4/2005 Ras Trokovic
Fibroblast Growth Factor Receptor 1 Signaling in the Early Development of the Midbrain- Hindbrain and
Pharyngeal Region
5/2005 Nina Trokovic
Fibroblast Growth Factor 1 in Craniofacial and Midbrain-Hindbrain Development
6/2005 Sanna Edelman
Mucosa-Adherent Lactobacilli: Commensal and Pathogenic Characteristics
7/2005 Leena Karhinen
Glycosylation and Sorting Of Secretory Proteins in the Endoplasmic Reticulum of the Yeast Saccharomyces
cerevisiae
8/2005 Saurabh Sen
Functional Studies on alpha2-Adrenergic Receptor Subtypes
9/2005 Tiina E. Raevaara
Functional Significance of Minor MLH1 Germline Alterations Found in Colon Cancer Patients
10/2005 Katja Pihlainen
Liquid Chromatography and Atmospheric Pressure Ionisation Mass Spectrometry in Analysing Drug Seizures
11/2005 Pietri Puustinen
Posttranslational Modifications of Potato Virus A Movement Related Proteins CP and VPg
12/2005 Irmgard Suominen
Paenibacillus and Bacillus Related to Paper and Spruce Tree
13/2005 Heidi Hyytiäinen
Regulatory Networks Controlling Virulence in the Plant Pathogen Erwinia Carotovora Ssp. Carotovora
14/2005 Sanna Janhunen
Different Responses of the Nigrostriatal and Mesolimbic Dopaminergic Pathways to Nicotinic Receptor Agonists
15/2005 Denis Kainov
Packaging Motors of Cystoviruses
16/2005 Ivan Pavlov
Heparin-Binding Growth-Associated Molecule (HB-GAM) in Activity-Dependent Neuronal Plasticity in
Hippocampus
17/2005 Laura Seppä
Regulation of Heat Shock Response in Yeast and Mammalian Cells
18/2005 Veli-Pekka Jaakola
Functional and Structural Studies on Heptahelical Membrane Proteins
19/2005 Anssi Rantakari
Characterisation of the Type Three Secretion System in Erwinia carotovora
20/2005 Sari Airaksinen
Role of Excipients in Moisture Sorption and Physical Stability of Solid Pharmaceutical Formulations
21/2005 Tiina Hilden
Affinity and Avidity of the LFA-1 Integrin is Regulated by Phosphorylation
22/2005 Ari Pekka Mähönen
Cytokinins Regulate Vascular Morphogenesis in the Arabidopsis thaliana Root
23/2005 J. Matias Palva
Interactions Among Neuronal Oscillations in the Developing and Adult Brain
25/2005 Michael Stefanidakis
Cell-Surface Association between Progelatinases and ß2 Integrins: Role of the Complexes in Leukocyte
Migration
26/2005 Heli Kansanaho
Implementation of the Principles of Patient Counselling into Practice in Finnish Community Pharmacies
Helsinki 2006
JULIA PERTTILÄ Expression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest Virus Replicase Proteins
Recent Publications in this Series:
ISSN 1795-7079 ISBN 952-10-2940-4
Expression, Enzymatic Activities and Subcellular
Localization of Hepatitis E Virus and
Semliki Forest Virus Replicase Proteins
JULIA PERTTILÄ
Institute of Biotechnology and
Department of Biological and Environmental Sciences
Faculty of Biosciences and
Viikki Graduate School in Biosciences
University of Helsinki
Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki
1/2006
1/2006
EXPRESSION, ENZYMATIC ACTIVITIES AND
SUBCELLULAR LOCALIZATION OF HEPATITIS E VIRUS
AND SEMLIKI FOREST VIRUS REPLICASE PROTEINS
Julia Perttilä
Institute of Biotechnology and
Department of Biological and Environmental Sciences
Faculty of Biosciences and
Viikki Graduate School in Biosciences
University of Helsinki
Academic dissertation
To be presented, with the permission of the Faculty of Biosciences of the University of
Helsinki, for public criticism in the auditorium 1041 at Viikki Biocenter (Viikinkaari 5,
Helsinki) on February 23rd, 2006, at 12 noon.
Helsinki 2006
Supervised by
Professor Leevi Kääriäinen
Institute of Biotechnology
University of Helsinki
and
Docent Tero Ahola
Institute of Biotechnology
University of Helsinki
Reviewed by
Professor Timo Hyypiä
Department of Virology
University of Turku
and
Docent Sirkka Keränen
VTT Biotechnology
Opponent
Docent Maarit Suomalainen
Haartman Institute
University of Helsinki
ISBN 952-10-2940-4 (sid.)
ISBN 952-10-2941-2 (PDF)
Gummerus Oy
Saarijärvi 2006
To my family
CONTENTS
LIST OF ORIGINAL PUBLICATIONS
ABBREVIATIONS
ABSTRACT .................................................................................................................. 1
INTRODUCTION ........................................................................................................ 2
1. ALPHAVIRUS-LIKE SUPERFAMILY .......................................................... 2
1. 1. Plant alpha-like viruses ......................................................................... 3
1. 2. Animal alpha-like viruses ....................................................................... 4
2. ALPHAVIRUSES ............................................................................................. 4
2. 1. Replication cycle ...................................................................................... 4
2. 2. Properties of nonstructural proteins ...................................................... 6
2. 2. 1. NsP4 ............................................................................................. 6
2. 2. 2. NsP3 ............................................................................................. 7
2. 2. 3. NsP2 ............................................................................................. 7
2. 2. 4. NsP1 ............................................................................................. 8
3. HEPATITIS E VIRUS ...................................................................................... 8
3. 1. General aspects of infection ................................................................... 8
3. 2. Classification and genetic heterogeneity ............................................... 9
3. 3. Virion structure and genome organization. ......................................... 10
3. 3. 1. ORF1 ......................................................................................... 11
3. 3. 2. ORF2 ......................................................................................... 12
3. 3. 3. ORF3 ......................................................................................... 12
3. 4. Experimental propagation, expression and replication ...................... 13
3. 5. Vaccine candidates against hepatitis E ............................................... 14
4. mRNA CAPPING ........................................................................................... 15
4. 1. Viral and cellular capping enzymes ..................................................... 16
AIMS OF THE STUDY ............................................................................................. 19
MATERIALS AND METHODS ............................................................................... 20
1. Cells and viruses .............................................................................................. 20
2. Bacterial strains and plasmids ........................................................................ 20
3. Infection and transfection ............................................................................... 20
4. Radioactive labelling and cell fractionation ................................................... 20
5. Flotation analysis ............................................................................................. 21
6. Antisera and cell-staining reagents ................................................................. 22
7. Immunoprecipitation and immunoblotting ..................................................... 22
8. Enzyme assays .................................................................................................. 22
9. Immunofluorescence microscopy .................................................................... 22
RESULTS ................................................................................................................. 24
1. The RNA capping activity of HEV .................................................................. 24
2. Membrane association of HEV capping enzyme ............................................ 25
3. Intracellular distribution and proteolytic processing of HEV
nonstructural polyprotein .................................................................................... 26
4. Membrane association and subcellular localization of SFV
nonstructural polyprotein .................................................................................... 27
DISCUSSION ............................................................................................................. 29
1. A unique cap formation pathway specific for alphavirus-like superfamily ... 29
2. Proteolytic processing of HEV nonstructural polyprotein ............................ 30
3. Membrane association and intracellular distribution of HEV and SFV
replicases .............................................................................................................. 32
4. Inhibitors of viral replication .......................................................................... 33
ACKNOWLEDGEMENTS ...................................................................................... 36
REFERENCES ........................................................................................................... 38
ORIGINAL PUBLICATIONS
This thesis is based on the following articles, which are referred to in the text by their
Roman numerals and on unpublished data presented in the text.
I
Magden J, Takeda N, Li T, Auvinen P, Ahola T, Miyamura T, Merits A,
Kääriäinen L. 2001. Virus-specific mRNA capping enzyme encoded by
Hepatitis E virus. J Virol. 75:6249-6255.
II
Salonen A, Vasiljeva L, Merits A, Magden J, Jokitalo E, Kääriäinen
L. 2003. Properly folded nonstructural polyprotein directs the Semliki
Forest virus replication complex to the endosomal compartment. J Virol.
77:1691-1702.
III
Perttilä J, Spuul P, Nordman N, Miyamura T, Kääriäinen L, Ahola T.
Expression and subcellular localization of Hepatitis E virus replication
proteins. Manuscript.
IV
Magden J, Kääriäinen L, Ahola T. 2005. Inhibitors of virus replication:
recent developments and prospects. Appl Microbiol Biotechnol. 66:612621.
ABBREVIATIONS
aa
BaMV
BMV
BNYVV
BSA
CP
CPVI
ER
GDP
GFP
GT
GTP
HCV
HEL
HEV
Huh-7
IPTG
kb
kDa
m7GMP
MT
MVA
MW
nsP
nt
NTPase
ORF
P15
PAGE
PBS
PFA
PFU
PNS
POL
PRO
RC
RdRp
RUB
S15
SDS
SFV
SIN
ss
TMV
ts
VLP
wt
amino acid
bamboo mosaic virus
brome mosaic virus
beet necrotic yellow vein virus
bovine serum albumin
capsid protein
cytoplasmic vacuole type I
endoplasmic reticulum
guanosine diphosphate
green fluorescent protein
guanylyltransferase
guanosine triphosphate
hepatitis C virus
helicase
hepatitis E virus
human hepatoma cells
isopropyl-β-D-thiogalactopyranoside
kilobase
kilodalton
7-methyl-GMP
methyltransferase
modified vaccinia virus Ankara
molecular weight
nonstructural protein
nucleotide
nucleoside triphosphatase
open reading frame
membrane fraction after centrifugation at 15 000 x g
polyacrylamide gel electrophoresis
phosphate buffered saline
paraformaldehyde
plaque forming unit
post nuclear supernatant
polymerase
protease
replication complex
RNA-dependent RNA polymerase
rubella virus
soluble fraction after centrifugation at 15 000 x g
sodium dodecyl sulfate
Semliki Forest virus
Sindbis virus
single-stranded
tobacco mosaic virus
temperature sensitive
virus-like particle
wild type
Abstract
ABSTRACT
Positive-strand RNA viruses are divided
into large groups termed superfamilies, the
members of which share common features
in their encoded replicase proteins, genome
organization and replication strategies. The
purpose of this work was to characterize
the properties of hepatitis E virus (HEV)
and Semliki Forest virus (SFV) replicase
proteins. These distantly related viruses
belong to the alphavirus-like superfamily
of positive-strand RNA viruses.
In the first part of this study, an Nterminal fragment of hepatitis E virus
replicase was shown to possess both
methyltransferase and guanylyltransferase
activities, required for capping of viral
mRNA. Hepatitis E virus capping enzyme
utilizes a unique mechanism of cap
formation. The capping enzyme of HEV
first acts as a methyltransferase, catalyzing
the transfer of a methyl group from Sadenosylmethionine to GTP to yield 7methyl-GTP. The capping enzyme then
acts as a guanylyltransferase, transferring
the methylated guanosine to mRNA via
a covalent complex. This virus-specific
pathway is conserved within members of
the alphavirus-like superfamily. Therefore,
these capping reactions offer attractive
targets for the development of antiviral
drugs.
The second part of this work deals
with the Semliki Forest virus replication
complex. We studied the role of Semliki
Forest virus nonstructural polyprotein
in the formation and targeting of the
replication complex by expressing its
cleavable and uncleavable intermediates.
The uncleaved polyproteins possessed
properties previously identified for the
individual nonstructural proteins. We
demonstrated that only polyproteinderived nonstructural proteins were able to
assemble into a complex. The study verified
that nonstructural protein nsP1 alone was
responsible for the membrane association
of the nonstructural polyprotein.
The same methods were used to study
expression and membrane association
of the HEV large nonstructural protein
encoded by ORF1 of the genome RNA.
We expressed the full-length pORF1 and
its shorter fragments in mammalian cells.
By subcellular fractionation experiments
and immunofluorescence microscopy we
observed that HEV replicase is tightly
associated with the membranes and is
localized in the perinuclear region of the
cell. Results indicated that the localization
signal, which directs HEV polyprotein to
a specific membrane region in the cell, is
located in its N-terminal part.
In the last part of the thesis we describe
already existing antiviral compounds
and propose novel potential targets. All
steps in the virus life cycle are, in theory,
equally appropriate for the development
of antiviral drugs. Major research efforts
have been focused on the enzymes that
mediate viral replication. Among them,
proteases and polymerases have been the
most popular targets of antiviral research
during the past years. The present work
describes the capping enzyme of alphalike viruses that is a novel target for the
development of specific inhibitors against
viral replication.
1
Introduction
INTRODUCTION
1. ALPHAVIRUS-LIKE
SUPERFAMILY
Viruses with single-strand positive-sense
RNA genomes represent the largest class
of viruses. This diverse group of viruses
includes major pathogens of vertebrate
and invertebrate animals, plants, fungi
and bacteria. The high mutation rate is
typical for RNA viruses and must have
played an important role in the evolution
of viral populations and their adaptation
to environmental changes (Domingo et al,
1999). Progress in sequencing techniques
and
computer-assisted
comparative
studies has led to an understanding of
the relationships within this class of
viruses. Positive-strand RNA viruses can
be categorized into large groups termed
superfamilies, the members of which
share common features in their encoded
replicase proteins, genome organization
and replication strategies (reviewed in
Koonin and Dolja, 1993). Such conserved
proteins include RNA-dependent RNA
polymerase (POL), RNA helicase (HEL),
chymotrypsin-like
and
papain-like
proteases (PRO) and methyltransferases
(MT). A comparative analysis of amino acid
sequences of viral proteins suggests that
there are three superfamilies of positivesense RNA viruses, i.e., the picornaviruslike, alphavirus-like and flavivirus-like
(reviewed in van der Heijden and Bol,
2002).
Amongst the members of the
alphavirus-like superfamily are animal
viruses from the family Togaviridae
(genera Alphavirus and Rubivirus) and
Hepeviridae (Hepatitis E virus), insect
viruses of the Tetraviridae family, as well as
numerous groups of plant viruses, including
Bromoviridae, Closteroviridae, and the
2
genera Tobamo-, Tobra-, Hordei-, Furo-, Beny-,
Capillo-, Tymo-, Carla- and Potexvirus, and
other plant virus groups. Members of this
group of viruses feature many similarities.
The three conserved domains are always
organized in the same order MT-HELPOL (reviewed in Kääriäinen and Ahola,
2002; van der Heijden and Bol, 2002).
The methyltransferase (or recently termed
capping enzyme) is the hallmark of the
alphavirus-like superfamily, as it is present
in all members of the family (Rozanov et
al., 1992) (Fig.1). In addition, members
of the alphavirus-like superfamily share
capped 5′ end of positive-strand mRNAs,
membrane-associated replication complex,
an extra untemplated G residue at the 3′
end of minus strand, asymmetric RNA
synthesis with an excess of plus-strand
RNA, and transcription of one or more
subgenomic RNAs. Many viruses utilize
translational readthrough of a termination
codon to synthesize the RNA polymerase
(reviewed in Strauss and Strauss, 1994).
There are also many differences among
the viruses that might have appeared at
later stages of evolution. For example,
the genome can consist of a single RNA
or be segmented. In alphaviruses, the
nonstructural proteins are proteolytically
processed by virus-encoded papain-like
protease, but this function is not present in
most of the plant viruses. On the other hand,
plant viruses possess genes for movement
within the plant that are not present in
the alphavirus genome. The structural
proteins are of different origin, which
leads to viruses with various structures
(Strauss and Strauss, 1994, Kääriäinen
and Ahola, 2002). The degree of sequence
conservation between homologous viral
proteins is relatively low. As a rule,
among distant groups of viruses, only
short amino acid sequence motifs that are
directly involved in enzymatic function
Introduction
are conserved (reviewed in Koonin and
Dolja, 1993). Nevertheless, the conserved
order of capping enzyme, helicase and
polymerase domains suggests that the
composition of the replication complexes
is similar among the members of the
alphavirus-like superfamily.
and complete replicase proteins by (i)
ribosomal readthrough or frameshifting,
(ii) expression from two different RNAs,
or (iii) proteolytic modification (reviewed
in van der Heijden and Bol, 2002).
Tobacco mosaic virus (TMV), type
member of the genus Tobamovirus
expresses a polyprotein (183 kDa)
containing all three domains MT, HEL
and POL in the same sequence after the
readthrough of an amber stop codon before
the POL domain. The major translation
product is smaller (126 kDa) and contains
only the MT and HEL domains. The
tobamoviruses share this strategy with
viruses from the genera Tobravirus,
Furovirus, Pomovirus and Pecluvirus (van
der Heijden and Bol, 2002).
1. 1. Plant alpha-like viruses
Plant viruses from the alphavirus-like
superfamily use several different strategies
to express their replicase proteins. For
example, Bamboo mosaic virus (BaMV),
a member of genus Potexvirus, expresses
a single 155-kDa polypeptide that has all
three domains methyltransferase, helicase
and polymerase. Other viruses regulate the
production of different amount of partial
ANIMAL VIRUSES
MT
MT
MT
PRO?
X
HEL
X
HEL
X
PRO
PRO
HEL
POL
hepevirus
POL
alphavirus
POL
rubivirus
INSECT VIRUSES
HEL
POL
betatetra/omegatetraviruses
MT
HEL
POL
potex, idaeo, allexi, foreaviruses
MT
HEL
POL
tobamo, tobra, furo, pomo, pecluviruses
MT
HEL
MT
PRO
MT
HEL
MT
PLANT VIRUSES
HEL
PRO
POL
bromo, cucumo, alfamo,
ilar, olea, hordeiviruses
POL
tymo, carlaviruses
POL
benyviruses
Figure 1. Schematic representation of the genome organization of alpha-like viruses (modified
from van der Heijden and Bol, 2002). Methyltransferase (MT), helicase (HEL) and polymerase
(POL) domains are present in all members of the family. An additional X domain is present
in animal viruses. Some viruses encode the POL domain via the read-through of a leaky stop
codon (▼).
3
Introduction
Brome mosaic virus (BMV), type
member of the genus Bromovirus,
expresses two replicase proteins. Protein
1a is similar to TMV p126 and contains
capping enzyme and helicase domains.
BMV protein 2a contains only the POL
domain (Ahola and Ahlquist, 1999). This
strategy for expression of replicase proteins
from two different RNAs is also used by
other members of the Bromoviridae family
and by the hordeiviruses.
Like alphaviruses, tobacco mosaic
virus (Merits at al., 1999), brome mosaic
virus (Ahola and Ahlquist, 1999) and
bamboo mosaic virus (Li et al., 2001)
synthesize cap for their mRNAs in
different manner than their host cells. The
cap formation pathway is described in
detail later in this study.
1. 2. Animal alpha-like viruses
Animal infecting viruses from the
alphavirus-like superfamily include viruses
from genera Alphavirus and Rubivirus of
the Togaviridae family and hepatitis E virus
of the Hepeviridae family (Koonin at al.,
1992). All animal viruses from the alphalike supergroup encode their proteins in a
single genomic RNA. Characteristic for
animal alpha-like viruses is the presence
of a conserved domain, known as the X
domain, the function of which remains to
be determined. The X domain is associated
with papain-like proteases in alphaviruses
and Rubella virus (RUB) and is preceded
by a proline-rich region in RUB and HEV.
Another conserved domain with unknown
function, so-called Y domain, resides
downstream of the methyltransferase
domain in rubella and hepatitis E virus,
but is not found in alphaviruses (Koonin at
al., 1992, Koonin and Dolja, 1993).
4
2. ALPHAVIRUSES
Alphaviruses are enveloped positive-strand
RNA viruses that are widely distributed
throughout the world. They are transmitted
to vertebrate hosts by mosquitos. The
alphavirus genus consists of about 30
members, some of which are pathogenic
to humans or domestic animals, causing
serious central nervous system infections
or milder infections with symptoms like
fever, rash and arthritis (Strauss and
Strauss, 1994).
The best characterized alphaviruses,
Sindbis virus (SIN) and Semliki Forest
virus, have been extensively studied
for the last 30 years. Alphaviruses
have been used as molecular probes to
study the modification and transport of
glycoproteins, as well as endocytosis and
membrane fusion events (Helenius, 1995,
Ellgaard and Helenius, 2003). Alphaviruses
have been successfully used as expression
vectors, due to their broad host range and
efficient cytoplasmic gene expression
in a variety of cell types (Liljeström and
Garoff, 1991).
As a result of increased understanding
of the molecular events during infection,
alphaviruses together with the wellstudied plant alpha-like viruses, primarily
Brome mosaic and Tobacco mosaic
viruses, became one of the most advanced
model systems to study the replication of
positive-strand RNA viruses (Kääriäinen
and Ahola, 2002).
2. 1. Replication cycle
The alphavirus particles are icosahedral
with a diameter of 700 Ả. The virion
consists of one copy of RNA (11.5 kb for
SFV) enclosed in a capsid surrounded
by an envelope. The virus envelope is
built by heterodimers of transmembrane
glycoproteins E1 and E2, which form
Introduction
80 morphologically distinct spikes. In
SFV a third glycoprotein, a peripherally
bound E3, is associated with the E1E2 dimer. The envelope surrounds the
nucleocapsid, which is formed by 240
copies of a capsid protein arranged in
pentamers and hexamers (Strauss and
Strauss, 1994). The attachment of the
virus particle to the cell surface is mainly
directed by E2, while E1 is required for
the membrane fusion between the viral
envelope and target membrane (Marsh and
Helenius, 1989, Helenius, 1995). In order
to infect a variety of hosts, alphaviruses
must use different receptor molecules for
attachment. Laminin receptor has been
identified as a receptor for SIN (Wang et
al., 1992). Other receptors are very poorly
characterized. The virus enters the cell
via receptor-mediated endocytosis, which
is followed by conformational changes
in the envelope triggered by low pH. As
a result, nucleocapsid is released into the
cytoplasm, where uncoating of the genome
RNA is mediated by ribosomes (Singh and
Helenius, 1992).
The alphavirus RNA replication takes
place in the cytoplasm of the infected cells
in association with membranes (Ranki and
Kääriäinen, 1979). The single-stranded
positive-sense genome RNA contains
a 5′ end cap and 3′ end polyA tail and is
used directly as mRNA (Kääriäinen and
Söderlund, 1978). The 5′ two-thirds of the
SFV 42S RNA genome is translated into
a large polyprotein of 2432 aa, designated
P1234, which is autocatalytically processed
to yield nonstructural protein nsP4 and
P123 (Fig.2). This is the “early RNA
polymerase” responsible for the synthesis
of full-size complementary RNA (42S
RNA minus strand). After further cleavage
of P123 into nsP1, nsP2 and nsP3, the
minus strand in turn acts as a template for
the synthesis of new 42S RNA plus strand,
as well as subgenomic 26S RNA of the
structural proteins (Kim et al., 2004).
The translation of 26S RNA leads
to production of alphavirus structural
proteins; which are synthesized as a
C-p62-6K-E1 polyprotein precursor.
The amino-terminal capsid protein C
autoproteolytically cleaves itself from
the nascent chain and interacts with the
genomic RNA in the cytoplasm to form
a nucleocapsid. The envelope proteins
are cotranslationally inserted into the
endoplasmic reticulum (ER) and are
transported to the plasma membrane via the
Golgi complex. During transit, the p62-E1
heterodimeric complexes are cleaved into
mature E1 and E2 heterodimers, which are
then oligomerized into spikes (reviewed
in Garoff et al., 2004). The formation of
an envelope around the viral nucleocapsid
and the release of virus particles from the
infected cells by budding are driven by
the interaction of the virus spike proteins
and the nucleocapsid (Suomalainen et al.,
1992).
Replication of all positive-strand
RNA viruses of eukaryotes takes place
in association with membranes. Different
viruses utilize various intracellular
membrane compartments and cause
diverse modifications and rearrangements
of the target membrane (Salonen et al.,
2005). Viruses of family Togaviridae
use membranes from endo- and
lysosomal compartments. Semliki Forest
virus replicase proteins together with
synthesized RNA were found in vacuoles
defined as cytopathic vacuoles type I
(CPV-1) (Grimley et al., 1968; Salonen
et al., 2005). Rubella virus replicates in
cytoplasmic structures morphologically
analogous to the alphavirus CPVs
(Kujala et al., 1999). Plant viruses of the
alphavirus-like superfamily replicate on
various intracellular membranes. For
5
Introduction
example, brome mosaic virus and tobacco
mosaic virus use the ER membrane as the
site of their replication, alfalfa mosaic
virus replicates on the vacuolar membrane
and turnip yellow mosaic virus on the
chloroplast envelope (Restrepo-Hartwig
and Ahlquist, 1996; Mas and Beachy, 1999;
van der Heijden et al., 2001, Prod′homme
et al., 2001).
2. 2. Properties of nonstructural proteins
2. 2. 1. NsP4
The size of nsP4 varies between 607614 aa in alphaviruses and it is the most
conserved of replicase proteins. It is
considered to be the catalytic subunit of
RNA polymerase. The C-terminal region
of nsP4 (approximately aa 280-530)
contains sequence elements typical for
all RNA-dependent RNA polymerases,
including the universal GDD motif (Poch
et al, 1989; Koonin, 1991). Furthermore,
the large C-terminal part of the protein (aa
110-610) is related to replicase proteins
from the alphavirus-like superfamily
(Koonin and Dolja, 1993). Genetic data
from temperature sensitive (ts) mutants
supports the role of nsP4 as the RNA
polymerase. In SIN ts6 (mutation G153E),
all viral RNA synthesis stops when cells are
shifted to the nonpermissive temperature
at any stage of infection, but synthesis is
reversible upon shift to the permissive
temperature (Keränen and Kääriäinen,
1979; Sawicki et al., 1981). The amount
of nsP4 in alphavirus infected cells is low
in comparison to the other nsPs. In most
alphaviruses nsP4 is only produced as a
result of the translational read-through
Figure 2. Translational strategy and proteolytic processing of SFV nonstructural and structural
proteins
6
Introduction
of an opal codon (UGA). Additionally,
nsP4 is rapidly degraded by an N-end rule
pathway in proteosomes (de Groot at al.,
1991, Takkinen at al., 1991). Presumably
only the nsP4 fraction, which is assembled
into replication complex, is stable and
protected from proteosomal degradation.
2. 2. 2. NsP3
The specific biochemical functions of
nsP3 (SFV 482 aa) are still unknown,
although genetic studies demonstrated that
it is essential for the alphavirus replication
(Hahn et al., 1989). It has been reported
to affect minus-strand and subgenomic
RNA synthesis (LaStarza et al., 1994;
Wang et al., 1994). The N-terminal part of
nsP3 is conserved among the alphaviruses
and homologous sequences were found
in rubella and hepatitis E viruses, and in
coronaviruses as well (Koonin and Dolja,
1993). Remarkably, the same domain
was found also in bacteria, archae and
eukaryotes (Pehrson and Fuji, 1998,
Karras et al., 2005). The conservation of
this domain during evolution suggests
its importance, however its function
remains to be determined. In contrast,
the C-terminal part of nsP3, rich in
serine, threonine and acidic residues,
is not conserved in length or sequence
(Strauss and Strauss, 1994). This region
is phosphorylated heavily on serines and
less heavily on threonines (Peränen at al.,
1988, Vihinen and Saarinen, 2000). The
SFV mutant encoding nonphosphorylated
nsP3 was infectious, but the level of RNA
synthesis was significantly decreased.
The mutant virus pathogenicity in mice
was also greatly reduced (Vihinen at al.,
2001).
2. 2. 3. NsP2
NsP2 (SFV 799aa) has multiple roles
in RNA replication and polyprotein
processing. It has two distinct functional
domains: the N-terminal part contains
sequence motifs typical for RNA and
DNA helicases, and the C-terminal part
is homologous to papain-like cysteine
proteinases (Gorbalenya et al, 1991;
Koonin and Dolja, 1993). The nucleoside
triphosphatase (NTPase) and RNA helicase
activities were verified by biochemical
studies (Rikkonen et al., 1994; Gomez de
Cedron at al., 1999). Both activities were
associated with the N-terminal part (1-479
aa) of nsP2. Additionally, the N-terminal
half of nsP2 has been identified as an
RNA triphosphatase, catalyzing the initial
step of the virus-specific mRNA capping
reaction (Vasiljeva et al., 2000). The Cterminal domain of nsP2 (residues 458799 for SFV) functions as the nonstructural
proteinase (Strauss and Strauss, 1994;
Vasiljeva et al., 2001). The activity is
responsible for all proteolytic cleavages
in the polyprotein processing, albeit with
different efficiencies. Site between nsP3
and nsP4 (3/4) was cleaved most readily,
site 1/2 fairly efficiently and site 2/3 poorly.
A mutation in the catalytic site of nsP2
protease (Cys478) abolished the proteolysis
of nonstructural polyprotein (Vasiljeva et
al., 2001). NsP2 plays also a role in the
regulation of subgenomic RNA synthesis.
Experiments with ts mutants demonstrated
that nsP2 directly or indirectly binds to
the 26S RNA promoter. The proteolytic
activity of nsP2 also regulates the levels of
production of minus strands vs plus strands
(Sawicki and Sawicki, 1993; Suopanki et
al., 1998). NsP2 has multiple locations in
cells. Only part of nsP2 is assembled with
other nsPs in the replication complex. In
infected cells, about half of the protein
is found in the nucleus and the rest is
distributed throughout the cytoplasm
(Peränen at al., 1990). When expressed
alone, nsP2 is localized exclusively in the
7
Introduction
nucleus. The nuclear targeting signal was
mapped by mutagenesis to the C-terminal
domain of nsP2 (Rikkonen at al., 1992).
2. 2. 4. NsP1
Both genomic and subgenomic RNAs
of alphaviruses have m7G(5′)ppp(5′)N
(cap0 structure) at their 5′end. NsP1
(SFV 537 aa) is a capping enzyme, which
catalyzes the formation of cap-structure.
The N-terminal half of nsP1 contains the
conserved domain, which is found in all
alphavirus-like superfamily members
(Rozanov et al., 1992). NsP1 possess two
enzymatic activities. Firstly, it acts as a
methyltransferase catalyzing the transfer
of a methyl group from S-adenosylmethionine (AdoMet) to GTP (Mi and
Stollar, 1991; Laakkonen at al., 1994).
Secondly, it is a guanylyltransferase
that transfers the m7GMP to the 5′
terminus of RNA via the formation of
intermediate complex nsP1-m7GMP
(Ahola and Kääriäinen, 1995). NsP1
catalyzes both capping reactions in a
different manner than cellular enzymes,
where guanylyltransferase first transfers
the GMP to the mRNA and only after
that methylation at position 7 takes place
(Ahola and Kääriäinen, 1995, Shuman,
1995, Shuman, 2001). In both pathways,
these reactions are preceded by RNA 5′
triphosphatase, which removes the 5′ γphosphate from the nascent viral RNA
chain. NsP2 has been demonstrated to
catalyze this reaction in alphaviruses
(Vasiljeva et al., 2000).
NsP1 is the membrane anchor for the
replication complex (this study II; Salonen
et al., 2005). It is tightly associated with
membranes both in alphavirus infected
cells and when expressed alone. The tight
binding was due to palmitoylation of
cysteine residues 418-420 (Peränen et al.,
1995, Laakkonen et al., 1996, Ahola et al.,
8
2000). When these residues were mutated
to alanines, nsP1 was still membraneassociated and enzymatically active.
However, it could be released from the
membranes by a high salt treatment. Thus,
the elimination of palmitoylation converted
nsP1 from an “integral” to a “peripheral”
membrane protein (Laakkonen at al.,
1996). Another mechanism responsible for
membrane association is a direct interaction
of nsP1 with the anionic phospholipids.
The interaction is mediated by amino acids
245-264, with both positively charged and
hydrophobic residues required for binding
(Ahola et al., 1999; Lampio et al., 2000).
The nsP1 polypeptide segment (245-264)
is highly conserved within alphaviruslike superfamily (Rozanov et al., 1992)
and could play a role in the membrane
association of other superfamily members.
3. HEPATITIS E VIRUS
3. 1. General aspects of infection
Hepatitis E, previously known as enterically
transmitted non-A, non-B hepatitis, is
an infectious viral disease with clinical
and epidemiological features of acute
hepatitis. It is transmitted by the fecaloral route mainly through contaminated
drinking water. The first documented
outbreak of hepatitis E occurred in New
Delhi, India, in 1955 and consisted of 29
000 cases (Purcell and Emerson, 2001).
Hepatitis E virus is the major causative
agent of acute hepatitis in young adults
living in developing countries with poor
sanitary conditions and high population
density. The mortality rate among hepatitis
E patients is usually 1-3 %, but it can reach
up to 20% for infected pregnant women,
especially during the third trimester (Tam
et al, 1999; Worm et al., 2002). Hepatitis
E is also diagnosed in industrialized
countries, although most of the cases are
Introduction
linked to travel history to endemic areas.
There is also a possibility for zoonotic
reservoir, since the virus is found in both
wild and domestic animals, e.g. non-human
primates, pigs, cows, chickens, sheep,
goats and rodents (Meng, 2000; Favorov
et al., 2000). The first animal strain of
HEV was isolated from a pig in the United
States (Meng et al., 1997). Cross-species
infections of HEV between swine and
nonhuman primates were experimentally
demonstrated, suggesting that hepatitis E
may be a zoonotic disease (Meng et al.,
1998).
Humans are the natural hosts for
hepatitis E virus. Several primate species,
including chimpanzees, cynomolgus
macaques, rhesus monkeys, tamarins
and African green monkeys, are found
to be susceptible to HEV infection and
have served as experimental models for
hepatitis infection (Bradley et al., 1987;
Purcell and Emerson, 2003). HEV enters
the host by oral ingestion and reaches
the liver by unknown mechanisms. HEV
replicates primarily, if not exclusively,
in hepatocytes, although, several studies
in rats and swine model suggested that
extrahepatic tissues such as peripheral
blood monocytes, spleen, lymph nodes
and small intestine may be involved in
HEV replication (Maneerat et al., 1996,
Williams et al., 2001). Hepatitis E infection
usually becomes apparent within 2-9
weeks of exposure, the average incubation
period being 6 weeks. Early symptoms of
infection, such as fever, nausea, vomiting,
diarrhea, headache and abdominal
enlargement can occur a week before
the onset of jaundice. Fecal excretion of
virus particles begins during this period,
reflecting replication of viruses within
hepatocytes. Viremia usually precedes the
major peak of alanine aminotransferase
activity (Tam et al, 1999). Anti-HEV IgM
appears in the serum of the patients early
during the infection and remains detectable
for 2-3 months. Anti-HEV IgG antibodies
can be detected after IgM response and
usually persist for years. Generally, the
period of recovery from hepatitis E can
take up to 14 weeks. HEV infection
never progress to chronicity (Purcell and
Emerson, 2001).
3. 2. Classification and genetic
heterogeneity
From 1988 to 1998, based on
physiochemical properties, hepatitis E
virus was classified into the Caliciviridae
family. However, sequence comparison
and genome organization analysis revealed
little, if any, homology between HEV and
caliciviruses. At present, HEV is classified
in to the new family Hepeviridae as a
separate genus Hepevirus (Emerson et
al., 2004a). Phylogenetic analysis of
HEV nonstructural regions shows that it
is related more closely to the alphaviruslike superfamily, in particular to rubella
virus and Beet necrotic yellow vein virus
(BNYVV), a plant benyvirus (Koonin et
al., 1992).
First sequence of entire HEV genome
from an outbreak in Myanmar (Burma)
became available more than a decade ago
(Reyes et al., 1990; Tam et al., 1991).
The genomes of several HEV strains
from different parts of the world became
available since, e.g. Pakistan (Tsarev et al.,
1992), China (Bi et al., 1993) and Mexico
strains (Huang at al., 1992). However, the
majority of HEV isolates have only been
sequenced partially. Sequence comparisons
and phylogenetic analyses show that the
genome sequence of HEV is quite stable.
The nucleotide identity of strains isolated
in single outbreak is very high (Arankalle et
al., 1999). However, the genome of strains
isolated from geographically distinct
9
Introduction
locations is generally more diverse. It has
been suggested that HEV isolates segregate
into four major genotypes: Genotype 1including the isolates from South-East
Asian (Burma, India), North and Central
Asian (China, Pakistan, Kyrgyzstan and
India) and North African strains; Genotype
2-comprising a single North American
(Mexico) isolate; Genotype 3-consisting
of the US human and swine isolates; and
Genotype 4-including several isolates
from China and most isolates from Taiwan
(reviewed in Wang and Zhuang, 2004).
Of these, the Mexican isolate cloned
from the Telixtac outbreak is the most
divergent genetically. The Asian strains
are well-conserved among themselves,
with nucleotide sequence identity between
93 and 100% in the coding regions and
amino acid sequence identity between
98 and 100 % (Tam et al., 1999). In spite
of the geographical diversity of HEV
isolates, only one serotype is recognized
(Schlauder and Mushahvar, 2001). That
suggests a possibility for the development
of a broad range vaccine against hepatitis
E infection.
3. 3. Virion structure and genome
organization
HEV is an icosahedral, non-enveloped
particle with spike-like protrusions visible
on the surface of the virus. The average
diameter of particles is about 32nm
(Balayan et al., 1983). The fact that HEV
can survive in the gastrointestinal tract
suggests that the virus is relatively stable to
acid and mild alkaline conditions (Purcell
and Emerson, 2001). However, insufficient
amount of virus has been available for
purification and direct chemical analysis
to yield reliable information. Recent
studies show that recombinant hepatitis
E virus capsid protein self-assembles into
virus-like particles (VLP), when expressed
by the use of a recombinant baculovirus
vector in an insect cell system (Xing et
al., 1999; Li at al., 2001; Maloney et al.,
2005). The 60 copies of a single capsid
protein cluster as 30 dimers into a T=1
lattice. The recombinant particles share
morphological and antigenic properties
with native virions that, together with
high levels of production, make rHEV
particles an ideal candidate for a hepatitis
E vaccine.
Hepatitis E virus genome is enclosed
within a capsid and contains a singlestranded, positive-sense, polyadenylated
RNA molecule of approximately 7.2 kb
in length (Fig. 3). The genome of the
prototype strain of HEV, recovered from
the outbreak in Myanmar, consists of 5′
noncoding (NC) region of 27 nucleotides,
followed by an ORF1 of 5079 base
pairs, which is postulated to encode the
nonstructural polyprotein. A second ORF
(ORF2) is separated from ORF1 by 38 nt
and consists of 1980 nucleotides. A 65nucleotide noncoding region is contiguous
ORF1 (nt 28-5106)
m7G
5′ NTR
(nt 1-27)
MT
Y PRO?
X
ORF2 (nt 5147-7126)
HEL
POL
ORF3 (nt5106-5474)
CP
An
3′ NTR
(nt 7127-7194)
Figure 3. Organization of HEV genome. The m7G cap and polyA tail are shown at the 5′ and
3′ terminus, respectively. The methyltransferase (MT), Y, protease (PRO), X, helicase (HEL),
RNA-dependent RNA polymerase (POL) and capsid (CP) domains are indicated.
10
Introduction
with the termination of ORF2 and is
terminated by a 3′ stretch of 150-200
adenosine residues. A third ORF (ORF3),
369 nucleotides in length, overlaps ORF1
by 1 nucleotide at its 5′ end and extends
328 nucleotides into ORF2. Thus, the HEV
genome consists of three ORFs and utilizes
all three of them (Tam et al., 1991).
3. 3. 1. ORF1
The functions of proteins encoded
by hepatitis E virus genome have
been predicted by computer-assisted
comparative analysis of HEV sequences
with proteins of other positive-strand RNA
viruses. Starting from the 5′ end of ORF1
the following conserved motifs have
been identified: (i) a methyltransferase,
suggesting that the 5′ end of the viral
genome is capped; (ii) an Y domain of
unknown function, found also in rubella
virus; (iii) a papain-like cysteine protease,
related to protease of alphaviruses and
rubella virus; (iv) a proline-rich region
that is thought to be important for the
flexibility of the molecule and that contains
a hypervariable sequence motif; (v) an X
domain of unknown function; (vi) an RNA
helicase possibly promoting the unwinding
of RNA required for genome replication
and transcription; (vii) an RNA-dependent
RNA polymerase (Koonin at al, 1992).
The molecular biology of HEV
replication is not well understood, mainly
because HEV does not grow efficiently
in cell culture. Much of the knowledge
concerning HEV has been obtained through
the expression of recombinant proteins in
vitro. So far, studies led to identification
of two active viral enzymes: an RNAdependent RNA polymerase (Agrawal
et al., 2001) and a capping enzyme (this
study).
Putative motive for a methyltransferse
is located in the N-terminal part of the
genome of all alphavirus-like family
members, being the hallmark of this
group of viruses (Rozanov et al., 1992).
The ability of a monoclonal antibody to
recognize 7-methylguanosine (m7G) in
RNA extracted from virions of different
HEV genotypes suggested that HEV RNA
is capped (Kabrane-Lazizi at al, 1999). The
enzymatic activity of methyltransferase
was experimentally demonstrated for Nterminal fragment of HEV polyprotein,
produced in insect cells using recombinant
baculovirus vector (I). This study describes
the properties of HEV as a capping
enzyme, which catalyzes both guanine-7methyltransferase and guanylyltransferase
activities required for capping of viral
mRNAs (see Results).
The RNA-dependent RNA polymerase
activity of HEV has been demonstrated
for a 63kDa protein, expressed in E.coli.
This recombinant HEV RdRp was able
to synthesize RNA in vitro using 3′ end
HEV RNA with poly(A) stretch as the
template in a primer-independent manner
(Agrawal et al., 2001). The polarity of the
newly synthesized product was confirmed
to be negative. The same study showed
the specific binding of HEV RdRp to the
3′ end of the viral genome. This region
with a number of stem-loop structures at
the extreme 3′ end shows a high degree
of nucleotide conservation among several
viral isolates and is believed to play
a crucial role in the initiation of viral
replication (Agrawal et al., 2001; Emerson
et al., 2001).
The replication cycles of many
positive-strand RNA animal viruses
studied include a proteolytic processing of
a polyprotein precursor by virus-encoded
proteases (Gorbalenya et al., 1991; Ropp
et al., 2000). The presence of papain-like
cysteine protease in hepatitis E virus was
shown by computer-assisted analysis,
11
Introduction
although the amino acid similarity
between the putative HEV protease and
other viral PCPs was weak (Koonin et al.,
1992). Contradictory results were reported
concerning the processing of the HEV
ORF1 polyprotein (Ansari et al., 2000;
Ropp et al., 2000; Panda et al, 2000).
We have studied this matter further (see
Results and Discussion).
3. 3. 2. ORF2
ORF2 codes for the major structural
protein (pORF2) of 660 amino acids (88
kDa). The N-terminal part of pORF2
contains N-linked glycosylation sites and
a putative endoplasmic reticulum (ER)directing signal peptide. It is synthesized
as a precursor which is cotranslationally
translocated into the ER, where it is
processed into the mature protein through
signal sequence cleavage (Jameel at al.,
1996). This protein is also glycosylated in
the ER at all three possible glycosylation
sites, which were mapped by mutational
analysis to asparagines residues (Asn137,
Asn310 and Asn562). Interestingly, the
pORF2 amino-terminal signal sequence
and all three N-linked glycosylation sites
are universally conserved in all isolates
of human HEV, suggesting functional
significance for glycosylation (Zafrullah
et al., 1999). Recent study showed that
ORF2 encoded protein has an RNA
binding activity (Surjit et al., 2004). The
ORF2 protein can interact specifically
with the 5′ end of the HEV genome.
Particularly, a 76-nucleotide region at the
5′ end of the viral RNA is responsible for
binding the ORF2 protein. Remarkably,
this 76-nt region includes the 51-nt HEV
sequence conserved among alphaviruses
(Niesters and Strauss, 1990). Structural
proteins of positive-strand RNA viruses are
known to use their RNA binding activity
for promotion of viral replication and
12
packaging of its genome into the capsid
during viral assembly. Since pORF2 is
the major capsid protein, it is the most
likely candidate to bind the genomic RNA
for its encapsidation (Surjit et al, 2004).
The ORF2 protein is also being explored
as a potential vaccine against hepatitis E
(discussed later in this study).
3. 3. 3. ORF3
Open reading frame 3 encodes a protein
of 13.5 kDa (called pORF3) with
unknown function. The ORF3 protein is
phosphorylated at a single serine residue
(Ser80) by a cellular mitogen-activated
protein kinase (MAPK) (Zafrullah et al.,
1997). Interestingly, this serine residue is
conserved among all HEV isolates with
the exception of more divergent Mexican
isolate (Huang et al., 1992). In addition,
pORF3 contains a C-terminal proline-rich
region that interacts with Src homology 3
(SH3) domains of several cellular proteins
involved in signal transduction (Korkaya
et al., 2001). Furthermore, ORF3 protein
can activate an extracellular signalregulated kinase (ERK), a member of the
MAPK superfamily (reviewed in Panteva
et al, 2003; Kar-Roy et al., 2004). The
ORF3 encoded protein has been shown
to self-associate into homodimers and to
interact with full-length, nonglycosylated
form of the capsid protein (Tyagi et al.,
2001; Tyagi et al., 2002). These results
clearly indicate that pORF3 must have
activities that involve interaction with
host cell proteins. Recent study by Tyagi
and colleagues (2004) demonstrated
that ORF3 protein specifically interacts
with α1-microglobulin/bikunin precursor
(AMBP) and enhances its export from
the hepatocytes. AMPB is a liverspecific protein that is processed into
two mature proteins, α1-microglobulin
and bikunin, which both possess general
Introduction
immunosuppressive properties (Tyagi et
al., 2004). Additionally, pORF3 associates
with the cytoskeleton and this association
requires the N-terminal hydrophobic
domain (aa 1-32) of the protein (Zafrullah
et al., 1997). The ability of pORF3 to
form a complex with the major capsid
protein pORF2 suggests that one possible
function of pORF3 within the HEV life
cycle is to serve as a cytoskeletal anchor
at which pORF2 can assemble the viral
nucleocapsid. However, the mechanisms
of viral assembly and transport are not
known at present.
3. 4. Experimental propagation,
expression and replication
The replication and transcription strategy
of HEV are poorly understood, mainly
because HEV does not replicate efficiently
in the cell culture. Nothing is known
about attachment, entry and uncoating
of the virus. The mechanisms of virus
assembly and transport from the host cell
also remain to be determined. The fact that
HEV replicates naturally in human and
monkey hepatocytes, sets limitations to
utilization of this system due to difficulties
associated with animal experimentation.
Due to the absence of cell culture system
and a small animal model, the knowledge
concerning HEV on the molecular level
has been obtained through the expression
of recombinant proteins. HEV genes have
been cloned and expressed in bacterial,
animal cell, baculoviral and yeast
expression systems (reviewed in Worm et
al., 2002).
An in vitro propagation and production
of HEV from the primary hepatocytes
of experimentally infected cynomolgus
macaques has been reported (Tam et
al., 1996, Tam et al., 1997). Both the
positive-sense and the negative strands of
HEV RNA were detected in hepatocytes,
indicating that replication proceeds via
synthesis of intermediate negative-strand
RNA, which in turn serves as a template
for synthesis of more genomic RNAs, as
well as subgenomic RNAs. At least two
subgenomic polyadenylated RNAs of
2 and 3.7 kb were identified (Tam et al.,
1991, Yarbough et al., 1991). However,
replication in this system is inefficient,
since reverse transcription-PCR is required
to monitor it. In another study, a fulllength HEV cDNA clone was constructed
and used for in vitro transcription of
RNA, which was transfected into HepG2
cells. Viral replication was detected for 6
passages (33 days) with strand-specific
PCR (Panda et al., 2000).
In vitro synthesized transcripts from
two full-length cDNA clones of the Sar55 strain (Pakistan) differing by two
nucleotides, were able to initiate infection
in rhesus monkeys and chimpanzees
after intrahepatic inoculation (Emerson
et al., 2001). Only capped transcripts
infected chimpanzees, indicating that
cap is required for viral viability. This
study demonstrated that a single mutation
located at nucleotide 7106 at the 3′ end of
the capsid gene abolished the infectivity of
a recombinant genome for rhesus monkeys
and attenuated it for chimpanzees. The
attenuation occurred even though the
mutation did not change the encoded amino
acid, suggesting that the mutation must be
located within a cis-reacting element. This
single nucleotide mutation is located in a
stem-loop structure predicted by Agrawal
et al. (2001), which is important for binding
of the RNA polymerase.
Recently, construction of HEV
replicon from the infectious cDNA clone
of the Sar55 strain has been reported
(Emerson et al., 2004b). In this replicon
the ORF2 and ORF3 genes were replaced
with the green fluorescent protein (GFP),
13
Introduction
enabling the detection of the replication
by the fluorescence microscopy. The
transfection with capped transcripts resulted
in the appearance of green fluorescent
cells at 2 to 4 days post transfection. As a
parallel experiment various cell lines were
transfected with the full-length genome, to
determine the degree of permissiveness for
HEV genomic replication. Four cell lines
out of seventeen supported the replication:
three human liver cell lines (Huh-7, HepG2
and PLC/PRF/5) and human intestinal
cell line Caco-2. Neither the replicon nor
the viral genome, were able to replicate
in any of the nonprimate cells tested,
suggesting that species-specific factors
may be required. Similar numbers of cells
were positive when transfected either with
full-length genome or with GFP replicon,
the efficiency of transfection staying
consistently under 15 %. Interestingly,
the single mutation at nt 7106 which had
attenuated Sar55 for nonhuman primates,
substantially reduced the efficiency of
replication, when introduced into the
replicon (Graff et al., 2005). Similar HEV
replicon with fused reporter genes was
constructed by Thakral and colleages
(2005). The replication of hepatitis E virus
genome in HepG2 cells was detected until
the seventh passage (50 days). Despite
numerous efforts, an efficient system to
study HEV replication still remains to be
developed.
3. 5. Vaccine candidates against
hepatitis E
There is no specific treatment for hepatitis
E infection at present. The development
of an attenuated or killed vaccine is not
currently possible, because an efficient
cell culture system to grow the virus has
not been discovered. Therefore, various
recombinant proteins have been explored as
vaccine candidates (reviewed in Emerson
14
and Purcell, 2001). ORF2 encodes the
major capsid protein, and it has been the
focus of the vaccine development. Many
different ORF2 antigens have been shown
to induce antibody, however in most of the
cases they were not neutralizing antibodies.
Only one neutralization epitope has been
identified so far. This epitope is located
between amino acids 578 and 607 of the
capsid protein (Schofield et al., 2000;
Meng et al., 2001).
The first candidate vaccine consisted
of a recombinant fusion protein comprising
tryptophan synthetase and the carboxyl
two thirds of the putative capsid protein of
Burmese strain, expressed in E. coli (Purdy
et al., 1993). Two cynomolgus monkeys
vaccinated with this protein were protected
against disease following challenge with
the homologous virus, and a third monkey
was protected when challenged with the
heterologous Mexican strain.
Truncated
recombinant
capsid
proteins expressed from baculovirus
vectors in insect cells have been studied
more extensively. A recombinant 50 kDa
ORF2 protein of Myanmar (Burmese)
strain expressed in insect cells was
secreted into the medium and assembled
into virus-like particles (Li et al., 1997).
These VLPs induced both systemic and
mucosal anti-HEV response in mice when
administered orally (Li et al, 2001). In a
recent study cynomolgus monkeys were
orally inoculated with purified VLPs. They
developed protection against infection or
developing hepatitis, when challenged
with native HEV by intravenous injection
(Li et al., 2004). These results suggest that
recombinant HEV VLPs are a significant
candidate for an oral hepatitis E vaccine
(Maloney et al., 2005).
So far, only one HEV vaccine
candidate has progressed to the stage of
clinical trials. A recombinant baculovirus-
Introduction
expressed vaccine, containing amino acids
112-607 of the 660-aa protein encoded
by ORF2 of a Pakistan strain, has been
developed at the National Institute of
Health, USA (Emerson and Purcell, 2001).
This candidate vaccine contains previously
identified immunodominant neutralization
epitope located between aa 458-607 in
ORF2 protein (Zhou et al., 2005). It has
just entered a phase II-III efficacy trial
in Nepal, where hepatitis E is endemic
(Purcell et al., 2003; Wang and Zhuang,
2004).
4. mRNA CAPPING
Eukaryotic pre-mRNAs are transcribed
by RNA polymerase II (Pol II) and
undergo several processing events
before becoming mature messenger
RNA. These modifications, including 5′
capping, splicing and polyadenylation, are
completed in the nucleus before mRNA is
transported to the cytoplasm and translated.
Cap formation occurs co-transcriptionally
during the initial stages of elongation,
when the nascent RNA transcript is only
25-30 nucleotides long (Shatkin and
Manley, 2000). Co-transcriptional capping
is facilitated by recruitment of the capping
enzyme machinery to the phosphorylated
C-terminal domain of RNA polymerase II,
a domain that serves as a “platform” for
macromolecular assemblies that regulate
mRNA synthesis and processing (Cho
et al., 1997, Shuman, 1997). The 5′ cap
A
B
(1) pppN1N2N3...
ppN1N2N3... + Pi
(2) GTP + E
GMP-E + PPi
(3) GMP-E + ppN1N2N3...
GpppN1N2N3... + E
(4) GpppN1N2N3... + AdoMet
m7GpppN1N2N3... + AdoHcy
Figure 4. Structure and synthesis pathways of the mRNA cap (modified from Gu and Lima,
2005). A) chemical structure. The cap consists of N7-methyl guanosine linked by an inverted
5′-5′ triphosphate bridge to the first nucleoside of the mRNA chain (base N can be adenine, guanine, cytosine or uracil. B) enzymatic synthesis of the mRNA cap in eukaryotic cells and DNA
viruses. See text for references and explanations.
15
Introduction
structure plays an essential role in the
mRNA cycle. It is required for accurate
and efficient splicing and nuclear export
of mRNAs (Shuman, 1995). Cap structure
is essential for translation of most
eukaryotic mRNAs, as it is recognized
by the eukaryotic initiation factor eIF4E.
Cap also increases mRNA stability by
protecting it against 5′→3′ exonucleotic
degradation (Furuichi and Shatkin, 2000).
Capping occurs by a series of three
enzymatic reactions (Fig. 4). First, the 5′
triphosphate end of the nascent pre-mRNA
transcript is hydrolysed to a diphosphate
by RNA 5′ triphosphatase (TP). Second,
the diphosphate RNA end is capped with
GMP by RNA guanylyltransferase (GT).
Finally, the GpppN cap is methylated by
RNA (guanine-N7) methyltransferase
(MT), completing the m7GpppN, or cap0,
structure (Banerjee, 1980). Methylation of
the cap generally becomes more complex
as organisms evolve. For example,
in higher eukaryotes mRNAs may be
further modified by 2′-O-methylation of
the riboses of the first and second bases
(cap1 and cap2, respectively). Transfer
of GMP from GTP to the 5′-diphosphate
terminus of RNA occurs in a two stage
reaction involving a covalent enzymeGMP intermediate. The GMP is linked
to the enzyme through a phosphoamide
(P-N) bond to the ε-amino group of a
lysine residue within a conserved KxDG
motif (reviewed in Shuman and Schwer,
1995; Shuman and Lima, 2004). Although
mRNA cap structures are conserved in
all eukaryotic organisms that have been
examined so far, the organization of
genes, subunit composition and catalytic
mechanisms of the component enzymes
differ significantly among species
(reviewed in Shuman, 2001; Gu and Lima,
2005). This diversity makes RNA capping
an attractive target for drug design.
16
4.1. Viral and cellular capping enzymes
The structures and mechanisms of the
triphosphatase and guanylyltransferase
components have been under intensive
study during the past decade, leading to
several high resolution crystal structures
of viral, fungal and mammalian capping
enzymes (Håkansson et al., 1997; Lima et
al, 1999; Changela et al., 2001; Fabrega
et al., 2003; Sawaya and Shuman, 2003).
The guanine-N7-methyltransferase is the
least understood component of the capping
apparatus. Recently, the X-ray structure of
the cellular cap methyltransferase Ecm1
in complex with AdoMet, AdoHcy and
the cap guanylate has been determined
(Fabrega et al., 2004).
Viruses adopt various types of
strategies for their parasitic replication
and proliferation in infected cells. With
a few exceptions, viral mRNAs contain
the same cap structure as cellular mRNAs
(Furuichi and Shatkin, 2000). Therefore,
viruses have played a significant role in
defining the structure of mRNA cap and
the biochemistry of cap formation.
The enzymes responsible for capping
have been initially isolated from vaccinia
virus, a large DNA virus that replicates
within the cytoplasm of the infected
cells (Martin and Moss, 1975; 1976).
Vaccinia virus mRNA capping enzyme
is a multifunctional protein with RNA
triphosphatase, RNA guanylyltransferase
and methyltransferase activities. The
protein is a heterodimer of 95 kDa and 33
kDa subunits encoded by the vaccinia virus
D1 and D2 genes, respectively (Barbosa
and Moss, 1978; Shuman and Morham,
1990). The 95 kDa polypeptide contains
both TPase and GTase, whereas the 33
kDa subunit is required for RNA (guanine7) methyltransferase activity (Shuman and
Moss, 1990). The guanylyltransferase
component of the enzyme catalyzes two
Introduction
sequential nucleotidyl transfer reactions
involving a covalent enzyme – (lysylGMP) intermediate. The active-site Lys260
residue is situated within a sequence
element KxDG, which is conserved
among guanylyltransferases, and is also
found, in addition to five other conserved
motifs, in DNA and RNA ligases (Cong
and Shuman, 1993; Shuman and Schwer,
1995). Sequence conservation suggests
that the GT-dependent capping enzymes
together with ATP- and NAD+-dependent
DNA ligases, and ATP-dependent RNA
ligases comprise a superfamily of covalent
nucleotidyltransferases, which evolved
from a common ancestral enzyme (Wang
et al., 1997). Crystal structures of five
superfamily members highlight a common
fold consisting of N-terminal nucleotidyl
transferase domain and a C-terminal OB
fold domain with typical five-stranded
antiparallel β-barrel and an α-helix
(Shuman and Lima, 2004). The 330-amino
acid Chlorella virus enzyme is the smallest
guanylyltransferase known and most
probably represents the minimal catalytic
domain (Ho et al., 1996). Crystal structure
of Chlorella virus guanylyltransferase in
GTP-, lysyl-GMP- and GpppG-bound
states revealed important conformational
changes during substrate binding and the
chemistry of the reaction, especially the
opening and closing of the cleft between
the nucleotidyl transferase and OB domains
(Håkansson et al., 1997; Håkansson and
Wigley, 1998). The capping enzyme must
FUNGI AND PROTOZOA
MT
GT
TP
S. cerevisiae
E. cuniculi
T. brucei
GT
C. elegans
Arabidopsis thaliana
TP
Chlorella virus
PLANTS AND METAZOA
TP
MT
DNA VIRUSES
MT
GT
MT
GT/TP
Poxviruses
(Vaccinia)
RNA VIRUSES
MT/GT
TP
Alphaviruses
Figure 5. Comparative scheme of mRNA capping apparatus in eukaryotes and viruses. MTmethyltransferase domain, GT-guanylyltransferase domain, GT/MT-protein or domain possessing both MT and GT activities, TP-RNA triphosphatase.
17
Introduction
be in closed conformation to catalyze
formation of the enzyme-guanylate
complex. An open guanylyltransferase
configuration is presumed to be required
at three phases of the catalytic cycle: for
GTP binding, for binding of ppRNA and
for release of the capped RNA product
(Håkansson et al., 1997). The reverse
direction of the guanylyltransferase
reaction is blocked by the addition of the
methyl group to the 5′ guanine nucleoside
by the guanine-7-methyltransferase. The
vaccinia virus enzyme prefers mRNA
substrates, but also catalyzes, albeit less
efficiently, methylation of GpppN, GTP,
dGTP, GMP and guanosine (Martin and
Moss, 1976).
Cellular capping enzymes are
larger and structurally more complex.
The enzymes that catalyze yeast mRNA
18
capping are encoded by three separate
genes, all essential for cell viability or
growth. The RNA capping enzyme of
yeast Saccharomyces cerevisiae exists as
a heterodimeric, bifunctional complex of
two polypeptides of 80 and 52 kDa. The
RNA triphosphatase is encoded by the 80
kDa subunit, whereas a 52 kDa subunit
contains a guanylyltransferase activity (Itoh
et al., 1984; Shibagaki et al., 1992). The
first metazoan gene coding for a capping
enzyme was obtained in Caenorhabditis
elegans (Takagi et al., 1997). In contrast
to the yeast capping enzyme, the metazoan
and the mammalian capping enzymes both
contain a single polypeptide with an Nterminal RNA triphosphatase domain and
a C-terminal guanylyltransferase domain
(Yue et al, 1997; Shuman, 2002).
Aims of the Study
AIMS OF THE STUDY
1) To identify and characterize the enzyme responsible for capping of hepatitis E
virus mRNA
2) To study the localization, membrane association and functions of Semliki
Forest virus non-structural polyprotein
3) To study expression, intracellular localization, membrane association and
processing of hepatitis E virus replicase
4) To review existing antiviral compounds and consider novel potential targets
19
Materials and Methods
MATERIALS AND METHODS
1. Cells and viruses
The origin and cultivation of Modified
Vaccinia Ankara virus (MVA) and HeLa
cells were described earlier (Wyatt et
al., 1995 and II). Cultivation of the
SFV prototype strain and BHK21 cells
was done as descibed by Keränen and
Kääriäinen (1974). Human hepatoma
Huh-7 cells, kindly provided by R.
Bartenschlager (University of Heidelberg,
Germany), were cultivated as described
in (Lohmann et al., 2003 and III). The
origin and preparation of HEV cDNA
constructs are described in article I. The
Bac-to-Bac Recombinant Baculovirus
system and the plasmid vector pFastBac1
(Gibco BRL) were used for the expression
of nonstructural proteins. Construction
of the recombinant baculovirus clones
expressing SFV and HEV non-structural
proteins has been described in (Merits et
al., 2001) and in article I, respectively.
Baculoviruses were cultivated in High
Five BTI-Tn-5B1-4 (Tn5) cells in High
Five medium (Invitrogen) according to
manufacturer′s instructions.
2. Bacterial strains and plasmids
Escherichia coli DH5α (Bethesda Research
Laboratories) was used as a host for
propagation of the plasmids and molecular
cloning. The recombinant proteins were
expressed in E.coli strain BL21(DE3)
(Novagen). DNA manipulations were
done according to standard procedures
described in Sambrook and Russell
(2001). The list of the plasmids used for
HEV protein expression in this study is
given in Table 1.
3. Infection and transfection
To express nonstructural proteins in HeLa
or Huh-7 cells, the cells were first infected
20
with 20 PFU of vaccinia virus (MVA)
per cell. Virus stock was diluted into
minimal essential medium supplemented
with bovine serum albumin (MEM + 0.2
% BSA). MVA-infected cells, expressing
T7 RNA polymerase, were transfected
with pSP73 plasmids. Transfections were
done in OPTIMEM® (Invitrogen) using
Lipofectamine 2000 (Invitrogen) or
ExGen500 (MBI Fermentas) transfection
reagents according to manufacturer′s
instructions. After six hours, the
transfection mixture was replaced with
cell-specific full medium and then treated
as explained in II and III. BHK cells were
infected using 50 PFU of SVF or 5-20
PFU of recombinant adenovirus per cell.
SVF- and adenovirus infected cells were
overlaid with fresh medium after removing
the virus inoculum and incubated as
specified in II. Insect cells were infected
with recombinant baculoviruses (10 PFU/
cell) in High Five medium and incubated
for 4 hours before changing medium to
fresh one. Cells were collected at 44 hours
post infection and treated as described in I.
4. Radioactive labelling and cell
fractionation
S-adenosyl[methyl- 3 H]methionine,
[3H]palmitic acid, [35S]methionine and
[α-32P]GTP (Amersham) were used as
described in I, II and III. The post nuclear
supernatant (PNS) fraction used in many
experiments was prepared as follows: cells
expressing non-structural proteins were
collected by scraping into cold phosphate
buffered saline (PBS) and pelleted at 250
x g for 10 minutes. The cell pellet was
resuspended in 5 volumes of hypotonic
buffer (10 mM Tris-HCl, pH 8.0 and 10
mM NaCl, supplemented with protease
inhibitor cocktail; Roche), left on ice for
15 min to swell and disrupted in a Dounce
homogenizer with 40 strokes. PNS was
Materials and Methods
prepared by removing the nuclei and intact
cells by centrifugation at 500 x g for 10
minutes. For some experiments PNS was
further centrifuged at 15 000 x g for 20
minutes to obtain membrane (P15) and
supernatant (S15) fractions.
5. Flotation analysis
Membrane association of nonstructural
proteins was established by flotation
assay in a discontinuous sucrose gradient
as described by Laakkonen et al. (1996).
Briefly, PNS fractions were adjusted
to a final concentration of 60 % (w/w)
and layered on top of a 67 % sucrose
cushion in polyallomer tubes. Samples
were overlaid by 50 % and 10 % sucrose
and ultracentrifuged over night at
50 000 x g, during which cytoplasmic
CONSTRUCT
pGEM3P110
pET30aHEVORF1
pBATP50
pET21aHEVX-HEL
pET21aHEVPOL
pSP73ORF1
pSP73MT496
pSP73MT712
pSP73P110
pSP73X-HEL-POL
pSP73X-HEL
pSP73POL
AcP110
AcMT361
AcMT496
AcMT589
membranes and membrane-associated
proteins float to the top of the tube. Nine
fractions were collected from the top and
immunoprecipitated with specific antisera
as described below. Precipitates were
analysed by SDS-PAGE followed by
immunoblotting or autoradiography.
To study the mode of membrane
association, the membranes from the top
fractions were pooled, sedimented and
resuspended in buffers containing 1 M
KCl, 50 mM EDTA, or alkaline sodium
carbonate at pH 11.5 or 12. Membranes
were resedimented and analyzed together
with the respective supernatants by SDSPAGE and Western blotting to see if the
treatment could release the protein from
the membranes (I and II).
DESCRIPTION
REFERENCE
aa 1-979 of HEV ORF1, pGEM3 vector, T7 promoter, prokaryotic
expression
full-length sequence of HEV ORF1, pET30a(+) vector, T7 promoter,
prokaryotic expression
aa 1-464 of HEV ORF1, pBAT vector, T7 promoter, prokaryotic
expression
aa 779-1209 of HEV ORF1, pET21a(+) vector, T7 promoter,
prokaryotic expression
aa 1179-1693 of HEV ORF1, pET21a(+) vector, T7 promoter,
prokaryotic expression
full-length sequece of HEV ORF1, pSP73 vector, T7 promoter,
eukaryotic expression
aa 1-496 of HEV ORF1, pSP73 vector, T7 promoter, eukaryotic
expression
aa 1-712 of HEV ORF1, pSP73 vector, T7 promoter, eukaryotic
expression
aa 1-957 of HEV ORF1, pSP73 vector, T7 promoter, eukaryotic
expression
aa 775-1693 of HEV ORF1, pSP73 vector, T7 promoter, eukaryotic
expression
aa 775-1206 of HEV ORF1, pSP73 vector, T7 promoter, eukaryotic
expression
aa 1206-1693 of HEV ORF1, pSP73 vector, T7 promoter, eukaryotic
expression
aa 1-979 of HEV ORF1, pFastBac1 vector, polyhedrin promoter,
eukaryotic expression
aa 1-361 of HEV ORF1, pFastBac1 vector, polyhedrin promoter,
eukaryotic expression
aa 1-496 of HEV ORF1, pFastBac1 vector, polyhedrin promoter,
eukaryotic expression
aa 1-589 of HEV ORF1, pFastBac1 vector, polyhedrin promoter,
eukaryotic expression
III
III
I
Peränen et al., 1996
III
III
III
III
III
III
III
III
III
I
unpublished
unpublished
unpublished
unpublished
Table 1. HEV constructs used in this study.
21
Materials and Methods
6. Antisera and cell-staining reagents
The preparation of polyclonal rabbit and
guinea pig antisera against SFV proteins
was described previously (Kujala et al.,
2001). The preparation and characterization
of immune antisera against HEV proteins
is described in detail (I and III). Briefly,
the proteins were produced in E.coli BL21.
After cell breakage with a French press
and centrifugation, the inclusion bodies
were washed successively with buffer W
(20 mM Tris-HCl, pH7.5, 10 mMEDTA,
1 % Triton X-100, 500 mM NaCl, 2 M
Urea) and PBS. Inclusion bodies were
resuspended in PBS containing 0.5 %
SDS. The immunization of rabbits and
guinea pigs was carried out essentially as
described previously (Kujala et al., 1999).
A variety of organelle specific markers
used for the characterization of cellular
origin of HEV replicase listed in III.
7. Immunoprecipitation and
immunoblotting
The immunoprecipitation of the proteins
with specific antibodies was performed as
described by Peränen et al., (1996). Samples
(PNS, P15, S15 fractions, cell lysate or
flotation fractions) were denatured with
1% SDS prior to immunoprecipitation and
then incubated overnight in the presence
of antibody in NET buffer (50 mM TrisHCl, pH 8, 150 mM NaCl, 1 % NP-40)
at +4 °C. For enzymatic studies PNS was
concentrated by immunoprecipitation
under native conditions.
For immunoblotting, proteins were
first separated in SDS-PAGE gel and
subsequently electrotransferred to a
nitrocellulose membrane (Hybond-Cextra, Amersham). Filters were blocked
with 5% milk powder in PBS supplemented
with 0.1 % Tween 20 (PBST) and probed
with specific antibody in the same buffer.
PBST was used for the washing and during
22
incubation with secondary antibody (antirabbit horseradish peroxidase, DAKO).
The proteins were visualized using
enhanced chemiluminescence (ECL)
detection system (Amersham).
8. Enzyme assays
Guanine-7-methyltransferase
activity
was assayed as described in detail in
I. The enzyme source, PNS from cells
expressing nonstructural proteins, was
immunoprecipitated under non-denaturing
conditions using relevant antibody before
assays. As a substrate we used GTP,
and as a methyl donor we used AdoMet
labeled with tritium in the methyl group
(S-adenosyl[methyl-3H]methionine). The
formation of a covalent guanylate complex
was detected using [ -32P]GTP in the
presence of unlabelled AdoMet and GTP.
NTPase and RNA triphosphatase assays,
used to study the enzymatic activities of
ns polyproteins in II, were performed as
described by Vasiljeva et al., 2000. Cell
lysate, used as an enzyme source, was
immunoprecipitated using relevant antinsP antibody before the assay.
9. Immunofluorescence microscopy
For the microscopic analysis cells were
grown over night on glass coverslips. HeLa
cells were transfected with plasmids in
combination with MVA infection (II, III)
or infected with SFV or with adenoviruses
(II). Huh-7 cells, used to study HEV
proteins, were transfected using MVA-aided
transfection system (III). Mock-infected
and/or untreated cells were included as
controls in all experiments. Cells infected
with SFV or transfected using MVA were
fixed with 4 % PFA at 5 and 6 hours p.i,
respectively (II and III). Expressions
based on recombinant adenoviruses were
fixed after 24-28 hours of incubation
(II). Cells were permeabilized using
Materials and Methods
0.1 % Triton X-100 before subjecting
to the antibody treatments. For inderect
immunofluorescence the cells were
stained with anti-nsP (II), with anti-HEV
antibodies (III) or in combination with
organelle-specific markers. This was
followed by treatment with species-specific
secondary antibodies conjugated to either
red-fluorescent Alexa Fluor 568 or greenfluorescent Alexa Fluor 488 (Molecular
Probes). The samples were analyzed with
scanning confocal microscopy (Leica
Microsystems).
23
Results
RESULTS
1. The RNA capping activity of HEV
The 5′ end of eukaryotic messenger RNA
molecule contains a m7G(5′)ppp(5′)N cap
structure, which is required for the efficient
initiation of translation and stability of
mRNA. It has been predicted earlier that
HEV belongs to the alpha-like superfamily
of viruses, which contain a conserved
methyltransferase or capping domain
(Rozanov et al., 1992). Later, the presence
of cap at the 5′ end of HEV RNA has been
verified experimentally (Kabrane-Lazizi
at al, 1999).
To study the capping reactions of
hepatitis E virus, we have used constructs
derived from the HEV Burma strain (I). A
protein of 50 kDa (aa 1-464 of ORF1) was
expressed in E.coli and used for production
of HEV-specific antibody (designated
α-MT). Since this protein was easily
aggregated during expression in E.coli,
a recombinant baculovirus expression
system was used in further experiments.
An N-terminal fragment of HEV ORF1
protein (aa 1-979) was expressed in Tn5
insect cells. The obtained protein of
110 kDa was tested for the presence of
predicted guanylyltransferase activity.
The flotation-purified membranes from
Tn5 cells expressing HEV P110 were
incubated with [α-32P]GTP followed by
immunoprecipitation with anti-HEV
antibody, as described in Materials and
Methods. The complex was detected as a
radioactive band, but only in the presence
of AdoMet. Two bands were visualized
after immunoprecipitation: one of 110kDa designated as P110 and another band,
presumably a proteolytic product of P110,
with an apparent molecular mass of 80 kDa
(P80) (I, Fig.4). The complex formation
was specific for guanine nucleotides,
GDP being the best substrate, followed by
24
GTP. Tritium-labeled methyl group from
was
S-adenosyl[methyl-3H]methionine
found in the covalent enzyme-guanylate
complex, indicating that complex formation
took place between P110 and m7GTP (I,
Fig.5). No incorporation of methyl group
to ATP, CTP or UTP was detected. In
addition to AdoMet, the reaction required
the presence of divalent cations (Mg2+ and
Mn2+) (I, Fig.3). An inhibitory activity of
several cap analogs on P110 was tested
and, as expected, both m7GTP m7GDP
had inhibited the incorporation of methyl
group from AdoMet to GTP. The reaction
was not inhibited by treatment with
deoxycholate or Triton X-100. Therefore,
immunoprecipitation in the presence of
detergents was used to obtain significantly
pure preparation of enzymatically active
P110 (I, Fig.5). Based on these results,
we propose an order of HEV capping
reactions:
(1) RNA triphosphatase
pppN1N2N3…→ ppN1N2N3…+ Pi
(2) Guanine-7-methyltransferase
GTP + AdoMet → m7GTP + AdoHcy
(3) RNA guanylyltransferase
m7GTP + P110 → m7GMP-P110 + PPi
(4) RNA guanylyltransferase
m7GMP-P110 + ppN1N2N3…→
m7GpppN1N2N3…+ P110
The capping enzyme of HEV first acts
as a methyltransferase, catalyzing the
transfer of a methyl group from Sadenosylmethionine to GTP to yield
m7GTP. It then acts as a guanylyltransferase,
transferring the methylated guanosine
to mRNA via a covalent complex. Both
capping reactions are virus-specific and
differ from those described for eukaryotic
Results
cells, but resemble those of the alphaviruslike superfamily (see Discussion).
To localize the activities more
precisely, three shorter constructs,
designated as MT361 (aa 1-361), MT496
and MT589, were made and produced in
insect cells with recombinant baculovirus
vectors (Fig. 6). The shortest construct did
not show any enzymatic activity, whereas
MT496 and MT589 had similar properties
as P110. Thus, sequence required for HEV
methyltransferase and guanylyltransferase
activities extends beyond the N-terminal
361 amino acids.
A
P110
MT369
MT496
MT589
IgG
B
Figure 6. Formation of enzyme-guanylate
complex in Tn5 insect cells with Ac-HEV. A)
Coomassie blue staining after immunoprecipitation of P15 fraction from baculovirus infected Tn5 cells. B) Covalent complex formed
after incubation of immunoprecipitated P110,
MT496, MT589 with [α-32P]GTP in the presence of 100 μM of AdoMet.
2. Membrane association of HEV capping
enzyme
Membrane association of the N-terminal
fragments of HEV ORF1 was studied
by a flotation assay, which separates
membranes from soluble and aggregated
components (see Materials and Methods).
All N-terminal fragments of HEV ORF1:
P110, MT361, MT496 and MT589 were
expressed by recombinant baculovirus
vector. All four proteins were associated
with the cytoplasmic membranes floating
to the top of the gradient (Fig. 7). This
result suggests that the membrane-binding
region is located within N-terminal 361
amino acids, a region corresponding to
alphavirus nsP1.
To probe the mode of membrane
association, the P15 fraction or the
floated membrane fraction from Tn5
cells expressing P110 was subjected to
various treatments. HEV P110 was not
released by high-salt or EDTA treatments,
which releases most peripheral membrane
proteins. P110 was quite tightly associated
to the membranes and was only partially
released by alkaline solutions at pH 12
(data not shown). There was no evidence
for palmitoylation in labelling experiments,
indicating that this membrane association
was not due to the palmitoylation of P110,
as is the case for alphavirus nsP1 protein.
Membrane association of the HEV
replicase was also studied using an in
vitro translation system. No significant
binding was detected, when HEV ORF1
and its truncated N-terminal derivatives
were translated in the presence of
liposomes containing anionic membrane
phospholipids (data not shown).
25
Results
3. Intracellular distribution and proteolytic processing of HEV nonstructural
polyprotein
To study the proteolysis of HEV
nonstructural polyprotein we made a
plasmid expressing the full-length ORF1
under the control of T7 promoter. HEV
ORF1 was expressed in vitro using
coupled transcription/translation system.
A 186 kDa protein, the predicted size of
the ORF1 primary translation product,
was present after 30 minutes and was not
increased significantly during 90 minutes
time course (III, Fig. 2). Two other faint
bands of approximately 130 and 56 kDa
were observed in addition to the full-size
translation product. Immunoprecipitation
with anti-HEV specific antibodies
revealed that upper band of 130 kDa
was an N-terminal fragment of HEV
ORF1 and a lower band (56 kDa) was a
C-terminal fragment (III, Fig. 2). The
intensity of these two bands was variable
1
2
3
4
5
6
7
in different experiments, and addition of
cycloheximide or divalent cations did
not have any effect on the course of the
reaction (III, Fig. 3).
The expression of HEV ORF1 in
vivo was carried out using a recombinant
vaccinia virus-aided transfection. One
strong band of 186 kDa was observed after
a standard labelling period (1 to 4 hours)
done 7 hours posttransfection (III, Fig.
4). A number of lower faint bands were
also detected. One of them was identified
by the immunoprecipitation as ~120 kDa
N-terminal fragment of HEV ORF1. The
result was reproduced in two cell lines:
HeLa and Huh-7 human hepatoma cells.
Prolonged labelling period (24 hours)
did not have any effect on the expression
pattern.
Localization of HEV nonstructural
polyprotein inside the cell was
studied by subcellular fractionation,
immunofluorescence analyses and double8
9
P110
10 % sucrose
MT589
50 % sucrose
MT496
sample ~60 % sucrose
67 % sucrose
MT361
Figure 7. Membrane association of HEV capping enzyme. Post-nuclear supernatant was
subjected to flotation in discontinuous sucrose gradient. Fractions were collected from the top
and subjected to SDS-PAGE followed by immunoblotting with α-MT antibody. The top of the
gradient is on the left.
26
Results
label confocal laser scanning microscopy
(see Materials and Methods). Biochemical
studies have shown that an N-terminal
fragment HEV P110 expressed in insect
cells was tightly membrane-associated
and present in the mitochondrial pellet
fraction P15 (I). Subsequent flotation in a
discontinuous sucrose gradient confirmed
tight membrane association of HEV P110.
Similar result was obtained with a shorter
fragment MT496 expressed under the
same conditions (this study). When the
full-length ORF1 was expressed in HeLa
cells via vaccinia virus-aided transfection,
the major pool of the polyprotein was also
found associated with P15 membranes
(III, Fig. 5). Further experiments revealed
that more than half (54 %) of the protein
was floating with the membranes in a
sucrose gradient. Immunofluorescense
microscopy with HEV-specific antibodies
confirmed membrane association of HEV
polyprotein. The full-length pORF1 was
concentrated in the perinuclear region of
the cell and showed a punctate distribution
(III, Fig. 6). Double-staining with antiHEV antibodies in combination with
organelle-specific markers was performed
to explore the subcellular localization
of pORF1 in more detail. HEV pORF1
was found in close proximity to Golgi
apparatus, but did not colocalize with
the cis-Golgi matrix protein GM130 (III,
Fig. 6). No colocalization was observed
with ER markers PDI (protein disulfide
isomerase) and BAP. A significant amount
of pORF1 was found closely interspersed
with staining of the ERGIC-53, a marker
for the endoplasmic reticulum-Golgi
intermediate compartment, but colocalized
only to a small extent. Similar results were
obtained with a shorter fragmet P110 (aa
1-957). The N-terminal fragment MT496
appeared as small globular masses (III,
Fig. 6). The protein was distributed
throughout the cytoplasm with slightly
greater accumulation around the nucleus.
All three C-terminal fragments X-HEL,
POL and X-HEL-POL showed a similar
localization pattern and were dispersed
evenly throughout the cytoplasm. These
results suggest that a specific localization
signal, which directs the polyprotein to a
restricted region in the cell, is located in
the N-terminal part of the pORF1.
4. Membrane association and subcellular
localization of SFV nonstructural
polyprotein
To study the role of the P1234 polyprotein
and its cleavage intermediates in the
membrane association and targeting of the
SFV replication complex, we expressed
the polyprotein precursor and its cleavage
products (P123, P12, P23 and P34) as
well as individual nonstructural proteins
nsP1 to nsP4 in insect and mammalian
cells (II, Fig.2). To prevent the processing
of the polyprotein, we created constructs
for expression of uncleaved nonstructural
polyproteins by mutating the active-site
cysteine 478 of nsP2 protease domain
to alanine. The uncleaved polyproteins
(designated P12CA34, P12CA3, P12CA
and P2CA3) had properties previously
identified for the individually expressed
nsPs, suggesting their proper folding
in the precursor state. Flotation assays
showed that uncleaved P12CA34, P12CA3,
P12CA behaved similarly to nsP1 expressed
alone, whereas P2CA3 and P34 did not
float, indicating that presence of nsP1 is
required for the membrane association
of the polyprotein (II, Fig. 3). The
tight binding of the polyproteins to the
membrane was due to the palmitoylation.
The polyproteins P12CA34, P12CA3 and
P2CA3 were also phosphorylated, like
nsP3 (II, Fig. 3). The polyproteins, which
contained nsP1 and nsP2 had enzymatic
27
Results
activities specific for authentic nsPs. We
have shown previously that nsP1 possess
a unique methyltransferase activity,
methylating GTP at position 7 of the
guanine to produce m7GTP. The same
protein acts also as a guanylyltransferase,
which forms a covalent complex m7GMPnsP1 in the presence of AdoMet and [α32
P]GTP (Ahola and Kääriäinen, 1995).
Polyproteins P12CA34, P12CA3, P12CA, all of
which contain nsP1, had methyltransferase
as well as guanylyltransferase activity (II,
Fig. 4). Polyproteins P12CA34, P12CA3,
P12CA and P2CA3 had NTPase and RNA
triphosphatase activities typical for nsP2.
To study the role of nonstructural
proteins in the intracellular targeting of
the replication complex, the wild type
polyproteins and their uncleaved forms
were expressed in Hela cells. Expression
of wild type P12 resulted in separation
of nsP1 and nsP2. NsP1 was found at
the plasma membrane and filopodia-like
extensions, whereas nsP2 was transported
to the nucleus (II, Fig. 5). In contrast,
expression of uncleavable P12CA resulted
in the accumulation of the protein at
28
the plasma membrane and filopodia,
indicating that the effect of nsP1 overrides
the nuclear targeting of nsP2 domain in
P12CA. Expression of P123 resulted in the
dissociation of nsPs from each other. NsP1
was localized at the plasma membrane,
nsP2 was transported to the nucleus, and
nsP3 was found in the cytoplasmic vesiclelike structures. A different staining pattern
was seen for P12CA3 (II, Fig. 5). The
polyprotein was localized in large vesicle
structures under the plasma membrane.
Co-staining with anti-lamp-2 showed that
some of these vesicles are of endosomal
origin. No staining of filopodia similar to
those in P12CA –expressing cells was seen.
The localization of P12CA34 was similar to
that shown for P12CA3. Thus, the presence
of the nsP3 domain in the nonstructural
polyprotein directed it to a new intracellular
localization. These results suggest that
nsP1 alone is responsible for membrane
association of the polyprotein, whereas
nsP3 together with nsP1 enables targeting
of the replication complex to the surface
of the endolysosomal vesicles.
Discussion
DISCUSSION
1. A unique cap formation pathway
specific for alphavirus-like superfamily
Viruses that replicate in the cytoplasm and
are unable to use the nuclear transcription
machinery of the host cell have developed
their own transcription and capping
systems. Although the structure of mRNA
cap is identical, a variety of RNA viruses
have diverse pathways for cap formation.
It has been previously shown that both
Sindbis virus and Semliki Forest virus nsP1
have guanine-7-methyltransferase activity
(Mi and Stollar, 1991; Laakkonen et al.,
1994). Next, it was demonstrated that this
activity preceded a guanylyltransferase
reaction, where nsP1 formed a covalent
enzyme-guanylate complex (Ahola and
Kääriäinen, 1995). These two reactions
were part of a virus-specific capping of the
mRNA. Since hepatitis E virus replicase
proteins have a distant homology with those
of alphaviruses, we wanted to test whether
they share a similar capping mechanism.
As a result of the present work, a unique
mRNA capping pathway that is specific
for hepatitis E virus and other members of
the alphavirus-like superfamily has been
characterized. We propose a following
order of capping reactions catalyzed by
viral enzymes, encoded by alpha-like
viruses. 1) GTP and AdoMet bind to the
enzyme at close proximity to each other.
2) The methyl group from AdoMet is
transferred to GTP resulting in formation
of m7GTP and AdoHcy. 3) The covalent
intermediate enzyme-m7GMP complex
is formed via phosphoamid bond with an
unidentified amino acid. 4) The m7GMP is
transferred to the 5′ end of nascent RNA,
whose γ-phosphate has been removed
by RNA-triphosphatase activity, to form
the 5′ cap structure. This distinctive
mRNA capping reaction is catalyzed by
a group of distantly conserved AdoMetdependent
guanylyltransferases
and
methyltransferases that were identified
in Semliki Forest virus (Ahola and
Kääriäinen, 1995), tobacco mosaic virus
(Merits at al., 1999), brome mosaic virus
(Ahola and Ahlquist, 1999), bamboo
mosaic virus (Li et al., 2001) and hepatitis
E virus (this study).
Semliki Forest virus replicase protein
nsP1 possess both activities required for cap
formation: guanine-7-methyltransferase
and guanylyltransferase (Ahola and
Kääriäinen, 1995). Nsp1 is able to form
a covalently bound enzyme-guanylate
complex typical for guanylyltransferase.
The complex consists of m7GMP, rather
than GMP linked to nsP1. The synthesis
of the alphavirus-specific complex
differs from the previously characterized
guanylyltransferases - GMP complexes
in its absolute requirement for a methyl
donor S-Adenosylmethionine.
Methyltransferase
activity
and
covalent complex formation exclusively
with m7GMP have been demonstrated
for replicase protein p126 of tobacco
mosaic virus (Merits et al., 1999). TMV
p126 is able to synthesize m7GTP using
S-Adenosylmethionine as the methyl
donor and catalyze the formation of
covalent guanylate-p126 complex in
the presence of AdoMet. Similar virusspecific guanine-7-methyltransferase and
covalent m7GMP binding activities were
shown for brome mosaic virus protein
1a (Ahola and Ahlquist, 1999). Analysis
of reaction products by nuclear magnetic
resonance spectroscopy demonstrated
that methylation takes place only on the
7-position of guanylate. The N-terminal
442 amino acids of a 155kDa replicase of
bamboo mosaic virus have been postulated
to encompass an mRNA capping enzyme
(Li et al., 2001). An AdoMet-dependent
29
Discussion
guanylyltransferase activity and critical
residues required for the capping reaction
were characterized (Huang et al., 2004;
Huang et al., 2005).
NsP1-related
proteins
encoded
by members of the alphavirus-like
superfamily do not contain a conserved
lysine residue, which forms covalent
linkage with GMP in cellular enzymes.
Instead, an absolutely conserved histidine
(His38 for SFV, His81 for TMV, His80 for
BMV, His68 for BaMV and His64 for HEV)
(Fig. 8) is a good candidate for covalent
binding with m7GMP, since the mutation
of this residue completely abolished GT
reaction without destroying MT activity
(Ahola et al., 1997; Ahola and Ahlquist,
1999; Merits et al., 1999; Huang et al.,
2004). Together with other characteristics
such as their membrane-association nature
and distinctive protein primary structures,
this group of methyltransferases and
guanylyltransferases represents a novel
mRNA capping enzyme different from
those of DNA viruses, metazoans and
fungi. It is likely that all members of
the alphavirus-like superfamily share
a similar conserved mechanism of cap
formation. Since it differs from cellular
RNA capping, it offers a target for virusspecific inhibitors of RNA replication, as
discussed in IV.
2. Proteolytic processing of HEV
nonstructural polyprotein
The majority of positive-strand RNA
viruses studied so far encode proteases
HEV 60 EVFWNHPIQRVIH
SFV 33 VTPNDHANARSFS
BMV 75 QYHAPHSLAGALR
TMV 76 TQNAVHSLAGGLR
BaMV 63 TKLHTHAAAKALE
30
that mediate viral polyprotein processing.
However, an extensive search in the
HEV polyprotein sequence for segments
resembling all known types of viral and
cellular proteases revealed no significant
similarity. The only region with very
modest conservation was found among
HEV polyprotein and putative cysteine
papain-like proteases (PRO) of rubella
virus and a plant benyvirus (Koonin at al,
1992).
At present, the manner in which
processing of the HEV ORF1 polyprotein
into individual proteins occurs is not
known. The proposed papain-like cysteine
protease is presumed to play role, but host
cell proteases may also be involved in the
process. The papain-like cysteine protease
is characteristic of alphaviruses and is also
found in members of other plus-strand
RNA virus families. Two residues, Cys481
and His558, were shown to be members
of the catalytic dyad in alphaviruses
(Strauss et al., 1992). The mutagenesis of
corresponding residue Cys478 in Semliki
Forest virus nsP2 abolished the proteolysis
of nonstructural polyprotein (Merits et
al., 2001). In addition to alphaviruses,
rubella virus and a beet necrotic yellow
vein virus utilize the papain-like cysteine
protease in the processing of their
nonstructural polyproteins. In rubella
virus the nonstructural ORF codes for a
240 kDa polyprotein, which is cleaved by
a virus-encoded protease to yield a 150
kDa N-terminal product and a 90 kDa Cterminal product. When cysteine residue
Figure 8. Amino acid sequences encompassing the conserved histidine residue of several
capping enzymes within members of alphavirus-like superfamily. HEV, SFV, BMV, TMV
and BaMV are abbreviations for Hepatitis E
virus, Semliki Forest virus, Brome mosaic virus, Tobacco mosaic virus and Bamboo mosaic
virus, respectively.
Discussion
(Cys1151) proposed as the catalytic residue
of this protease was mutated to a Gly, only
the full-length polyprotein was produced
(Marr et al., 1994). The BNYVV RNA1
contains a single ORF that encodes for a
220 kDa nonstructural polyprotein, which
is autocatalytically processed into a150
kDa N-terminal protein and a 60 kDa Cterminal protein (Hehn et al., 1997).
Although the presence of papainlike cysteine protease in hepatitis E virus
has been predicted by computer-assisted
analysis, the amino acid alignment
between the putative HEV protease and
other viral proteases was weak (Koonin et
al., 1992). The catalytic residues predicted
by computer alignment for HEV protease
are Cys483 and His590. The cysteine residue
is conserved among all HEV isolates,
whereas His590 is not (Ropp et al., 2000).
In Myanmar strain of HEV a tyrosine is
found at position 590 and in Mexico strain
this residue is a leucine. Thus, His590 is
unlikely to be a catalytic residue. In the
study by Ropp and colleagues, the fulllength HEV ORF1 was expressed in vitro
using a coupled transcription/translation
system and in vivo using a vaccinia virusaided transient expression system. Both in
vitro and in vivo expression under standard
conditions yielded only the full length 185
kDa polyprotein. However, prolonged
expression in vivo (24-36 hours) led to
two potential processing products: an Nterminal 78 kDa fragment containing the
MT and PRO motifs and a C-terminal
protein with the X, HEL and POL domains.
The same result was observed in all four
cell lines used in the experiment (HeLa,
Vero, HepG2 and Chang Liver cells). The
site-specific mutagenesis of the Cys483
residue failed to abolish this cleavage,
suggesting that either HEV polyprotein is
not processed or it is cleaved at one site
by a virus-encoded protease novel among
alphavirus-like superfamily members.
There is also a possibility that the cleavage
observed after prolonged expression was
mediated either by vaccinia virus vector or
by a cellular enzyme.
Another study demonstrated that
no proteolytic processing of HEV
nonstructural polyprotein (~ 186 kDa) was
detected when only ORF1 was expressed
in E. coli, in a cell free translation system
or in HepG2 cells (Ansari et al., 2000).
However, when the full-length genome
of hepatitis E virus was expressed in the
HepG2 cells, the presence of viral proteins
pORF2, pORF3 and processed components
of the ORF1 polyprotein was detected
(Panda et al., 2000). In this experiment
no ORF1 size protein was seen. Instead
anti-polymerase antibody precipitated
a protein with a molecular weight of 36
kDa. Additionally, 35 and 38 kDa proteins
were detected by anti-methyltransferase
and anti-helicase antibodies, respectively.
Our studies of proteolytic processing
of HEV pORF1 suggest that the predicted
papain-like cysteine protease either is not
active or does not exist. In this respect HEV
is similar to plant potexviruses, which
express a single polyprotein responsible for
methyltransferase, helicase and polymerase
functions. The absence of protease
within HEV nonstructural polyprotein
is not characteristic for alphavirus-like
superfamily, since other animal virus
members (alphaviruses and rubella virus)
use papain-like cysteine protease to
catalyze cleavage of their polyproteins.
However, our results obtained from the
study of Semliki Forest virus polyprotein
indicate that it does not have to be fully
processed to mediate viral replication. In
fact, the uncleaved polyprotein possessed
enzymatic activities and other properties
specific for the individual nonstructural
proteins.
31
Discussion
3. Membrane association and intracellular
distribution of HEV and SFV replicases
Compared to the alphaviruses our
current knowledge of hepatitis E virus
nonstructural proteins is limited and
based mainly on the sequence homology
analyses (Koonin and Dolja, 1993). It is
known that all positive-strand RNA viruses
replicate in the cytoplasm of the infected
cells in association with various organellespecific membranes. The membranes
play a structural and functional role by
offering a suitable microenvironment for
viral RNA synthesis and facilitate the
recruitment of membrane-associated host
factors required for the viral replication. In
many cases, viral infection has a dramatic
effect on the structure and function on
the host cell membrane system. Different
families of positive-strand RNA viruses
appear to carry out RNA replication at
different membrane sites or compartments
of the cellular secretory and endocytic
pathways. Alphaviruses and rubiviruses
replicate in cytoplasmic structures defined
as cytopathic vacuoles type I (CPV-I),
which morphological features are quite
well characterized by immunofluorescence
and electron microscopy studies (Grimley
et al., 1968; Froshauer et al., 1988). CPVs
are modified endosomes and lysosomes
with a diameter of 200 to 1 000 nm. The
cytosolic surface of CPVs consists of small
invaginations with a diameter of about 50
nm, which have been designated spherules
(Froshauer et al., 1988). Previously
it has been demonstrated that all four
nonstructural proteins of Semliki Forest
virus were associated with spherules
together with nascent RNA, indicating
that they are the sites of synthesis of plusstrand RNA molecules (Kujala et al.,
2001). In the present study we have also
shown that nsP1 alone is responsible for
the membrane association of the replication
32
complex, whereas the P123 intermediate
is required for targeting to endosomes and
lysosomes.
Several plant viruses from the
alphavirus-like superfamily replicate at
the ER-derived membranes. For example,
tobacco mosaic virus replication takes
place in large structures, which originate
from the ER and involve association
with microfilaments, microtubules and
virus-encoded, heavily phosphorylated
movement protein (Mas et al., 1999).
Brome mosaic virus RNA replication
complexes colocalize with the ER markers,
but not with the markers of Golgi or later
compartments of the cellular secretory
pathway (Restrepo-Hartwig and Ahlquist,
1996). These macromolecular complexes
are generated early during infection and
consist of viral RNA, replicase proteins,
host cell-derived membranes and host
cell proteins. Studies of individual
nonstructural proteins, the components
of the RNA replicase, have revealed that
the replication complexes are associated
with the membranes and targeted to the
respective organelle by the ns proteins
rather than by RNA (Salonen et al.,
2005). Results obtained from our studies
on membrane association of hepatitis E
virus nonstructural polyprotein suggest
that a localization signal, which directs the
polyprotein to a specific membrane region
in the cell, is located in its N-terminal
part. By use of immunofluorescence
and confocal scanning microscopy, we
observed that HEV replicase was localized
in the perinuclear area of the cell and
showed a reticular/punctate distribution.
This staining pattern suggests that hepatitis
E virus replicase polyprotein is most
probably localized on the endoplasmic
reticulum (ER) or ER-derived membranes.
In this respect HEV is similar to another
causative agent of viral hepatitis, hepatitis
Discussion
C virus, a positive-strand RNA virus from
the Flaviviridae family. The developmemt
of effective replicon system enabled
characterization of HCV replication
complex in detail (Lohmann et al., 1999).All
nonstructural proteins of hepatitis C virus
were associated with the ER membranes,
indicating that ER compartment is the
site of membrane-associated HCV
RNA replication (Mottola et al., 2002).
Furthermore,
electron
microscopy
revealed the loss of the ER organization
and appearence of morphological
alterations such as convoluted ER
cisternae and paracrystalline structures.
The development of effective cell culture
system, which supports replication of
hepatitis E virus, should allow us to
achieve deeper level of understanding of
events during viral replication.
4. Inhibitors of viral replication
Viral hepatitis is the most common
inflammatory liver disease with millions
of new cases emerging worldwide every
year. The disease is caused by one of five
primarily hepatotropic agents that have
been designated the hepatitis viruses A,
B, C, D and E (Chen, 2003). Although
the acute disease caused by these five
viruses is clinically undistinguishable, the
agents themselves are totally different,
belonging to five different virus families.
Hepatitis viruses A and E are spread
via the enteric route and usually cause
a self-limiting hepatitis followed by
complete recovery. Viruses B, C and D
are transmitted by parenteral or mucosal
exposure to infected blood. They are often
associated with chronic liver disease that
can lead to the development of cirrhosis
and hepatocellular carcinoma. Currently,
two therapeutic agents are used for the
treatment of the hepatitis B infection:
antiviral immunomodulator interferon-
α and lamivudine, an inhibitor of DNA
polymerase and reverse transcriptase
(Lok and McMahon, 2001). Alternatively,
adefovir dipivoxil has been used for
treatment of lamivudine-resistant chronic
hepatitis B infection (Ocama et al., 2005).
Effective immunization against hepatitis
viruses A and B is now available. One
vaccine candidate against hepatitis E
infection has progressed to the stage of
clinical trials and is currently evaluated in
Nepal.
Despite numerous efforts, no vaccine
is available against hepatitis C virus.
Problems in the development of new antiHCV therapies include the persistence
of the virus, genetic diversity during
replication in the host and the development
of drug-resistant mutants. Major research
efforts have been aimed primarily at
specific elements and enzymes, which
mediate viral replication, with viral
protease and polymerase being the most
attractive targets. Several peptidomimetric
protease inhibitors are at different stage of
development. However, RNA-dependent
RNA polymerase remains to be the most
popular drug discovery target. Several
nucleoside analogues that inhibit synthesis
of viral RNA were identified. They act
as chain terminators upon incorporation
into the nascent RNA molecule by viral
polymerase. Furthermore, different class
of agents that target the same viral enzyme
were characterized. These non-nucleoside
inhibitors block the polymerase, preventing
a conformational transition required for
initiation of RNA synthesis (De Francesco
and Migliaccio, 2005).
However, in spite of encouraging
results, the current therapy for hepatitis
C virus still remains pegylated interferon
in combination with ribavirin (Moradpour
et al, 2002). Since its discovery in
1972, ribavirin has been used against a
33
Discussion
broad range of RNA and DNA viruses.
Ribavirin is a synthetic purine nucleoside
analogue with a structure closely related
to guanosine. A number of possible
mechanisms of action were proposed for
ribavirin. For instance, it has been shown
to inhibit host cell inosine monophosphate
dehydrogenase, an enzyme that catalyzes
a rate-limiting step in synthesis of GTP.
This leads to a decreased pool of GTP
levels in the cell, indirectly affecting
the transcription of viral genomes and
replication of RNAviruses (Tan et al., 2002).
Ribavirin has been also shown to have an
inhibitory effect on viral polymerases by
competitively inhibiting the binding of the
nucleotides. Incorporation of ribavirin into
viral RNA drives the viruses beyond the
critical mutation rate, thus forcing viruses
into “error catastrophe” (Crotty et al.,
2001). It has been previously suggested
that ribavirin has a negative effect on
the capping of viral RNA transcripts
(Goswami et al, 1979). Interestingly, the
mutations in the Sindbis virus genome that
confer resistance to ribavirin were mapped
to the RNA guanylyltransferase encoding
region (Scheidel and Stollar, 1991).
Recent study demonstrated that ribavirin
triphosphate can be used as a substrate by
the vaccinia virus RNA capping enzyme
and can form a covalent RMP-guanylate
complex analogous to the classical GMPenzyme intermediate. Furthermore, it can
be transferred to the diphosphate end of
an RNA transcript to form the unusual
RpppN structure (Bougie and Bisaillon,
2004). This study showed that the RNA
transcripts blocked with ribavirin could
not be methylated and therefore, were not
recognized by the translational machinery,
leading to a decreased protein synthesis.
Thus, ribavirin can directly interfere with
viral RNA capping. Since cap structure
is essential for mRNA translation and
34
stability, viral enzymes involved in cap
formation are attractive targets for the
development of antiviral drugs (IV).
A number of cap analogs have been
developed in recent years. In the study by
Lampio and colleagues (1999) over 50
guanosine analogs were tested for their
effect on guanine-7-methyltransferase and
guanylyltransferase activities of Semliki
Forest capping enzyme nsP1. The most
effective inhibitors of SFV capping enzyme
contained a methyl group at position 7 of
the guanine ring and one methyl or ethyl
group at the position 2. Many cap analogs
tested in this study appeared to be efficient
inhibitors of translation by competing with
the 5′ cap of mRNAs for the binding to
initiation factor eIF4E. Our current study
described inhibitory effect of several cap
analogs on the capping activity of hepatitis
E virus enzyme. As expected, m7GTP and
m7GDP were efficient inhibitors of the
HEV methyltransferase activity, which
is in accordance with the properties
of GTP and GDP as substrates for the
methyltransferase (I). Interestingly, the
double-substituted guanylate analogs were
less efficient inhibitors. It is likely, that
the phosphorylation status of the analogs
is more important than the substitutions
of the guanylate moiety. The present
work provides a novel approach for
development of specific inhibitors against
hepatitis E infection. As described above,
the RNA capping pathway is conserved
among all members of the alphavirus-like
superfamily. Therefore, it may be possible
to design compounds with broad-range
antiviral properties.
Indeed, promising results with RNA
capping inhibitors were obtained very
recently in experiments with human
respiratory syncytial virus (RSV), a
negative-strand RNA virus (Liuzzi et
al., 2005). In the case of RSV, viral
Discussion
polymerase is responsible not only for the
synthesis of both genomic and subgenomis
mRNAs, but also for cotranscriptional
capping and polyadenylation of mRNAs
5′ and 3′ ends, respectively. In this study
compounds that block synthesis of RSV
mRNAs by preventing the guanylation of
viral transcripts were characterized. It was
shown that this class of RSV polymerase
inhibitors increased the appearance of
short uncapped RNA transcripts. Based
on the results, it has been proposed that
the inhibitors prevented cotranscriptional
capping of viral mRNAs required for
full elongation of transcripts. Thus,
these inhibitors represent a novel class
of nonnucleoside antiviral agents with a
unique mechanism of action.
Another potential target for antiviral
research is the membrane association
of replication complexes, which is
common to all positive-strand RNA
viruses. The membrane association
provides a structural framework for
replication process and offers protection
for the viral RNA molecules against host
defence mechanisms (Salonen at al.,
2005). Different viruses utilize various
intracellular membrane compartments,
causing diverse modifications and
rearrangements of the target membrane.
The membranes can be derived from the
ER, mitochondria, chloroplasts, or from
the endo-lysosomal compartment (Salonen
at al., 2005). However, in many cases the
mechanisms of membrane binding and
targeting of the replication complexes to
specific intracellular organelles are poorly
understood. Alphaviruses represent the
first example of structurally characterized
membrane binding (Kääriäinen and Ahola,
2002). In recent years, this field of research
has attracted an increased interest, which
may lead to better understanding of the
events taking place in the infected cells
during viral replication. This knowledge,
in turn, will provide new approaches for
the development of antiviral drugs.
35
Acknowledgements
Acknowledgements
This work has been carried out at the Institute of Biotechnology, University of Helsinki, during years
1999-2005, with the financial support from Academy of Finland, Sigrid Juselius Foundation and
Viikki Graduate School in Biosciences.
I wish to express my deepest gratitude to my supervisors, professor Leevi Kääriäinen and
docent Tero Ahola, for their guidance and support during these years. I am especially grateful to
Leevi who was the driving force behind this whole endeavor. Without his enthusiasm and constant
encouragement this thesis would never be completed.
I would like to thank professor Mart Saarma, the director of the Institute of Biotechnology,
for providing excellent research and core facilities. I am grateful to professor Marja Makarow, the
Head of the Viikki Graduate School in Biosciences, for providing top-level education and for her
interest in my work. I also thank professor Timo Korhonen, the Head of the Division of General
Microbiology, and Dr. Ritva Virkola for their flexible attitude towards my studies. My special thanks
to Drs Eeva Sievi and Nina Saris for taking care of the Ph.D. bureaucracy.
I thank professor Timo Hyypiä and docent Sirkka Keränen for their careful reviewing of this
work and constructive comments.
I wish to thank Dr. Andres Merits for pleasant collaboration and for numerous cloning tips.
I am grateful to Dr. Petri Auvinen for his help and advice during my first years in the virology lab. I
thank Dr. Nisse Kalkkinen, the member of my follow-up group, for his interest in my work.
All the former and present members of the SFV-group, including Tarja, Helena, Pekka,
Hannu, Nana, Pia, Peter, Javier, Pirjo, Maarit, Leena and Giuseppe are all warmly thanked for
help and advice. I wish to express my sincere gratitude to Airi for her invaluable help and for her
ability to create such a warm atmosphere in the lab. My special thanks to Lidia and Anne, my fellow
students, for the great time we had together in and out of our shared office. I thank Andrey and Sveta
for enjoyable lunches that helped keep me fluent in Russian. I also wish to thank Katri for being such
a pleasant travel companion. Caroline Griggs is acknowledged for revising the language and Timo
“Tinde” Päivärinta for the layout of this thesis. I am truly grateful to all my friends outside of work
for the fun we had together and just being there.
I want to express my deepest gratitude to my parents, Mila and Igor Magden, who have
loved and supported me throughout my entire life. I thank my husband Jani for his love, support,
endless patience and the “occasional pair of shoes”. My golden boy Ram is acknowledged for taking
36
Acknowledgements
me out for a walk every day. My biggest hugs and kisses are for my daughter Elli, who showed me
what really is important in life.
Helsinki, January 2006
37
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