encoding region
and the P1-encoding region. This eliminated the possibility of specific RNA
structural involvement in the inhibition of
translation.
3. Co-translational CP-CP interactions
inhibit viral RNA translation
Early on, CP was suggested to interfere with
viral RNA translation via CP-RNA
translation. Nonetheless, this follow up study
precludes the direct involvement of CP-RNA
interaction and instead suggest CP-CP
interactions are the driving force behind this
inhibition. Co-expression of RLuc-CP-3UTR
and PVAwt with CPwt caused about 300-fold
and 100-fold inhibition of reporter gene
expression respectively which persisted 10
days post inoculation. However, when an inframe stop codon was inserted upstream the
CP cistron in these constructs, the inhibition
was only about 3-fold and was indifferent 10
days after inoculation (Figure 5 A, B&C, II).
It is worth noting that insertion of a stop
codon upstream the CP cistron in PVAwt
rendered this construct (PVASTOPCP) noninfectious and considerably reduced its RNA
levels, nonetheless, consistent with the
reporter activities, there was no significant
difference in RNA levels in plants co-
30
Summary of results and discussion
interaction was confirmed by Zilan E and
Maiss E and the authors showed that the
interacting domains are found between amino
acids 1-221 of PPV CP (Zilian, Maiss 2011).
This was contrary to an earlier report from
another potyvirus, Soybean mosaic virus, in
which this domain was thought to locate to
the CP C-terminus between amino acids 171285 (Kang et al. 2006). In both cases, these
domains were thought to be necessary for CP
subunit interactions and were associated to
virion assembly.
(Maia, Haenni & Bernardi 1996, Guo, Merits
& Saarma 1999). Subsequent studies with
Lettuce mosaic virus (LMV), PPV and PVY
showed that HC-Pro interacts with viral CP
in planta (Roudet-Tavert et al. 2002) and this
might be necessary for other biological
functions than aphid transmission. A novel
function of HC-Pro in assembly was recently
identified (Valli et al. 2014). The authors
suggested that HC-Pro probably stabilizes cognate CP and newly synthesized
virus particles during the late stages of infection. The involvement of CP-HC-Pro
interactions in this novel function is yet to be
established. During CP-mediated inhibition, viral RNA translation of the entire
genome was blocked as evidence by western blot analysis (Figure 6, II) which means
that HC-Pro was probably also negatively
affected. The reduced amount of HC-Pro in
the cell might have resulted in unstable CP
and virus particles and delayed their appearance.
4. Possible regulation of the shift from
viral RNA translation to encapsidation
by CP
CP regulates viral processes in a
concentration dependent manner. In Rubella
virus for example, lower CP concentrations
stabilize viral RNA while higher
concentrations inhibit replication in order to
initiate encapsidation (Chen, Icenogle 2004,
Ilkow et al. 2008). We believe that PVA CP
interferes with viral RNA translation to
recruit this RNA for encapsidation via
cotranslational
CP-CP
interactions.
Virus particles similar in length and diameter to native virions were formed when
PVAwt was expressed with CPwt (Figure 7
A, II) and these virus particles
encapsidated the same RNA molecule as
native virions. However, virus particles
obtained from plants expressing PVAwt
and CP were detected 10 days after inoculation while those of native virions were
detected three days earlier, that is 7 days
post inoculation. Potyviral proteins are
synthesized in equimolar ratios and the
protein amounts are regulated depending on their necessity at the different stages
during infection. HC-Pro is a multifunctional protein involved in several stages
during the virus life cycle (Maia, Haenni &
Bernardi 1996). Earlier studies showed PVA
HC-Pro and CP do not interact directly with
each other in vivo but both coordinate the
aphid transmission activity of potyviruses
Post-translational modifications of the CP are
important during potyvirus infection to for
example enhance CP stability and infectivity
via CP O-GlcNAcylation (Perez Jde et al.
2013) or to prevent its interaction with viral
RNA via CP phosphorylation (Ivanov et al.
2001). Proteosomal degradation mediated by
HSP70/CPIP (Hafren et al. 2010) also
downregulate CP amounts during the early
stages of infection when it is not required.
CPIP is a co-chaperone of HSP70 which interacts with CP (Hafren et al. 2010) and is
important for potyvirus infection (Hofius
et al. 2007). Based on our results, we believe that the CP- mediated effect, mimics
the final stages during viral infection
when CPs become saturating in the cell
for encapsidation and propose a mechanism through which CP is regulated by
competing CP-CP and CP-CPIP interactions
(Figure 8, II). During the early stages of infection when CP is not required in excess,
CPIP binds CP to form CP-CPIP complexes and takes it to HSP70 which directs it toward the ubiquitination pathway
31
Summary of results and discussion
(Hafren et al. 2010). Potyvirus RNA
encapsidation by CP occurs in vitro via an
octameric ring formed by the interaction
between the N- and C-terminal of the CP
(Anindya, Savithri 2003). We speculate that
during late infection, CP-CP interactions
occurring between two CP populations from
in cis and in trans translations, overwhelm
the system overshadowing CP-CPIP interactions. Because in trans CP interacts
with newly synthesized CP in cis, the
translating RNA is immediately recruited for
encapsidation in this way. An octameric ring
is probably formed around this RNA
molecules somewhere within the CP cistron.
Section 2: Building functionalized potyvirus-based nano-carriers
already established protocol (Browning et al.,
1995). Nicotiana benthamiana plants were
infected by infiltrating Agrobacterium
suspensions containing PVA genome. PVA
particles were purified with high purity
(Figure 3A) and the yield was about 7.6 mg
obtained from 502 g of infected leaf material.
The yield is quite low compared to other
commonly utilized viruses for example TMV
or CCMV in which about 2 g can be obtained
from 1 kg infected leaf material. The low
yield might be because Nicotiana
benthamiana is not a natural host of PVA.
Virus particles were analyzed using TEM
after brief staining with uranyl acetate
(Figure 3B).
Viruses due to their ability to self-assemble
into defined nano-structures consisting of a
highly symmetrical protein backbone have
surge the interest in their usage in the
development of enzyme-functionalized nanodevices. These nano-carriers can be utilized
for example as intermediate scaffolds to
direct the controlled positioning of enzymes
on solid supports necessary for studying
single or multi-enzyme kinetics. Two
potyviruses, ZYMV and PVA, were
exploited as enzyme nano-carriers in this
study. These are flexible filamentous viruses
with large aspect ratio for the presentation of
biomolecules. In this study, ZYMV particles
were purified from zucchini squash leaves (I)
while PVA particles were purified from
Nicotiana benthamiana leaves following an
Figure 3. Purification of PVA virions from Nicotiana benthamiana leaves. A. SDS-PAGE analysis of
purified PVA virions. B. TEM imaging of purified PVA virions.
32
Summary of results and discussion
(supplementary data, I) indicating that this
modification probably did not occur. Apart
from the protein chimera, all proteins were
expressed in soluble and active form in E.
coli (Figure 2B, I; Figure 2A, III). An inadvertent mutation was introduced into STS
during the cloning process which resulted
in S276P, however it was ignored as it was
not situated in the active sight of this
chalcone synthase- like enzyme (Jez, Noel
2000, Suh et al. 2000). mYFPz, z4CL2His
and zSTSHis maintained their activity after
fusion to the z33 peptide (Figure 7, Table
2,
I).
The
protein
chimera,
z4CL2::STSHis,
accumulated
in
inclusion bodies. This protein was purified from inclusion bodies but remained
inactive in spite of all refolding attempts
(Figure 3A, III). The z33 peptide in the
protein chimera was not affected by denaturing purification as its affinity for PVA
IgGs was unaltered (Figure 3, III).
4CL2, STS and a fusion protein
4CL::STS containing the same GSG linker,
have previously been expressed in soluble form in E. coli with an N-terminal
His-tag (Zhang et al. 2006, Wang et al. 2011).
The C-terminal location of the 6x His-tag in
the z4CL2::STSHis protein chimera in
addition to the N-terminal z33-tag might
have been the major cause of the aggregation
of this protein into inclusion bodies. The
activities of z4CL2His / zSTSHis and that
of the less abundant soluble fraction of
z
4CL2::STSHis were tested from clarified
soluble cell lysates and MS/MS analysis
showed resveratrol was synthesized from
these lysates (Figure 2, III).
5. Via IgG binding peptide, z33 (I, III)
5.1. Expression of z33-tagged enzymes
A non-covalent versatile approach was
developed to decorate native virus particles
with functional biomolecules (Figure 1, I).
This approach utilized the z33 peptide, a 33
amino acid peptide originating from the
protein A (SpA) region of the bacteria
pathogen, Staphylococcus aureus (Braisted,
Wells 1996, Cedergren et al. 1993). This
peptide binds to the Fc region of IgGs with
high affinity, a process which is enthalpy
mediated (van Eldijk et al. 2014). Due to its
robustness, it has found application in
antibody purification (van Eldijk et al.
2014), gene therapy (Tanaka et al. 2006,
Kawashima et al. 2011) and directed
gene transfer (Volpers et al. 2003). In
this study, z33 peptide was used to mediate the association of enzymes on
ZYMV and PVA virus particles via specific antibodies raised against respective
viruses. The z33 peptide was fused to the Nterminus of mYFP, 4CL2, STS and a protein chimera 4CL2::STS to obtain
mYFPz, z4CL2His, zSTSHis and z4CL2::STSHis
respectively. A 6x His-tag was fused to the
C- terminus of these proteins and a five
amino acid flexible linker, GGGGS, was
fused to the C-terminus of the z33 peptide
before the protein domains for flexibility.
An additional three amino acid linker,
GSG, was inserted between the protein domains in the protein chimera to minimize
steric hindrance. This flexible linker has
been vastly used to separate the protein domains of fusion proteins and in
some instances improved the catalytic efficiencies of the proteins (Lu F and Feng
MG, 2008). However, the serine residues
in this linker is sometimes prone to οxylosylation (Spencer et al. 2013, Wen et
al. 2013). This was not specifically
investigated in this study. Nonetheless, the
molecular weights of two of the proteins,
mYFPz, z4CL2His, were unchanged as
revealed by MS and MALDI-TOF
5.2. z33-IgG parameters
After expression of the enzymes, their
binding affinities for IgGs were investigated.
The z33 peptide is able to bind both heavy
chains in an antibody and by mixing the
proteins and their respective antibodies, we
showed that a 5:1 ratio corresponding to 5
proteins to 1 antibody was optimal for
achieving full binding. This was shown with
mYFPz (Figure 3, I) and with the protein
33
Summary of results and discussion
chimera z4CL2::STSHis purified under
denaturing conditions (Figure 3A and B, III).
The z4CL2::STSHis chimera was inactive
after denaturing purification but the z33 was
fully functional enabling this protein to bind
to the antibody. Additionally, the N-terminal
location of the z33 peptide in the fusion
proteins caused about 10 to 20 % (KD ~ 860
nM) reduction in its affinity for the antibody
when assessed by surface plasmon resonance
(Figure 4, I) compared to previous results
obtained when it was attached to the Cterminus (Volpers et al. 2003).
external surface of PVA particles both in
solution and on carbon coated grids showing
the robustness of this method. The extent of
coverage of the particles by the protein
chimera was not examined however, EM
images showed an additional layer all
along the length of the particle. The increased size of the particles was a result of
enzyme coupling (Figure 3 C&D, III).
5.4.
Potyvirus particles
enzyme nano-carriers
as
multi-
Immobilization of 4CL and STS on
synthetic protein scaffolds increased product yield 5-fold compared to the same
free proteins (Wang, Yu 2012). The authors
also showed that the product yield from
these immobilized mono-enzymes was
2.7 fold higher than that achieved with
a fusion protein (4CL::STS ) in an earlier
publication (Zhang et al. 2006). This
showed that scaffolding enzymes is a more
efficient way of improving enzyme activity
than creating enzyme fusions. When
z
4CL2His was coupled to ZYMV particles in
solution and its kinetic parameters compared
to those of the enzymes free in solution
and a closely related isoenzyme 4CL1
from Arabidopsis thaliana, there was no
difference in specific activity between the
free enzymes and the enzymes coupled to
particles (Table 2, I). In spite of the high
concentration of the enzyme on the virus
surface, the activity was not enhanced.
5.3. Macromolecular assembly in solution
and on carbon coated grids
Macromolecular assemblies were built
from the z33-tagged proteins, the virus
specific antibody and the virus particles both
in solution and on carbon coated grids. Virus
particles attached to carbon grids were
subsequently decorated with active mYFPz
and z4CL2His. Secondary antibodies
conjugated to 10 nm gold beads were used to
visualize the proteins on the virus surface. On
average, about 15 gold bead were observed
per ZYMV particle (Figure 5, I). The same
experiment performed with the protein
chimera, z4CL2::STSHis, revealed even less
gold beads on the surface of PVA particles
(Figure 3D, III). Contrary to this observation,
densitometry analysis to identify the amount
of each constituent of the macromolecular
assembly in solution, showed that an 87
% coverage of ZYMV particle with mYFP
was achieved (Figure 6, I). A 1:1:5 ratio of ZYMV CP to ZYMV antibody to protein was used. Consistent with these
results, correlative
microscopy
using
grids compatible
with
epifluorescence
and transmission electron microscopy
showed that ZYMV particles were
completely covered with mYFPz. These assessments were based on its fluorescent activity (Figure 7, I). We were also able to
couple z4CL2::STS with a molecular
weight of about 107 kDa on the
A fusion protein obtained by genetic fusion of the protein domains of 4CL and STS
separated by the GSG linker enhanced
product yield 15-fold when compared to the
mono-enzymes in solution (Zhang et al.
2006). This study was the motivation behind
creating the protein chimera used in our
study. Because 4CL2 activity was not
enhanced when immobilized on ZYMV
particles probably because the particles were
floating in solution, PVA particles were
immobilized on a surface prior to
34
Summary of results and discussion
functionalzation to check if this would
enhance enzyme activity by favouring
channelling and concentration of the reaction
intermediates in a tight space. To this effect,
a bottom-up approach was used to capture
active z4CL2His and zSTSHis or z4CL2::STSHis
from clarified soluble lysates onto PVA
particles immobilized on polypropylene
tubes (Figure 4 & 5, III). The DAS-ELISA
method was used to trap these particles onto
the tube surface followed by the
other components in the assembly. This
method has been employed earlier to
immobilize virus particles for different
downstream applications (Fedorkin et al.
2000, Yang et al. 2012). Resveratrol synthesis was initiated from tubes containing
immobilized z4CL2His and zSTSHis or
z
4CL2::STSHis and after product extraction,
peaks identical to resveratrol standard
were identified (Figure 4A & 5, III). The
observed activity was specific to the enzymes grafted on the virus particles.
Binding of the z33-taged enzymes to the
virus ENCs only occurred in the presence
of antibodies raised against
potyvirus CP and there was no unspecific
binding to the first antibody layer or the
tubes (Figure 4B, III). The virus particles
efficiently blocked the first antibody
surface. Resveratrol was synthesized in
quite low amounts from the functionalized
particles. The activity of STS enzyme
varies depending on its source (Lim et al.
2011) and because the presence of the z33
peptide did not affect the activity of
4CL2 (Pille et al. 2013), we believe STS
limited the product yield. The low catalytic
efficiency of this system made it difficult to
compared the activity of the enzymes immobilized and free in solution and that
from the mono-enzymes and the protein chimera. Although several factors need
to be considered when making these comparisons, it would be interesting to be able
to do that with our systems. Several
contaminating components were associated with the macromolecular assemblies
after
SDS-PAGE
analysis (data not shown), showing a need for
improvement which could enable a cost
effective one-step method to immobilize and
purify enzymes during the design of nanodevices.
6. Leucine zipper strategy (unpublished
results)
An alternative strategy tested during this
study involved the use of complementary
acid/base heterodimeric leucine zippers (LZ)
described above. Two systems were chosen
for this study, a parallel Arg/Glu system
(Moll et al. 2001) and an antiparallel Kg/Eg
system (McClain, Woods & Oakley 2001).
Their sequences are given in Table 6 in the
Materials ad Methods section. As mentioned
earlier, this strategy has been successfully
applied to encapsulate GFP in CCMV
VLPs (Minten et al. 2009, Minten,
Nolte
&
Cornelissen
2010).
Heterodimeric leucine zippers were also
chosen here to prevent self-dimerization.
Baratova and co-workers (Baratova et
al. 2001) predicted that the first 15 amino
acids of PVA CP and amino acids 27-50
are exposed to the surface of PVA particles. In these exposed regions, there are 8
acidic amino acid residues and 7 basic
amino acid residues, making the PVA
particle surface slightly negative (one
negative charge). Consequently, we fused the
acidic LZs (Glu and Eg) to PVA CP and the
basic LZs (Arg and Kg) to the enzymes of
interest. An Xpress epitope followed by a
linker, (GGGGS)3, was inserted at the Cterminus of the LZ in the fusion proteins
to enable immuno-detection and flexibility
of the LZs respectively. The codon usage
of both acidic LZs sequences were optimized for
plant
expression
from
Nicotiana benthamiana
codon
usage
table (http://www.kazusa.or.jp/
codon/cgibin/showcodon.cgi?species=4100)
and
bacterial expression from E. coli codon usage
table
(http://bioinfo.weizmann.ac.il:3456/kegg/co
35
Summary of results and discussion
don_table/codon_eco.html) and synthesized
accordingly. The pEAQ-HT expression
vector
(Sainsbury,
Thuenemann
&
Lomonossoff 2009) was used for plant
expression and the pQE-30 vector was used
for bacterial expression. The expression
cassettes are shown in Figure 4 A and B.
LZGlu and LZEg were fused to the Nterminus of PVA CP and cloned into
pQE30 to obtain LZGluCP and LZEgCP. These
proteins were expressed in soluble form in
E.coli but the yields were very low (Figure
4C). LZGluCP was not detected on SDSPAGE after induction with 100 mM
IPTG
whereas LZEgCP levels were
indifferent before and after IPTG induction.
The insertion of this peptides to the
N-terminus
probably destabilized these
proteins. Potyviral CPs have been expressed as recombinant proteins in E. coli
and readily formed VLPs even when 36
amino acid residues were added to the Nterminus
(Jagadish
et
al.
1991,
Anindya, Savithri 2003, Hema et al. 2008)
and in planta (Cerovska et al. 2008). Hence,
the low expression levels were associated to
the presence of the leucine zippers. The
protein yield was not improved when a
different E. coli expression vector, pGEX-
6P-1, was used (data not shown). The LZGlu
optimized for plant expression was fused to
the N-terminus of PVA CP and cloned
into pEAQ-HT. Expression of this protein in Nicotiana benthamiana resulted in
low yield as shown on SDS-PAGE gel
(Figure 4D). These proteins did not assemble into VLPs (data not shown). The absence of VLPs was associated with
steric hindrance. Steric hindrance has
been avoided by assembling capsids from
wild type CP and fusion CP. This has been
achieved by either co-expressing fusion CP
with wild type CP or by inserting a 2A
peptide (described earlier) from foot-andmouth disease virus between the fusion CP
and the wild type CP. Inspired by these
approaches, we mixed different ratios of
Agrobacterium containing the fusion
LZGlu
CP and the CPwt for transient expression in plants. However, no VLPs were
formed in these plants even 10 post inoculation after TEM analysis (data not
shown).
36
Summary of results and discussion
Figure 4: Expression of LZ-CP fusion proteins in E. coli and in Nicotiana benthamiana. Expression cassette and plasmids for A) bacterial and B) plant expression. C) Analysis of crude extract after expressing
LZGlu
CP and LZEgCP fusion proteins in M15 (pREP4). Protein expression was induced by adding 1 mM
IPTG. CPwt expressed and purified from E. coli was used as a positive control. D) LZGluCP expressed in
Nicotiana benthamiana and leaf samples analysed 5 dpi.
7. PVA-binding
peptide
(unpublished results)
short term at – 20 °C. A peptide, P12
[FTTMAWRGGG], was selected after phage
display screening with these PVA particles.
This peptide was fused to the N-terminus of
P12
STSHis and P124CL2His and the yellow
fluorescent protein, P12YFPHis, containing a
C-terminal 6x His-tag and all proteins were
clone into the pET21a for expression in E.
coli. P12YFPHis was expressed in soluble form
in E. coli with reasonable yields (0.33 mg
protein from 500 mL culture) and was
purified by affinity chromatography using
Ni-NTA beads (Figure 5A). This showed that
the presence of the P12 peptide did not
interfere with it expression in E. coli.
P12
YFPHis was coupled to PVA virions on a
carbon coated grid and a secondary antibody
conjugated to 10 nm gold beads was used to
strategy
The third strategy involved the use of specific
PVA-surface binding peptides selected after
phage display screening. The protein
backbone of PVA permitted the screening of
peptide libraries for PVA-specific binders.
This strategy has been successfully employed
with other viruses and antigens (McCafferty,
Schofield 2015). For this purpose, wild type
PVA particles were isolated from Nicotiana
benthamiana infected leaves and further
purified through a 30 % sucrose cushion and
5 – 40 % sucrose gradient to ensure
uniformity in size (Figure 2 above). The virus
particles were stored in 0.1 M sodium
phosphate buffer, long term at – 80 °C and
37
Summary of results and discussion
visualize the protein on the surface of the
particles. TEM images of the decorated
particles revealed on average one gold bead
per virus particle (Figure 5B). There was no
visible increase in the size of the particle to
showcase its coverage with the protein as
seen with the z33 peptide (Figure 3D, III). In
agreement with these results, imaging of the
P12
YFPHis decorated particles on confocal
microscope showed no fluorescent activity
(data not shown). This could also suggest that
this protein was inactive prior to its coupling
to PVA particles. However, the fluorescent
properties of the P12YFPHis were clearly
visible through an eppendorf tube (data not
shown). It was concluded that the interaction
of this peptide with the virus particle
surface is not optimal enough for building
functional virus-based ENCs.
Figure 5: A) Expression of P12YFPHis fusion protein in E. coli BL21 cells with IPTG induction at OD600
0.5 and purified under native conditions using Ni-NTA agarose. The right panel shows coomassie stained
SDS-PAGE gel of purified P12YFPHis and the left panel shows nitrocellulose membrane of the purified
protein probed with α-His. Protein yield was about 0.35 mg protein from 500 mL culture. B)
Immunosorbent TEM images of PVA particles decorated with P12YFPHis. PVA virions were decorated with
P12
YFPHis and captured on a carbon coated grid. A secondary antibody conjugated to 10 nm gold beads was
used to visualize the protein on the surface of the particles. Grids were stained with uranyl acetate and TEM
images of the decorated particles revealed on average one gold bead per virus particle.
38
Concluding remarks and perspectives
CONCLUDING REMARKS AND PERSPECTIVES
In this study, CP-mediated inhibition of
potyviral RNA translation is shown to be
initiated
by
co-translational
CP-CP
interactions in vivo, a process mediated by
two CP populations. We propose this as a
mechanism through which the CP
specifically selects viral RNA to initiate its
encapsidation. The involvement of CP-CP
interactions in potyvirus assembly is evident.
Potyvirus CP can associate into VLPs in the
absence of viral RNA and this was shown to
proceed through an octameric ring in vitro.
This means that CP-CP interactions might
represent the main stabilizing force in these
particles. The direct involvement of potyviral
RNA in this process is yet to be elucidated.
RNA can act as a nucleating or stabilizing
agent during encapsidation. These studies
suggest it might act primarily to stabilize the
virus particle. Phosphorylation is a
posttranslational modification shown to
decrease the affinity of potyvirus CP for viral RNA. A Thr-242 residue located in the
C- terminal part of the core region of the CP
was found to be the phosphorylation site
(Ivanov et al. 2003). CP phosphorylation is
thought to play a role in virus assembly.
If potyviral RNA plays a role in stabilizing
the particle via electrostatic interactions,
an interesting question that arises is if
phosphorylation of Thr-242 affects the
overall charge of the interior cavity and
by so doing prevents or reduces its affinity for RNA via repulsion. This study
gives the first insight into the possible
initial
events
during
RNA
encapsidation and brings to mind more
interesting questions.
when excess is required for assembly. Subgenomic RNAs, ribosomal frameshifts or
internal ribosomal entry sites are some of the
strategies employed by RNA viruses to
regulate viral protein concentrations during
viral infection. An interesting question that
arises is how poyviruses synthesize excess
CP for the assembly process especially as CP
binding to RNA almost completely shuts
down the translation machinery.
Understanding the molecular events
underlying the different stages of potyvirus
infection is important for developing novel
resistance strategies in addition to exploiting
them as platforms for nanotechnology
applications. Potyviruses are an important
class of viruses with quite interesting
properties such as their charged exterior
surface, the surface exposed amino acids,
their stability, the availability of in vitro
assembly conditions to name a few that can
be exploited. As with other viruses, investing
on fundamental research in order to
understand the specific behaviour of this
dynamic nano-vessels is crucial for
developing
novel
virus-based
nanotechnologies. For example knowledge of
the specific role of potyviral RNA can be
useful for example in building RNA
mediated assemblies on supports or even in
mineralization. In addition, knowledge of the
co-translational CP-CP interaction involving
two CP populations can have potential for
example in multivalent displays applicable in
vaccine and biosensor technologies. The only
limiting factor as regards the use of
potyviruses, would be the virus yields which
although reasonable are quite low compared
to other most exploited viruses such as TMV
and CCMV.
Potyviral proteins are produced in equimolar
amounts in the cell and CP is down regulated
during the early stages by proteosomal
degradation via the CPIP/HSP70 pathway
(Hafren et al. 2010). However, to the best of
our knowledge there is no available
information on how they boost CP synthesis
The versatile approach for grafting
biomolecules on virus particles developed in
this study can be extended to any virus using
39
Concluding remarks and perspectives
virus specific antibodies. The specific
advantage of this functionalization method is
that no modification of the CP is required, avoiding assembly defects and although not tested in this study, this approach can be used to functionalized
VLPs as well. This approach was exploited in single and multi-enzyme displays with full particle coverage observed
in the former. The potential of this system
in one step purification and immobilization is
yet to be exploited and could provide a cost
effective and efficient way for displaying
functional biomolecules on viruses bringing together the top-down approach for immobilizing ENCs on solid supports and bottom up approach based on self-assemblies.
40
Acknowledgements
ACKNOWLEDGEMENTS
This work was carried out mostly at the
Department of Food and Environmental
Sciences at the University of Helsinki and
partly at the French National Institute for
Agricultural Research (INRA) during the
years 2010 to 2016. I thank everyone who
was a part of this journey, I definitely could
not have made it on my own.
programme, MBDP, thank you for allowing
me to be a part of your programme.
To my former colleagues and collaborators in
Finland and France: Dr. Anders Hafrén,
Andres Lõhmus, Dr. Daniela Cardinale,
Dr. Jocelyn Walter, Dr. Noelle Carette,
Amandine Barra, Dr. Maija Pollari, Dr.
Justine Charon, Dr. Katri Eskelin, Dr.
Kostantin Ivanov, Dr. Matti Wahlsten,
Shreya Saya, Swarnalok De, Dr. Gabriela
Chavez Calvillo, Marta Bašić, Dr. Taina
Suntio and Dr. Kimmo Rantalainen, I say
thank you. You each played a unique and
indispensable role during this journey. I
especially thank: Anders for the insights and
guidance at the beginning of my studies;
Kostya for the cloning and protein
purification tips, the useful scientific and
non-scientific discussions; Daniela, Noelle,
Amandine and Jos for the wonderful
collaboration and friendship over the years
and for making my stay in Bordeaux
memorable. Special thanks to Jos and Jean
Noel for the never ending support and
hospitality during our stay in Bordeaux. You
were an angel, thank you; Andres for your
friendliness, the fun discussions and your
willingness to stretch a helping hand each
time I needed one; Katri for the valuable
scientific and non-scientific discussions.
First, my sincere gratitude to Dr. Kristiina
Mäkinen and Dr. Thierry Michon for
giving me the opportunity to partake in such
a booming research area. Your continuous
guidance, great scientific input, critical
reading and commenting on all my
manuscripts and excellent mentoring have
played a great part in making this possible
and have moulded me into an independent
researcher. Kristiina, your friendly ways,
positive and caring attitude made work life
easy. Thank you! Thierry, thank you for
your assistance with most administrative
issues at the University of Bordeaux/INRA.
Special thanks to my thesis committee
members, Prof. Teemu Teeri and Dr. Harry
Boer for ensuring my studies proceeded in
the right direction. Thank you Prof. Teeri for
the exceptional fulfilment of your duties as
head of my major.
I thank my pre-examiners, Prof. Michel
Taliansky and Docent Vesa Hytönen and
my opponent Prof. Christina Wege, for
accepting to evaluate my dissertation and for
your critical comments.
In addition, thank you Taru Rautavesi for
ensuring we had everything we needed in the
lab; Dr. Sanna Koutaniemi for lending and
helping me with the gel filtration column and
runs; Mervi Lindman for your help with EM
related issues; Dr. Hany Bashandy for your
help with HPLC; Ritva Partanen for
assistance with administrative issues, Dr.
Seija Oikarinen and the University of
Bordeaux ‘co-tutelle team’ for coordinating
the joint degree process, Anita Tienhaara
for designing my thesis cover.
I thank the former Finnish Doctoral
Program in Plant Sciences (FDPPS), for
travel grant assistance and access to relevant
courses and Dr. Karen Sims-Huopaniemi
the coordinator of the program at that time for
the great assistance. Not forgetting my
current doctoral school, YEB and doctoral
41
Acknowledgements
Thank you again Kristiina and Jos for
your assistance with the Finnish and French
abstracts respectively.
My warmest gratitude to All my friends
and MECA family for continuous support
and encouragement.
To my family, I cannot thank you all enough.
Mum, my rock, Frida Besong, for your
endless love, the sacrifices you made to ensure I had a decent education, your support
through endless phone calls, prayers and
encouragement. Thank you. You are one
in a million. My siblings: Susan, Moses,
Samuel, Marvine and the rest of my family,
thank you. I appreciate all your love and
support over the years.
To my dearest husband, Dr. Joseph Ndika,
you’ve been there during the stressful and
joyful moments, showed me continuous
support and love and taken care of our
beautiful kids, Jaycie and Jodelle, during
my long hours in the lab and made time to
read and comment on some of my
manuscripts. You are SPECIAL and I thank
you. Jaycie and Jodelle, studies have not
been easy with you two but you are my
greatest motivation and rainbow during
the cloudy days. You are God given and
my blessings, love you dearly.
June 2016, Helsinki
Jane E. Besong-Ndika
42
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Population Genetics and Molecular Epidemiology of Campylobacter jejuni
1/2016 Hanna Help-Rinta-Rahko
The Interaction Of Auxin and Cytokinin Signalling Regulates Primary Root Procambial
Patterning, Xylem Cell Fate and Differentiation in Arabidopsis thaliana
2/2016 Abbot O. Oghenekaro
Molecular Analysis of the Interaction between White Rot Pathogen (Rigidoporus microporus)
and Rubber Tree (Hevea brasiliensis)
3/2016 Stiina Rasimus-Sahari
Effects of Microbial Mitochondriotoxins from Food and Indoor Air on Mammalian Cells
4/2016 Hany S.M. EL Sayed Bashandy
Flavonoid Metabolomics in Gerbera hybrida and Elucidation of Complexity in the Flavonoid
Biosynthetic Pathway
5/2016 Erja Koivunen
Home-Grown Grain Legumes in Poultry Diets
6/2016 Paul Mathijssen
Holocene Carbon Dynamics and Atmospheric Radiative Forcing of Different Types of Peatlands
in Finland
7/2016 Seyed Abdollah Mousavi
Revised Taxonomy of the Family Rhizobiaceae, and Phylogeny of Mesorhizobia Nodulating
Glycyrrhiza spp.
8/2016 Sedeer El-Showk
Auxin and Cytokinin Interactions Regulate Primary Vascular Patterning During Root
Development in Arabidopsis thaliana
9/2016 Satu Olkkola
Antimicrobial Resistance and Its Mechanisms among Campylobacter coli and Campylobacter
upsaliensis with a Special Focus on Streptomycin
10/2016 Windi Indra Muziasari
Impact of Fish Farming on Antibiotic Resistome and Mobile Elements in
Baltic Sea Sediment
11/2016 Kari Kylä-Nikkilä
Genetic Engineering of Lactic Acid Bacteria to Produce Optically Pure
Lactic Acid and to Develop a Novel Cell Immobilization Method Suitable
for Industrial Fermentations
ISSN 2342-5423
ISBN 978-951-51-2239-1
12/2016
Helsinki 2016
JANE E. BESONG-NDIKA Molecular Insights into a Putative Potyvirus RNA Encapsidation Pathway and Potyvirus Particles as Enzyme Nano-Carriers
Recent Publications in this Series
dissertationes schola doctoralis scientiae circumiectalis,
alimentariae, biologicae. universitatis helsinkiensis
JANE E. BESONG-NDIKA
Molecular Insights into a Putative Potyvirus RNA
Encapsidation Pathway and Potyvirus Particles as
Enzyme Nano-Carriers
DIVISION OF MICROBIOLOGY AND BIOTECHNOLOGY
DEPARTMENT OF FOOD AND ENVIRONMENTAL SCIENCES
FACULTY OF AGRICULTURE AND FORESTRY
DOCTORAL PROGRAMME IN MICROBIOLOGY AND BIOTECHNOLOGY
UNIVERSITY OF HELSINKI
12/2016