3

Molecular Virology of Hepatitis E Virus

R. Prasida Holla, MSc1,
Imran Ahmad, MSc1,
Zulfazal Ahmad, PhD1 Shahid Jameel, PhD1

1 Virology Group, International Centre for Genetic Engineering and Address for correspondence Shahid Jameel, PhD, ICGEB, Aruna Asaf
Biotechnology (ICGEB), New Delhi, India, Ali Marg, New Delhi 110067, India (e-mail: shahid@icgeb.res.in).


Both authors contributed equally to the article.

Semin Liver Dis 2013;33:314.

Abstract Hepatitis E virus (HEV) is the causative agent of hepatitis E. It is a nonenveloped virus
Keywords with a 7.2 kilobases positive-stranded RNA genome. The molecular virology of HEV is
hepatitis E getting better understood with the development of replicons and in vitro infection
HEV systems, and the discovery of related viruses that infect animal species other than
ORF1 humans. This review focuses on the virology of HEV and updates the current knowledge
ORF2 on the HEV genome and its constituent proteinsORF1, ORF2, and ORF3, and the viral

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ORF3 life cycle.
RNA replication

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an
acute viral hepatitis that is endemic to many resource-limited
regions of the world.1 According to the World Health Organi-
zation, there are approximately 20 million incident HEV
infections every year, with over 3 million acute cases of
hepatitis E and 70,000 hepatitis Erelated deaths.2 Most
of these are in high-prevalence areas in East and South Asia
where sanitary conditions are poor and there is frequent
contamination of drinking water with fecal matter. Large
outbreaks of hepatitis E have also been reported from these
areas, as from other areas of overcrowding and poor sanita-
tion such as refugee camps.
Hepatitis E was recognized to be caused by a distinct agent
in the 1970s from outbreak investigations undertaken by
Khuroo and colleagues in the Kashmir, India, and by Purcell
and colleagues who retrospectively studied the 19551956
outbreak of jaundice in New Delhi, India. The historical
aspects of hepatitis E and HEV are covered in greater detail
in a recent review.3 Viruses similar to HEV have been discov-
ered from pigs (swine HEV) and chickens (avian HEV), and
more recently from bats, rats, rabbits, and sh. The successful
transmission of swine HEV to macaques, which are a model
for human HEV transmission, and reports showing nucleotide
sequence identity between viruses recovered from uncooked
meat and humans who became infected after consuming the
meat, strongly support HEV as a zoonosis.
HEV is a nonenveloped virus, which is 27 to 34 nm in size
and is classied as a Hepevirus in the family Hepeviridae.4
Within this genus at least four genotypes of HEV are recog-
nized as a species, each dominant in a given geographic area
(Fig. 1). Genotype 1 includes strains from Asia and Africa;
genotype 2 includes a Mexican strain and few variants from
Africa; genotypes 3 and 4 include human and swine HEV
strains from industrialized countries and Asia (particularly
China), respectively.4,5 While genotypes 1 and 2 are restricted
to humans, genotypes 3 and 4 have a broader host range and
are zoonotic. The human and swine HEV strains show exten-
sive serological cross reactivity with a single serotype.
Studies on HEV transmission and pathogenesis as well as
preclinical vaccine development studies were performed in
nonhuman primates such as cynomolgus, rhesus and owl
monkeys, and chimpanzees.68 Pigs have also been used for
transmission and molecular studies.9 But a small animal
model for human HEV is still elusive, which has hampered
virological studies. Cell culture systems based on replicon
RNA transfection and those using the human, swine, and
rabbit viruses have recently become available, which are
discussed below. These are likely to result in a more complete
understanding of hepatitis E virology.
Hepatitis E Outbreaks
Many outbreaks of hepatitis E have been recorded from
endemic areas. The rst recorded outbreak in Delhi, India
in 1955 to 1956 with an estimated 29,000 cases was retro-
spectively conrmed about two decades later when stored
sera were tested negative for hepatitis A and B10; this was also
the case with a subsequent outbreak from Ahmedabad, India

Issue Theme Hepatitis E in 2013: Copyright 2013 by Thieme Medical DOI http://dx.doi.org/
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Krawczynski, MD, PhD Tel: +1(212) 584-4662.
4 HEV Virology Holla et al
Fig. 1 Global distribution of hepatitis E virus (HEV). The world map shows an updated distribution pattern of HEV genotypes among infected
humans as well as the distribution of swine HEV genotypes in infected pig populations.
in 1975 to 1976. The proposal that enteric non-A, non-B
hepatitis (ET-NANBH) was due to a distinct agent was made
following the investigation of a 1978 outbreak in Kashmir,
India.11 The viral inoculum was rst passaged into nonhuman
primates and into a human volunteer from a 1983 outbreak
among Soviet troops stationed in Afghanistan.12 Subsequent-
ly, two small outbreaks affecting approximately 200 people
were reported in 1986 to 1987 from Mexico.13 A prolonged
epidemic from late 1986 to 1988 was reported from the
Xinjiang Uyghur autonomous region in western China14; a
subsequent outbreak was reported from Nepal in 1988. In
1991, a large epidemic occurred in Kanpur, India in which
79,000 people were affected15; a massive outbreak in China in
the same year affected more than 100,000 people. Nepal saw
the recurrence of a hepatitis E outbreak in 1994, but the strain
responsible was different than the 1988 outbreak. Several
other outbreaks have been reported from Asia and Africa. The
Uganda outbreak of 2007 had almost 11,000 cases and 160
fatalities.16 In 2004, the refugee camps in Sudan and Chad
experienced hepatitis E, which were caused by viruses ge-
netically related to Asian outbreaks, but some isolates were
closer to the Mexican genotype 2 virus.17 Most recently, an
outbreak was reported from Bangladesh in June 2011 and
another in June 2012 from a village in Maharashtra, India,
which had more than 3000 cases and 18 fatalities. A common
feature of all hepatitis E outbreaks was contamination of
drinking water supplies with sewage or fecal material.
The HEV Genome
The genome of HEV is a single-stranded positive-sense RNA of
 7.2 kb, which has features typical of a eukaryotic mRNA,
including a 7-methylguanine cap at its 5 end and a poly(A)
stretch at the 3 end. Besides three partially overlapping open
Seminars in Liver Disease Vol. 33 No. 1/2013
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reading frames (ORFs) called orf1, orf2, and orf3, the viral
genome also has short 5- and 3-untranslated regions (UTRs),
a conserved 58-nucleotide region within orf1 and a sequence
closer to the 3 end of orf1, which is homologous to the
junction region found in alphaviruses (Fig. 2).18 These
structures are proposed to be important for HEV RNA repli-
cation.19 The region between the end of orf1 and start of orf3/
orf2 is complex and also contains regulatory elements.20
The cloning and sequencing of the HEV genome was rst
reported from cDNA libraries made from the bile of macaques
experimentally infected with stool samples from hepatitis E
patients.18,21 Full-length genomic sequences from various
genotypes and geographic isolates of HEV have since become
available.2224 Three RNA species of 7.2, 3.7, and 2 kb,
designated as the genomic and two subgenomic RNAs, re-
spectively, were detected in the liver tissue of macaques
experimentally infected with HEV.18 In the original model,
while the 7.2 kb RNA was proposed to be translated into the
ORF1 protein, the 3.7 and 2 kb subgenomic RNAs translated
into the ORF3 and ORF2 proteins, respectively.18 However,
other workers were unable to nd the 3.7 kb RNA in animal
livers or in cell culture models of HEV replication. In stable
Huh-7 cell lines made from functional HEV RNA replicons, the
genomic RNA and only one subgenomic RNA of 2.2 kb were
observed.25 The capped subgenomic RNA initiated at nucleo-
tide position 5122, which was downstream of the rst two
methionine codons in orf3, and was bicistronic for the
translation of ORF3 and ORF2 (Fig. 2). This model rational-
ized the reading frame differences observed in genotype 4
HEV isolates and was further conrmed by intrahepatic
inoculation of wild-type and mutant genotype 3 swine HEV
replicons into pigs26 and from PLC/PRF/5 hepatic cells that
were either transfected with an infectious genotype 3 RNA or
infected with HEV genotype 4.27 In both cases, only the 2.2 kb
 HEV Virology Holla et al 5

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Fig. 2 The hepatitis E virus (HEV) genome. The  7.2 kb positive sense RNA genome of HEV has a 7-Me-G cap at its 5 end and a poly A tail at its 3
end. There are short stretches of untranslated regions (UTR) at the 5 and 3 ends. The three open reading frames (ORFs) are shown. The orf1
(nucleotides 285109) encodes a nonstructural polyprotein that contains the methyltransferase (MeT), Y, papain-like cysteine protease (PCP),
variable (V), X(macro), RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) domains. The orf2 (nucleotides 51477129) and orf3
(nucleotides 51075477) are translated from a 2.2 kb subgenomic RNA generated during viral replication. The ORF2 protein has three N-linked
glycosylation sites (red dots) at residues 137, 310, and 547. The domain structure of the ORF3 protein and a phosphorylation site (red diamond) at
residue Ser-71 are shown.

subgenomic RNA species, whose 5 end mapped to nucleotide
5122, was observed. A recent review provides more details on
HEV RNA species, regulatory elements on the viral genome,
and their role in genome replication.20
Genetic Variants
Comparative analysis of the genomic sequences of multiple
HEV isolates has revealed extensive genomic diversity and the
identication of four major genotypes and several subtypes
within each genotype.28 A virus with a 6.6 kb RNA genome
and 50% nucleotide sequence identity with mammalian
HEVs, called avian HEV, was isolated from chickens affected
by the hepatitis-splenomegaly syndrome.29,30 Though ini-
tially considered as HEV genotype 5, avian HEV is now
proposed as a new species within the family Hepeviridae.4
Recently, HEV-like viruses were also isolated and character-
ized from Norway rats31 and ferrets.32 The rat HEV has a
sequence identity of 55 to 59% with HEV genotypes 1 to 4,
and ferret HEV has an identity of 72.3% to rat HEV. A unique
strain of HEV was identied from farmed rabbits in China,
which shared 74 to 79% nucleotide sequence identity to
existing HEV strains, and 46% identity to avian HEV.33 High
rates of HEV seropositivity were recently reported in rabbits
raised on farms in Inner Mongolia, China.34 Sequence analysis
showed 89.3 to 95.9% identity to rabbit HEV strains from
other sites in China, with the strains clustered into three
distinct groups within HEV genotype 3.34 Finally, a virus that
infects various species of trout sh was isolated from the
cutthroat trout; it bears only 18 to 27% sequence similarity to
avian or mammalian HEVs and may be classied as a new
genus within the family Hepeviridae.35
It is suspected that a high level of genomic intermixing
takes place in animals and in patients. Recombination in HEV
genomes was recently reported.36 Phylogenetic and recom-
bination analyses of complete HEV genomes showed three
potentially signicant intra- and intergenotype recombina-
tion events. Compartmentalization of HEV quasispecies be-
tween serum and cerebrospinal uid (CSF) was observed in a
kidney transplant patient with chronic hepatitis E.37 The
genetic variability among quasispecies from solid-organ
transplant patients who developed hepatitis E was found to
be lower in those who cleared the virus as compared with
those who progressed to chronicity.38
Chronic hepatitis E infection in immunocompromised
individuals has revealed the presence of viral strains carrying
insertions from human genes. These recombinants were
barely detectable in feces from patients, but were selected
with serial passage on cells in culture. The Kernow-C1 strain
of HEV was isolated from the feces of a chronically infected
HIV-positive patient, and was subsequently passaged on

Seminars in Liver Disease Vol. 33 No. 1/2013

6 HEV Virology Holla et al
HepG2/C3A cells.39 After six passages, the isolated viral
species were found to carry insertions from the host genome,
which included 174 nucleotides or 117 nucleotides from the
S17 or S19 ribosomal protein genes, respectively, or 114
nucleotides from a GTPase gene; point mutations were also
observed in the hypervariable region (HVR) within orf1.39 The
S17 insertion and point mutations conferred an adaptive
advantage in culture, and expanded the host range and
tropism of the virus.39 The S17 fragment from the Kernow
virus was also cloned into an infectious clone of the HEV
genotype 1 Sar-55 strain. Although the virus produced from
this clone did not show any adaptive advantage in HepG2/C3A
cells, it had a broader tropism and infected a hamster cell
line.40 The reasons for better growth in culture and expanded
tropism are unclear at this time.
HEV Proteins
The HEV genome encodes three proteins, which are called the
ORF1, ORF2, and ORF3 proteins. Although the kinetics of
expression of these proteins during the viral life cycle is not
fully understood, antibodies to these proteins in infected
humans and experimental animals is a conrmation of their
expression during HEV infection.41,42
The ORF1 Protein
The ORF1 nonstructural polyprotein of HEV is 1693 amino
acids (180 kilodaltons [kDa]) and contains several function-
al motifs and domains present in the nonstructural proteins
of other positive-stranded RNA viruses.43 These include the
methyltransferase (MeT), papain-like cysteine protease
(PCP), RNA helicase (Hel) and RNA-dependent RNA polymer-
ase (RdRp) (Fig. 2). Some uncharacterized domains homol-
ogous to other animal and plant positive-strand RNA viruses
are also found within the ORF1 protein. These include the Y
domain, the X domain (now called the macro domain), and a
polyproline or hypervariable region that shows high levels of
nucleotide (and amino acid) variation between HEV isolates
(Fig. 2). A bioinformatic analysis of the HEV ORF1 protein43
proposed many putative biochemical activities, some of
which have now been characterized in greater detail. It is
not entirely clear whether the ORF1 polyprotein is processed
into biochemically distinct units, as is the case with many
positive-strand RNA viruses. Expression of the ORF1 poly-
protein by itself showed some processing when expressed in
mammalian cells using recombinant vaccinia viruses44 or in
insect cells using recombinant baculoviruses,45 but not when
expressed in Escherichia coli or HepG2 hepatoma cells.46
However, processing was reported in HepG2 cells transfected
with a genomic replicon of HEV.23 No viral infection study has
so far addressed expression and processing of the ORF1-
derived nonstructural polyprotein.
Methyltransferase
An alignment of the ORF1 polyprotein suggested that amino
acids 60 to 240 might serve as the viral methyltransferase.43
Viral methyltransferases catalyze the capping of viral RNA,
and because the HEV genomic and subgenomic RNAs are
Seminars in Liver Disease Vol. 33 No. 1/2013
capped, it was expected that the HEV genome would encode a
methyltransferase. The capping of mRNA is well character-
ized in mammalian, yeast, and several viral systems, in which
it follows a common mechanism, which involves RNA tri-
phosphatase, guanyltransferase, and guanine-7-methyltrans-
ferase activities.47 A 110-kDa protein that included amino
acids 1 to 979 of ORF1 was shown to contain the guanyl-
transferase and guanine-7-methyltransferase activities.48
The RNA triphosphatase activity was suggested to be part
of the HEV helicase protein.49
Two reports have shown the presence of a 7-methylgua-
nine cap on HEV genomic RNA, and its requirement for
infectivity.24,50 This cap structure is required for efcient
binding of mRNAs to the ribosome, as well as for viral RNAs to
evade activation of interferon-mediated innate immunity.
The 5 triphosphate (5ppp) group on an uncapped RNA
molecule is a potent activator of the interferon response.51
Protease
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A papain-like cysteine protease (PCP) was also predicted
within the ORF1 polyprotein.43 This prediction was strength-
ened by conservation of the X domain in HEV, which in other
systems was found exclusively in association with the PCP.
Although this alignment placed the putative PCP domain in
the region of amino acids 433 to 592 of the ORF1 polyprotein,
no functional activity for the HEV protease has so far been
described. This was further conrmed recently based on
biochemical data and the lack of conservation of the catalytic
Cys-His dyad in a large number of HEV isolates.52 However, a
deubiquitination activity was recently reported in a Met-PCP
fusion protein, and is proposed to play a role in combating the
cellular antiviral response.53 Signicantly, the cellular ubiq-
uitin-proteosome system and the free ubiquitin pool also
appear to be important for HEV replication.54
RNA Helicase
Many positive-stranded RNA viruses encode a RNA helicase,
which is essential for replication of the viral genome of these
viruses. The putative RNA helicase of HEV was shown to
contain all the conserved motifs found in SF-1 type helicases
and to have the nucleotide triphosphatase (NTPase) as well as
RNA-binding domains.43 This was biochemically conrmed
recently for a region of the ORF1 protein encompassing amino
acids 960 to 1204.55 The enzyme hydrolyzed all rNTPs, as also
dNTPs albeit with a lower efciency, and showed unwinding
activity only on RNA duplexes with 5 overhangs. The HEV
helicase belongs to the 5!3 class of SF-1 family of helicases
that are also found in other positive-strand RNA viruses such
as alphaviruses, arteriviruses, coronaviruses, and rubiviruses.
RNA-Dependent RNA Polymerase
The RdRp replicates the genomic RNA through an antige-
nomic RNA intermediate, and is an essential enzyme encoded
by all RNA viruses. A strong homology of the HEV ORF1 region
encompassing amino acids 1207 to 1693 was observed with
the RdRp proteins of other positive-strand RNA viruses.43 The
RdRp region comprising nucleotides 3546 to 5106 of HEV was
expressed in E. coli and the puried recombinant protein

bound the 3 end of HEV genomic RNA.56 The recombinant
RdRp also used the 3 end of HEV RNA as a template to
synthesize the complementary strand in vitro. A  40 kDa C-
terminal region of the ORF1 polyprotein, expressed as a
fusion with the enhanced green uorescent protein, localized
to the endoplasmic reticulum (ER) and was able to copy HEV
plus-strand RNA transfected into these cells.57 These studies
also suggest that analogous to several other RNA viruses,
intracellular (ER) membranes are the likely sites for HEV
replicase localization and possibly RNA replication as well.
Macro Domain
Macro domains have homology to the nonhistone domain of
the histone macroH2A and are found in a variety of bacterial,
archaeal, and eukaryotic proteins, where they contribute to
ADP-ribose metabolism and posttranslational modica-
tions.58 These are also expressed in members of a few viral
families in which viral RNA is replicated in the cytoplasm in
membrane-associated replication complexes; these include
all members of Coronaviridae, rubella virus, and alphaviruses
(Togaviridae).59 Macro domain proteins hydrolyze ADP-ri-
bose 1-phosphate (ADPR-1P), a product of cellular pre-tRNA
splicing, and associate with poly(ADP-ribose) polymerase-1
(PARP-1),60 suggesting some role in cellular apoptosis. Two
reports describe the macro (erstwhile X) domain within
amino acids 775 to 960 of the HEV ORF1 polyprotein.61,62 It
is suggested that viral macro domains function as poly(ADP-
ribose)-binding modules and may have a role in viral RNA
replication and/or transcription.61 Because some of the mac-
ro domains, including that of HEV, can also bind poly(ADP-
ribose) in the presence of poly(A), they could recruit poly
(ADP-ribose)-modied cellular factors to the replication com-
plex while bound to viral polyadenylated RNA.
Polyproline Region
The polyproline region (PPR) or hypervariable region in the
ORF1 polyprotein is classied as an intrinsically disordered
region (IDR), rich in polar and charged amino acids. A recent
study63 shows the PPR mutation rate to be similar to the rest
of orf1, but due to a higher substitution rate within the PPR,
there is greater promiscuity in the rst and second codons.
Due to a preference for cytosine (C) in the rst and second
positions, there is increased frequency of proline residues in
this region. Sequence conservation and lower rates of substi-
tution in the PPR in genotype 1 compared with genotypes 3
and 4 may suggest an increased need for adapting to a wider
host range by zoonotic strains. In another recent study, a
three-dimensional model of the PPR region was used for
function prediction using gene ontology annotations.64 Sev-
eral putative functions were found, such as peptide cleavage
sites, sites modied by enzymes, and sites that bind to
proteins, nucleotides, and metal ions, indicating a potential
role in viral replication. These proposals await experimental
validation.
The ORF2 Protein
The ORF2 protein is the viral capsid protein of 660 amino
acids (Fig. 2), which was proposed to encapsidate the viral
HEV Virology Holla et al 7
RNA genome.19 When expressed in animal cells in culture,
nonglycosylated and glycosylated ORF2 proteins of 74 kDa
and 88 kDa were observed.65 The nascent protein contains a
N-terminal signal sequence that translocates it into the
endoplasmic reticulum, where it acquires N-linked glycosyl-
ation65 at conserved asparagine (Asn) residues 137, 310, and
562 (Fig. 2).66 This and the requirement of glycosylation
sites on the ORF2 protein for the formation of infectious virus
particles was subsequently shown using infectious repli-
cons67 and HEV that propagates efciently in cultured PLC/
PRF/5 cells.68 A genotype 3 ORF2 protein expressed using
recombinant vaccinia viruses was also glycosylated and lo-
calized to the ER, Golgi, and surface of infected cells.69
A N-terminal truncated ORF2 protein (aa 112660) of 58
kDa and a 50 kDa form were efciently secreted as virus-like
particles (VLPs) of 23 to 24 nm in the culture medium of
insect cells infected with recombinant baculoviruses.70 When
expressed similarly, the full-length ORF2 protein was proc-
essed into 63 kDa, 56 kDa, and 53 kDa proteins, which
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correspond to truncated proteins spanning amino acids 112
to 660, 112 to 607, and 112 to 578, respectively.71 Cryoelec-
tron microscopy and image reconstruction methods showed
these VLPs to contain protruding dimers at the icosahedral
twofold symmetry axes such that the 60 monomers were
organized as 30 morphological units.72 Subsequently, amino
acids 126 to 601 in the ORF2 protein were found to be
essential for the formation of secreted VLPs in this expression
system.73 X-ray crystal structures show the ORF2 protein
monomers to contain three distinct domains called the shell
(S), middle (M), and protruding (P) domains.74,75 These and
other structural details are covered in a recent review.76
Recombinant ORF2 proteins expressed in E. coli can also
assemble into higher-order structures, as exemplied by the
p239 (aa 368606), E2 (aa 394606) and E2a (459660)
proteins that predominantly occur as homodimers and that
model the dominant antigenic and neutralizing determinants
on the HEV surface.77,78 The crystal structure of the E2s
protein (aa 455602) shows it to form a tight homodimer
that is essential for HEV interaction with the host cell, and
contains the HEV neutralizing antibody recognition site.79
Various studies have characterized the neutralizing do-
mains of the ORF2 capsid protein through phage display
methods80 or with multiple overlapping peptides and trun-
cated ORF2 proteins.81 The minimal neutralizing domain was
mapped to residues 458 to 607.82 When combined with
structural details, these mapping studies show the P domain
of the ORF2 protein to contain its neutralization epitope.74,75
Immunological and structural studies on the ORF2 protein
have also provided a basis for hepatitis E vaccine develop-
ment. Two recombinant hepatitis E vaccines based on recom-
binant ORF2 proteins expressed as VLPs in insect cells83 or in
E. coli84 have undergone successful clinical testing in humans.
The ORF2 particles have also been used as an epitope presen-
tation system,85 which when combined with the ability of
these VLPs to render successful oral immunization,86 can
provide a powerful vaccine platform. A recent study com-
pared the cross-protective ability of N-terminal truncated
ORF2 proteins from swine, rat, and avian HEV in a pig
Seminars in Liver Disease Vol. 33 No. 1/2013
8 HEV Virology Holla et al
challenge model infected with genotype 3 human HEV.
Although a strong IgG anti-HEV response was observed, there
was only partial cross protection.87 This has important im-
plications for current and future vaccines, especially against
novel zoonotic strains of HEV.
Recombinant ORF2 capsid proteins have also been used to
model the interactions of HEV with its target cells. The
puried p239 protein, which antigenically and structurally
mimics the HEV capsomere can bind and penetrate cells
susceptible to HEV infection in vitro.88 In interaction studies,
p239 bound to cellular Grp78/BiP, -tubulin and heat shock
protein 90 (Hsp90), with Hsp90 binding being important for
its intracellular trafcking.89 The ORF2 VLPs bound Huh7
liver cells in an interaction dependent upon heparan sulfate
proteoglycans (HSPGs) present on the cell surface.90 Our
studies also show the p239 VLPs to bind cell lines susceptible
to HEV infection; these are being used to identify the cellular
receptor for HEV (R.P. Holla, Z. Ahmad, and S. Jameel, unpub-
lished results).
For nonenveloped viruses, the interactions of their capsid
proteins with cellular proteins are important for virus entry,
intracellular trafcking, and signaling,91 as well as for capsid
assembly and virus egress. When expressed in animal cells,
the ORF2 protein localizes to the ER, where it causes ER stress
and some of the protein is retrotranslocated to the cytoplasm
through an ER-associated degradation (ERAD) pathway.92 A
recent study shows ORF2-dependent phosphorylation of the
eukaryotic initiation factor 2a (eIF2a), increased expression of
the ATF-4 transcription factor, and activation of the CHOP
promoter.93 Even though this ER stress pathway leads to
apoptosis, none was observed in ORF2-expressing cells, pos-
sibly due to its interaction with heat shock protein 72
(Hsp72), which triggers an anti-apoptotic response.93 The
glycosylated form of the ORF2 protein also inhibited the
nuclear factor kappa B (NFB) pathway by reducing the
proteasome-mediated degradation of inhibitor of kappa B
(IB), which was due to ORF2 protein interaction with -TRCP,
a component of the ubiquitination complex.94 Whether these
interactions modulate HEV entry, intracellular trafcking,
assembly, or egress remains to be seen in an infection model.
The ORF3 Protein
The ORF3 protein is 114 amino acids long and is dispensable
for replication of HEV in vitro in cell lines,95 but is required for
infection in the macaque model of experimental infection.96
This suggests that the ORF3 protein functions as a viral
accessory protein, and is likely to affect the host response
to infection. Primary sequence analysis of the ORF3 protein
did not show homology to any other protein. It contains two
large N-terminal hydrophobic domains, D1 (aa 723) and D2
(aa 2853), and two proline-rich domains, P1 (aa 6677) and
P2 (aa 95111) (Fig. 2). In mammalian cells, the 13 kDa
protein was phosphorylated at a single serine residue within
the P1 domain by the cellular mitogen-activated protein
kinase (MAPK)97; this was also the case in cells transfected
with the HEV genomic RNA.23,95,96 Subcellular fractionation
showed the ORF3 protein to associate with the cytoskele-
ton,97 and confocal microscopy revealed punctate and la-
Seminars in Liver Disease Vol. 33 No. 1/2013
mentous intracellular distribution, attributed to its
localization to endosomes98 and its interaction with micro-
tubules,99 respectively.
The D1 domain is very cysteine-rich and is required for the
ORF3 protein to associate with the cytoskeleton,97 to bind
microtubules99 and a MAPK phosphatase.100 The P1 domain
contains a PMSP motif in which Ser-71 is phosphorylated by
MAPK, in transfected cells as well as in vitro97; whether the
ORF3 protein expressed during viral infection is also phos-
phorylated has not been tested. The P2 domain has two
overlapping PXXP motifs, which have been described in
many viral and cellular signal-transducing proteins and
bind the Src homology 3 (SH3) domains found in other
proteins. This domain was shown to be responsible for
interaction of the ORF3 protein with many cellular SH3
domain-containing proteins.101
The ability of the ORF3 protein to interact with multiple
cellular proteins suggested its potential role in optimizing the
cellular environment for viral infection and replication.
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Though most of the studies are based on ORF3 overexpression
in hepatic cell lines, they provide insights into the versatile
nature of this protein. The ORF3 protein activates the extra-
cellularly regulated kinase (Erk), a member of the MAPK
family,101 and this depends upon its ability to bind and
inactivate an Erk-specic MAPK phosphatase.100 Cells ex-
pressing the ORF3 protein also displayed higher levels of
hexokinase and oligomeric forms of the voltage-dependent
anion channel (VDAC) protein, which lead to attenuation of
mitochondrial death signaling.102 Subcellular localization of
the ORF3 protein to early and recycling endosomes, and its
interaction with CIN85, a multidomain adaptor protein im-
plicated in the downregulation of receptor tyrosine kinases,
delays postinternalization trafcking of the activated growth
factor receptors.98,103 The activation of Erk and effects on
growth factor receptors are proposed to promote cell survival
and proliferation. Another consequence of its effects on
trafcking is reduced levels of phospho-STAT3 (pSTAT3) in
the nuclei of ORF3-expressing cells.98 This results in de-
creased expression of acute-phase response proteins and
attenuation of a major inammatory pathway in the liver,
which would again create an environment supportive of viral
replication. Reduced levels of acute phase proteins are also
observed in the plasma of hepatitis E patients.104
Various other ORF3-interacting cellular proteins have been
identied through interaction screens. These include the -1-
microglobulin and bikunin precursor protein (AMBP) and its
constituent -1-microglobulin and bikunin, brinogen
chain, and hemopexin.105108 Increased secretion of -1-
microglobulin was observed from ORF3-expressing cells,
mediated by the tumor susceptibility gene 101 (Tsg101)
protein, a member of the endosomal-sorting complex, which
bound the ORF3 protein through a conserved PSAP motif in its
P2 domain.109 Because -1-microglobulin is immunosup-
pressive, this was proposed to protect virus-infected cells
in the liver.109 The urine of hepatitis E patients also showed
increased -1- microglobulin levels compared with hepatitis
B patients and healthy controls.104 Hemopexin is a heme-
binding acute-phase plasma protein, which protects cells

from hemoglobin-mediated oxidative damage and together
with transferrin and haptoglobin plays an important role in
iron homeostasis. The ORF3 protein binds hemopexin
through its D2 domain, and this is proposed to aid in viral
infection.107 Reduced levels of hemopexin and haptoglobin
have also been observed in the plasma of hepatitis E pa-
tients.104 The ORF3 protein also increased the expression of
many glycolytic enzymes through Erk-mediated stabilization
of the hypoxia inducible factor-1 (HIF-1) transcription fac-
tor.110 Finally, it modulates the phosphorylation and the
nuclear localization of hepatocyte nuclear factor 4 (HNF4),
and through it the expression of many cellular genes that are
regulated by this liver-specic transcription factor.111 These
studies support a prosurvival (or antiapoptotic) role for the
ORF3 protein, as also its effects on cellular energy homeosta-
sis, innate host responses and transcription of cellular genes,
which are likely to support HEV pathogenesis in the infected
host.
Some recent studies have shown the ORF3 protein to be
associated with virions and have proposed a role in virus
egress. An ORF3-decient infectious cDNA clone replicated
efciently in PLC/PRF/5 and A549 cells, but was defective in
virus release.68 In an immunocapture PCR assay, the ORF3
protein was found on the surface of cell culture generated
HEV, which also showed a lower density than the ORF3-
decient virus. These observations suggested the ORF3 pro-
tein to be present on the virion surface in association with
lipids, and to be important for HEV egress.68 Anti-ORF3
antibodies were also able to capture HEV from the serum,
but not the feces of hepatitis E patients, the former banding at
a lower density compared with the latter.112 The HEV in
serum appears to be associated with lipids, but this coat is
possibly shed during its passage through the gut.
The egress of a genotype 1 HEV from Caco-2 (intestinal)
and Huh-7 (hepatoma) cells depended upon the expression of
a functional ORF3 protein.113 This protein accumulated in
polarized Caco-2 cells at the apical surface from which the
virus was released.113 In this as well as another study,114 a
PSAP motif within the P2 domain of the ORF3 protein was
found to be important for virus egress, but not for its
accumulation at the apical end. One of the mutant viruses
showed no infection following intrahepatic inoculation in
macaques, but another was infectious and the recovered virus
was found to have completely reverted to a wild-type ORF3
sequence. These results support previous observations that
the ORF3 requirement for infection.96 In this study also, the
ORF3 protein, in association with lipids, was found to coat the
virion surface.
Recent studies show the requirement of an intact PSAP
motif for the formation of membrane associated HEV par-
ticles with the ORF3 protein on their surface,114 and this to be
mediated by the cellular Tsg101 protein115; this holds true for
avian HEV as well.116 Several RNA viruses bear proline-rich
sequences in their late domains, which are required for viral
budding. These include P(S/T)AP and PPXY (X, any amino acid)
motifs, which hijack host proteins in the vacuolar protein
sorting (VPS) pathway. This pathway gives rise to multi-
vesicular bodies (MVBs), which are topologically identical
HEV Virology Holla et al 9
to virus budding.117 Viral late domains interact with mem-
bers of the VPS pathway, and redirect the complexes to the
site of virus budding on the plasma membrane.117 Although it
is a nonenveloped virus, the association of HEV with lipids,
endosomal localization of the ORF3 protein, and the presence
of a PSAP motif that is required for viral egress, suggests that
HEV follows the VPS pathway for its release from infected cells
(Fig. 3).
HEV Replication and In Vitro Propagation
The HEV replication cycle is poorly understood due to its
inability to efciently propagate in vitro in cultured cells or to
infect small animal models. The original view of the HEV life
cycle118 was based on its genome analysis and analogy to
other positive-strand RNA viruses; it is still generally rele-
vant. However, in recent times in vitro expression and repli-
con studies have added some interesting details. An updated
model of the HEV life cycle is shown in Fig. 3.
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Various transformed cell lines, such as the hepatic cell lines
Huh7, PLC/PRF/5 and HepG2, the lung carcinoma cell line
A549, and the colon carcinoma cell line Caco-2, have been
shown to support HEV infection and replication. The binding
and entry of HEV is poorly understood as no cellular receptor
for the virus has been identied. The HSPGs present on cell
surface Syndecan type proteins are required as attachment
factors, but their role is largely to concentrate the virus on the
cell surface before its binding to and entry through a specic
receptor (Fig. 3, steps 1, 2).90 One recent report suggests
that HEV enters liver cells through receptor-dependent
clathrin-mediated endocytosis (Fig. 3, step 3).119 However,
alternate entry pathways are also possible (R.P. Holla and S.
Jameel, unpublished results). Following entry, Hsp90 and
tubulin appear to be involved in intracellular trafcking,89
but little else is known about this process. The location and
mechanism of uncoating to release viral RNA (Fig. 3, step 4)
is not known.
Once it is released in the cytosol, the viral RNA is directly
translated into the ORF1 polyprotein (Fig. 3, step 5). The
viral methyltransferase, RNA triphosphatase, helicase and
replicase activities are used to replicate the genomic RNA
into negative-sense RNA intermediates (Fig. 3, step 6),
which have been detected in replicon-transfected cells23 as
well as in the livers of experimentally infected macaques120
and pigs.9 The negative-strand RNA serves as a template for
the synthesis of genomic as well as subgenomic positive-
sense RNAs (Fig. 3, steps 7, 8), of which the latter are
translated into the ORF2 and ORF3 proteins (Fig. 3, step
9).2527 The ORF2 protein packages the genomic positive-
sense RNA into progeny virions (Fig. 3, step 10). Recent
evidence suggests that the ORF3 protein together with lipids
coats this particle, possibly during the budding process
(Fig. 3, step 11).
Although hepatocytes are the most likely cell type in which
HEV replicates, in vitro results also support infection and
replication in nonhepatic cell types such as A549 lung carci-
noma cells and in Caco-2 colon carcinoma cells. In pigs
experimentally infected with swine HEV, negative-sense
Seminars in Liver Disease Vol. 33 No. 1/2013
10 HEV Virology Holla et al

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Fig. 3 The hepatitis E virus (HEV) replication cycle. The viral particles are concentrated on the surface of target cells through heparin sulfate
proteoglycans acting as attachment factors (1) and subsequently bind a specic yet uncharacterized receptor (2), following which the particles are
internalized by endocytosis (3). The virus uncoats (4) to release genomic RNA that is translated in the cytoplasm into nonstructural proteins (5).
These nonstructural proteins include the RNA-dependent RNA polymerase that replicates the positive sense genomic RNA into negative sense
transcripts (6); the latter then act as templates for the synthesis of a 2.2 kb subgenomic RNA (7) as well as full-length positive sense transcripts (8).
The positive sense subgenomic RNA is translated into ORF2 (gray triangles) and ORF3 (blue circles) proteins (9). The ORF2 protein goes through
the endoplasmic reticulum and packages the genomic RNA to assemble new virions (10). The ORF3 protein is also associated with
endomembranes (11) and may aid in viral egress (12,13). Recent studies suggest that mature virions are associated with the ORF3 protein and
lipids (13), which are subsequently removed through a process that is not understood at present, to resume a fresh infection cycle.

replicative RNA intermediates were detected in the liver, but
also in the small intestine, lymph node, and colon.121 In
hepatitis E patients, HEV RNA, but no viral replication, was
also detected in peripheral blood mononuclear cells.122
Antiviral Targets in HEV
As in many viruses, multiple steps in the HEV life cycle can
also be potential targets for antiviral drug development. The
methyltransferase and guanyltransferase activities in the
ORF1 protein being strictly virus-specic are good targets
for antiviral development.48 The HEV RNA helicase is bio-
chemically characterized and is essential for viral RNA repli-
cation.54 The determination of its structure is important to
understand how distinct it is from human helicases before
being promoted as a good drug target. The HEV RdRp would
also be a potential target. However, only its binding to the
viral RNA genome,56 but not its biochemical activity, have so
far not been characterized. Because the RdRp enzyme is
unique to RNA viruses and is absent in vertebrate hosts,
panviral inhibitors targeting positive- or negative-strand
RNA viruses should be explored against this enzyme.
Interference with HEV RNA replication has been at-
tempted using ribozymes and small interfering RNAs.123,124
Although such approaches are feasible in vitro, the delivery
and targeting of such inhibitors in vivo would be the real
challenge. Several recent reports show the efcacy of Ribavi-
rin monotherapy in treating immunocompromised trans-
plant patients with chronic HEV infection,125127 severe
acute hepatitis E,128 and acute on chronic liver failure.129
Summary
Since its discovery in the early 1970s, molecular biological
tools have contributed signicantly toward understanding
the virology of HEV. Although the lack of efcient in vitro
culture systems and small animal models of viral infection
continue to be problems, recent work is beginning to over-
come some of these hurdles. Much of the early information
and models of the HEV life cycle and genome replication were
based on comparative information from better-characterized
positive-strand RNA viruses. Viral ORFs have been expressed
in various heterologous systems and the proteins have been
characterized for their structure and function. With the
recent availability of replicon- and infection-based in vitro
cell culture models, the molecular virology of HEV is becom-
ing increasingly clear. This review summarizes the current
knowledge on HEV virology.
Acknowledgments
The HEV work from the authors laboratory has been
funded by grants from the Wellcome Trust, UK, National
Institutes of Health, USA, Department of Biotechnology,
India, and from ICGEB internal resources. All are gratefully
acknowledged. Support in the form of Research

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 HEV Virology Holla et al 11

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