Biochem. J. (2009) 423, 303–314 (Printed in Great Britain)
303
doi:10.1042/BJ20091000
REVIEW ARTICLE
The hepatitis C virus and its hepatic environment: a toxic
but finely tuned partnership
Marie PERRAULT and Eve-Isabelle PÉCHEUR1
Institut de Biologie et Chimie des Protéines, UMR CNRS 5086, Université Lyon 1, IFR128 Lyon Biosciences Gerland, Lyon, France
Twenty years after its discovery, HCV (hepatitis C virus) still
infects 170 million people worldwide and cannot be properly
treated due to the lack of efficient medication. Its life cycle
must be better understood to develop targeted pharmacological
arsenals. HCV is an enveloped virus bearing two surface
glycoproteins, E1 and E2. It only infects humans through
blood transmission, and hepatocytes are its only target cells.
Hepatic trabeculae are formed by hepatocyte rows surrounded
by sinusoid capillaries, irrigating hepatic cells. Hepatocytes are
polarized and have basolateral and apical poles, separated by
tight junctions in contact with blood and bile respectively.
In blood, HCV remains in contact with lipoproteins. It
then navigates through hepatic microenvironment and extracellular matrix, composed of glycosaminoglycans and proteins.
HCV then encounters the hepatocyte basolateral membrane,
where it interacts with its entry factors: the low-density lipoprotein receptor, CD81 tetraspanin, and the high-density lipoprotein
(scavenger) receptor SR-BI (scavenger receptor BI). How these
molecules interact with HCV remains unclear; however, a
tentative sequence of events has been proposed. Two essential
factors of HCV entry are the tight junction proteins claudin-1 and
occludin. Cell polarity therefore seems to be a key for HCV entry.
This raises several exciting questions on the HCV internalization
pathway. Clathrin-dependent endocytosis is probably the route of
HCV transport to intracellular compartments, and the ultimate
step of its entry is fusion, which probably takes place within
endosomes. The mechanisms of HCV membrane fusion are still
unclear, notably the nature of the fusion proteins is unknown and
the contribution of HCV-associated lipoproteins to this event is
currently under investigation.
INTRODUCTION
THE HEPATITIS C VIRUS: FACTS AND FIGURES
Hepatocytes are the only targets of an insidious pathogen, HCV
(hepatitis C virus). This enveloped virus strictly infects humans,
and has the capacity to travel furtively in the blood from its initial
point of entry in the body, due to its association with lipoproteins.
At the approach to the liver, it diffuses through the hepatic
microenvironment, where it comes into contact or interacts with
several molecules of the extracellular matrix. Hepatocyte invasion
involves a set of HCV entry factors or receptors that most likely
act in a combination to accomplish virus cell entry. HCV is
internalized into endosomal compartments, and ultimately fuses
its lipid envelope with intracellular membranes to deliver its
genetic material to the cell cytosol, launching virus replication. In
the present review, we first place HCV in the context of the hepatic
microenvironment it has to cross, and of the cell it invades. The
human hepatocyte is a polarized cell, organized into a basolateral
pole and an apical pole. This delimits two membranes, separated
by TJs (tight junctions) composed of specific proteins. Since two
of the recently described entry factors of HCV are components of
the TJ, it appears that the polarity of the hepatocyte is a key for
HCV entry. We then summarize recent studies on HCV early
steps of entry, demonstrate a crucial role for a quartet of proteins in a finely tuned process, and we address the question of
HCV hepatotropism. Finally, we describe our current knowledge
about HCV membrane fusion.
HCV infects an estimated 3 % or 170 million of the world’s
population [1]. In Egypt, the HCV prevalence rate is the highest of
the world, with approx. 25 % of the population positive for HCV.
This is the unfortunate result of campaigns of intravenous mass
administration of tartar emetic against schistosomiasis, without
disposable needles and syringes [2]. HCV is transmitted mainly
through exposure to contaminated blood or blood derivatives, i.e.
by transfusion or intravenous drug administration in drug users
or by pricking with HCV-soiled needles. Some cases of sexual
transmission have also been reported [3,4]. HCV is a major cause
of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma,
and hepatitis C is also the most frequent indication for liver
transplantation. A protective vaccine is not available yet, and
therapeutic options are still limited. Current treatment consists in
the administration of pegylated recombinant IFN-α (interferon-α)
associated to ribavirin. However, sustained virological response
is achieved in roughly 50 % of patients and toxic side effects can
be extremely severe [5,6]. There is therefore an urgent need to
develop more effective and better tolerated therapies for chronic
hepatitis C. However, a more detailed understanding of the viral
life cycle is necessary for such an achievement.
HCV was identified in 1989 by immunoscreening of an expression library with serum from a patient with post-transfusion
non-A, non-B hepatitis [7]. HCV is the only member of the
Key words: cell polarization, hepatitis C virus (HCV), hepatocyte,
internalization, membrane fusion.
Abbreviations used: ECM, extracellular matrix; EWI-2wint, EWI-2 without its N-terminus; FRAP, fluorescence recovery after photobleaching; GAG,
glycosaminoglycan; HCV, hepatitis C virus; HCVcc, cell culture-produced HCV; HCVpp, HCV pseudotyped particle(s); HDL, high-density lipoprotein; HS,
heparan sulfate(s); LDL, low-density lipoprotein; LDL-R, LDL receptor; LEL, large extracellular loop of CD81; LPL, lipoprotein lipase; NCR, non-coding
region; SR-BI, scavenger receptor BI; TJ, tight junction; TRL, triacylglycerol-rich lipoprotein; VLDL, very-low-density lipoprotein; ZO-1, zonula occludens-1.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
304
Figure 1
M. Perrault and E.-I. Pécheur
A model of a putative HCV particle
In the absence of three-dimensional structures of the virion or any of its structural proteins
(the envelope proteins E1–E2 and the core protein), HCV is represented roughly as a sphere
without any symmetry. The E1–E2 glycoproteins (blue and pink respectively) are set in the
viral envelope (red) via their transmembrane domains. Short cytoplasmic regions are depicted,
forming putative luminal domains of E1 and E2. E1 is drawn approximately to scale with respect
to E2, and they are both in close apposition. Their distribution at the particle surface is left
loose on purpose, since currently available high resolution cryo-TEM (transmission electron
microscopy) examinations of HCVpp [40] or HCVcc [25] did not show any high density layer of
proteins on the lipid envelope. The internal layer is formed by the core protein (green) compacting
the viral RNA genome (black); no structural information is available about the nucleocapsid and
its arrangement (illustration by J.-F. Michel).
Hepacivirus genus. This genus belongs to the Flaviviridae
family, which includes human and animal pathogens (yellow
fever, dengue, West Nile and tick-borne encephalitis, human
flaviviruses, cattle pestiviruses and simian GB-virus B) [8]. HCV
infects only humans and chimpanzees, which sets this virus apart
from the other Flaviviruses. Another peculiarity of HCV is the exceptional low density of the virus particles resulting from the
association of the virus with serum lipoproteins [9]. Indeed,
the majority of HCV circulating in blood was found associated
with β-lipoproteins and VLDL (very-low-density lipoproteins)
and LDL (low-density lipoproteins) [9–11] (see below). A final
peculiarity of HCV is that it targets the liver, and among liver cells,
HCV infects exclusively hepatocytes, the polarized cells where
the virus replicates. However, HCV RNA has been observed in
other human tissues and cells, including B- and T-lymphocytes,
monocytes and dendritic cells. In spite of these observations, no
HCV replication could be detected in these cells. It is thus very
likely that blood cells do not support the full HCV infection cycle
[12]. It has been suggested that B-cells might constitute a reservoir
for HCV and help the virus to persist and to be transmitted to the
liver [13]. The question therefore arises about the specific tropism
of HCV towards liver cells.
HCV isolates from the serum of patients fall into three major
categories, depending on the degree of sequence divergence of
the HCV RNA: genotypes, subtypes and isolates. There are
six major HCV genotypes differing in their nucleotide sequence
by 30–35 %, and a seventh genotype has been discovered in 2008
[14–17]. Within an HCV genotype, several subtypes (designated
as a, b, c etc.) can be defined that differ in their nucleotide sequence
by 20–25 %.
HCV is an enveloped virus which contains a positive strand
RNA genome composed of a 5 -NCR (non-coding region) including an IRES (internal ribosome entry site), an open reading
frame encoding structural and non-structural proteins, and a 3 NCR. The structural proteins include the capsid protein C (core),
compacting viral RNA, and the envelope glycoproteins E1 and
E2, involved in viral entry and membrane fusion (Figure 1).
c The Authors Journal compilation c 2009 Biochemical Society
The non-structural proteins include the p7 ion channel, the
NS2–3 protease, the NS3 serine protease and RNA helicase,
the NS4A polypeptide, the NS4B and NS5A proteins, and the
NS5B RNA-dependent RNA polymerase (RdRp) (for reviews,
see [15,18]). All HCV proteins are anchored to or associated with
cell membranes, implying that the whole viral life cycle deals
with membranes, from the cell entry to the budding [19,20].
Structural proteins compose the viral particle, whereas nonstructural proteins are involved in viral replication and assembly
after cell invasion. To date, no three-dimensional structure of
the structural proteins is available, which seriously hampers our
comprehension of the molecular mechanism by which the virus
infects its target cells, how the nucleocapsid is assembled and,
consequently, of how the virion is formed. Similarly, no highresolution images of HCV are available to date, which would
allow a three-dimensional reconstruction of the whole virus.
However, HCV has been visualized by conventional techniques
in transmission electron microscopy, revealing viral particles
of approx. 60 nm in diameter [21–25] and the presence of the
envelope glycoproteins on its surface [21,26]. HCV envelope
glycoproteins E1 (∼ 31 kDa) and E2 (∼ 70 kDa) are anchored in
the viral membrane by their C-terminal transmembrane domains.
They form a heterodimer stabilized by non-covalent interactions.
This oligomer is most likely to be the structure present at the
surface of HCV particles which is involved in viral entry [27–
30]. Although the exact role of E1 still remains unclear, E2 was
shown to bind a number of entry factors such as glycosaminoglycans, the ubiquitous tetraspanin CD81 or the scavenger
receptor SR-BI (scavenger receptor BI; reviewed in [31–34]).
Therefore virus-associated E2 is likely to be directly involved in
the interactions important for virus attachment and the initiation
of productive infection.
As mentioned above, the density of HCV circulating in the
blood of patients is very heterogeneous, with very-low-, lowand high-density virus fractions. Very-low- and low-density
fractions of HCV are associated with TRL (triacylglycerol-rich
lipoproteins), positive for apolipoproteins B, CII, CIII and E
[11,35]. This heterogeneity, together with low viral titres in the
blood of patients, limited if not precluded the use of plasmaisolated viruses for biochemical analyses that would require a high
number of particles. The advent of HCV pseudotyped particles,
HCVpp, in 2003 was a milestone on the difficult pathway
towards the comprehension of HCV infection and particularly
of mechanisms of HCV entry [36–39]. HCVpp are composed of
a lipid envelope harbouring HCV E1–E2 proteins assembled on
to a retroviral nucleocapsid. We have visualized HCVpp at high
resolution by cryo-transmission electron microscopy (Figure 2)
[40]. They appear as regular 100-nm spherical structures
containing the dense nucleocapsid surrounded by the lipid bilayer.
E1–E2 glycoproteins were not readily visible on the membrane
surface. Although this model system allowed major breakthroughs
in the field of HCV entry research (reviewed in e.g. [31,32,41]), it
only helped to understand the early steps of HCV internalization,
since HCVpp are built around a retroviral core. Another major
milestone came in 2001 with the discovery and isolation by the
group of Wakita of a genotype 2a HCV clone from a patient with
a fulminant hepatitis [42]. This clone, called JFH1 (for Japanese
fulminant hepatitis 1), was able to replicate and produce infectious
viral particles in cell culture, the so-called HCVcc (cell-culture
produced HCV) [21,43,44]. The introduction of this model in
2005 therefore made it possible to study the whole HCV life
cycle in cell culture. From these milestones, the comprehension
of the HCV life cycle entered a new era, and studies dealing with
biochemistry and cell (patho)biology of HCV infection could be
conducted.
Hepatitis C virus and its hepatic environment
Figure 2 Morphology
microscopy
of
pseudoparticles
observed
by
electron
HCVpp (A) and no env pp (a construct without envelope proteins) (B) were observed by cryo-TEM
(transmission electron microscopy) and TEM after negative staining (insets). The central scheme
depicts the constitutive elements: the nucleocapsid (NuC), lipid membrane (LB) and envelope
glycoproteins (depicted at the top of the scheme). The nucleocapsids (black asterisks) and the
lipid bilayers (black arrows) are clearly visible. Glycoproteins are not detectable for HCVpp [40].
Scale bar, 50 nm. Scale bar in inset, 100 nm.
After a brief introduction to the architecture of the liver and
more specifically of hepatocytes, we will focus on the most
recent studies of the complex and subtle interplay between HCV,
hepatocytes and their microenvironment during virus internalization, with an emphasis on three main aspects: receptor recognition in the context of polarized cells such as hepatocytes,
early steps of HCV entry and, finally, the biochemical aspects of
its membrane fusion.
HCV EN ROUTE TO THE LIVER: A TRIP TO AND ACROSS THE
HEPATIC MICROENVIRONMENT
Since the target cell of HCV is the hepatocyte, HCV has to first
travel in the blood from the initial site of contamination. HCV
circulating in the blood can be separated on gradients into highand low-density fractions, the latter demonstrating the association of elements of the virus with the serum β-lipoproteins
VLDL and LDL [9–11,35]. These light fractions were found
to be extremely infectious to chimpanzees [45]. Similarly
for HCVcc, the highest infectivity was observed for lowdensity fractions [43], positive for apolipoproteins B and E
[46], which are components of LDL and VLDL [47]. The
notion that highly infectious HCV represents in fact a ‘lipoviro-particle’ (LVP or low-density fractions containing HCV
RNA) has therefore emerged [15,35,48,49]. As we will see
below, this might be of relevance for driving HCV entry into
hepatocytes.
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HCV approaches the liver in sinusoid capillaries, which form
a fenestrated endothelium composed of endothelial cells and
macrophages, present at the border and the Küpffer cells
(Figure 3A). Leaving the circulation through the fenestrae of
these capillaries, HCV must then cross the space of Disse lined
with hepatic stellate cells (or Ito cells), which play a major
role in the production of the hepatic ECM (extracellular matrix)
[50,51] (Figure 3A). A direct interaction was observed between
a soluble form of HCV E2 and the tetraspanin CD81 present
at the surface of hepatic stellate cells, leading to the activation
of the matrix metalloproteinase MMP-2 [52]. This proteolytic
enzyme is involved in ECM degradation and remodelling, and its
HCV-mediated activation could explain subsequent liver injuries
(inflammation, acute and chronic tissue damage, fibrogenesis).
The space of Disse is therefore a zone of active exchange between
blood and hepatocytes. Hepatic ECM, although forming a very
limited compartment within the normal liver (less than 3 % of
the relative area of a normal liver section [53]), is of major
importance in liver physiology through its scaffolding effect and
its role in biological functions such as cell proliferation, migration,
differentiation and gene expression. It is mainly composed of
collagens [54], of glycoproteins such as laminin, fibronectin,
tenascin and nidogen, and of GAGs (glycosaminoglycans) and
proteoglycans (formed by a ‘core’ protein on to which several
GAG chains are anchored) such as heparin, heparan-, dermatan-,
chondroitin-sulfates, perlecan, hyaluronic acid, biglycan and
decorin [55,56]. The overall density of the highly hydrated gel
formed by the ECM is low, which allows easy diffusion between
blood and liver cells. Interestingly, several viruses were found
to interact with elements of the ECM of their target tissue
[57,58]. Among Flaviviridae, the Dengue virus and the classical
swine fever virus bind to heparan sulfates [59–62]. Molecules
of the ECM were also shown to play a role in HCV adhesion
to the cell membrane, although with somewhat controversial
results. Pietschmann and co-workers showed a deleterious effect
of heparin on HCVcc infection of hepatoma Huh-7 cells [63]. This
agrees with the inhibition of the binding of plasma HCV to Vero
cells after treatment of this plasma with heparin [64], and with
the inhibition of HCVpp binding and infection of Huh-7 cells by
heparin [65,66]. Heparin was also shown to block HCVcc entry
into hepatoma cells at the early step of their membrane binding
[67]. Using purified HCV glycoproteins, both E1 and E2 were
shown to bind to heparin [65,66,68]. Thus heparin was used to
purify plasma HCV [69] and HCVcc [70] efficiently by affinity
chromatography. Conversely, Wakita and co-workers showed a
limited effect of the pretreatment of viral particles with heparin
on HCV infectivity, in spite of an inhibition by heparin of HCVcc
binding to Huh-7 cells. A similar conclusion was reached by
treatment of cells with heparinases [70]. HS (heparan sulfates; in
particular highly sulfated HS) were also reported to play a role in
HCV entry [65,68]. Indeed, synthetic peptides from E2 displayed
a similar affinity towards heparin, HS and dextran sulfate in a
solid-phase heparin-binding assay [71]. These controversial
results might come from the diversity of models used,
parameters observed and technologies applied to the considered
study.
LPL (lipoprotein lipase) is present in the blood and can be
bound to microvilli at the surface of hepatocytes (Figure 3B) [72].
It hydrolyses triacylglycerol from TRLs, and is involved in the
hepatic clearance of TRL-rich lipoproteins from the circulation.
LPL acts by forming a bridge between the lipoprotein and HS
at the cell surface [73]. The membrane-bound (via HS) and
catalytically active form of LPL was found to enhance plasma
HCV internalization into hepatoma cells, but to inhibit HCV
infection [74,75]. It was suggested that by interacting with the
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306
Figure 3
M. Perrault and E.-I. Pécheur
Architecture of a hepatic trabecula (A), a hepatocyte (B) and a simple epithelial cell (C)
TJs are highlighted (green) and delimit the basolateral from the apical membrane of the polarized cell (red).
lipoprotein moiety of the viral particle, LPL would stimulate a
non-productive route of HCV entry, leading to virus degradation.
HCV AND HEPATOCYTES: A FINELY TUNED TOXICITY
After its journey across the hepatic microenvironment, HCV finds
access to the hepatocyte, its target cell for productive infection.
Hepatocytes are the ‘chief’ functional cells of the liver and perform an astonishing number of metabolic, endocrine and secretory
functions. Hepatocytes contribute to roughly 80 % of the mass of
the liver. They are epithelial cells with an unique architecture
as compared with other epithelial cells. In three dimensions,
hepatocytes are arranged in plates that anastomose with one
another in trabeculae (Figure 3A). The cells are polygonal in shape
and their sides can be in contact either with sinusoids (through
their sinusoidal face) or neighbouring hepatocytes (through lateral
faces). A portion of the lateral faces of hepatocytes is modified
to form bile canaliculi [76]. Microvilli are present abundantly on
the sinusoidal face and project sparsely into bile canaliculi. This
biliary pole forms the apical membrane delimited by TJs and is
c The Authors Journal compilation c 2009 Biochemical Society
equivalent to the apical pole of simple epithelial cells (Figure 3B).
Owing to the particular structure of their apical pole, at least
two faces of hepatocytes are in contact with blood sinusoids and
form the basolateral or sinusoidal pole. This pole does not sit
on a basal lamina, in contrast with simple epithelia (Figure 3C),
but it is in contact with the hepatic ECM. At their basolateral
pole, hepatocytes capture nutrients, information from their
microenvironment and substances to be transported to the bile,
and several receptor or effector molecules are concentrated at this
membrane, such as integrins (receptors to elements of the ECM),
transporters of nutrients, transferrin or lipoprotein receptors
and tetraspanins. At the apical pole, hepatocytes secrete bile
components and re-absorb other molecules. Membrane proteins
such as transporters of bile acids and multi-drug-resistance
proteins (ATPase pumps for various substrates) are localized at
this pole [77]. The formation of TJs is contemporary with that
of the apical pole, and the development of membrane polarity
is integral to the process of hepatocyte differentiation through
cell–cell contact, cell–ECM contact or both [78,79]. Importantly,
the formation of such specialized membrane domains implies
different protein compositions at the apical and basolateral poles.
Hepatitis C virus and its hepatic environment
In this context, HCV probably ‘lands’ on the hepatocyte
basolateral membrane, where several molecules will ensure its
‘guidance system’ into the cell.
Integrins
RGE/RGD is a well known cell adhesion motif for integrin
recognition [80]. This motif is also present in the sequences of
flaviviral envelope proteins and involved in virus entry [81,82]. It
is also found in the HCV E2 glycoprotein and conserved among
various virus genotypes. However, its role in HCV entry through
the recognition of an integrin molecule could not be demonstrated
[83].
LDL-R (LDL receptor)
Since HCV is found associated to serum β-lipoproteins, the
LDL-R has long been considered a candidate molecule involved in
HCV entry [9,84]. Physiologically, LDL-R transports cholesterolrich LDL particles from the extracellular medium into cells
by clathrin-dependent endocytosis. The endocytosis of plasma
HCV mediated by LDL-R was established by Agnello et al.
[85], relying on the association of the virus to LDL and VLDL,
but not to HDL (high-density lipoprotein). However, this was
performed with hepatoma cells where HCV replication was
not fully supported. Primary cultures of highly differentiated
human hepatocytes are therefore likely to represent the most
physiologically relevant model to investigate serum-derived HCV
infection. Using plasma HCV and primary human hepatocytes
freshly isolated from HCV-negative liver biopsies, Maurel and
co-workers clearly demonstrated that the LDL-R is involved in an
early stage of HCV infection, through elegant approaches based
upon competition with LDL, peptides from LDL and antibodies
to the LDL-R [86]. However, LDL-R is not present only on
hepatocytes, and expressing this receptor ectopically does not
restore cell responsiveness to HCV. This indicates that other
‘players’ are required in the ‘toxic game’ to mediate HCV entry.
The HDL receptor, SR-BI
SR-BI is mainly present at the basolateral pole of hepatocytes,
but is also found at the apical membrane [87,88]. It was found
to interact directly with cholesterol by a photolabelling approach,
suggesting its localization in cholesterol-enriched domains of the
membrane, also called ‘rafts’ [87]. It behaves as an authentic
receptor for HDL. After binding of its ligand, SR-BI could
undergo a rapid internalization into early endosomes by clathrindependent endocytosis, leading to removal of cholesterol and
recycling of the protein part of HDL into recycling endosomes
[89]. However, another pathway of cholesterol uptake that did
not involve endocytosis has also been reported [90]. Recent data
indicate that SR-BI plays a role in the metabolism of VLDL, and
could be a receptor for these lipoproteins as well [91]. SR-BI
is highly expressed in steroidogenic cells as well as in the liver.
Its implication in HCV entry was first suggested by the group
of Vitelli in 2002, which showed that a soluble form of E2
(sE2) could bind to human hepatoma cells, and the isolated
receptor molecule was SR-BI [92]. The binding of sE2 to human
hepatoma cells could also be blocked by an anti-SR-BI serum
[36], and direct binding of sE2 to cells non-permissive for
HCV infection but expressing SR-BI was recently demonstrated
[93]. Concerning the whole virus, conflicting results have been
reported, some suggesting a glycoprotein-dependent SR-BI–
HCV interaction [93], other reporting an interaction between
307
HCV and SR-BI through the lipoprotein part of the viral particle,
namely apolipoprotein B [94]. This apparent discrepancy might
come from the use of viruses from different origins, HCVcc
derived from the plasma of mice transplanted with human
hepatocytes in the former case, HCV derived from the plasma
of infected patients in the latter.
Infection of Huh-7 cells [36] and of primary human hepatocytes
[95] by HCVpp was prevented by anti-SR-BI antibodies and SRBI gene extinction strategies. Similar conclusions were reached
using HCVcc and human hepatoma cells or primary human
hepatocytes [67,96]. Altogether, these results defined SR-BI as
an essential factor for HCV entry. The establishment of cell
lines where ectopic expression of SR-BI restored HCV infection
allowed the demonstration that the lipid transfer function of SRBI should be intact to convey HCV entry [97]. Since SR-BI is
mainly considered an HDL receptor, several investigations dealt
with the relationship between its capacity to bind HDL and
its involvement in HCV entry. Interestingly, HDL was found
to enhance HCV infectivity [96,98–101], although no direct
interaction between HCV and HDL could be observed [98,101]
(for a specific review, see [75]). Anti-SR-BI antibodies abrogated
this enhancing effect, through a subtle interplay between inhibition of HCV binding to SR-BI, inhibition of HDL binding to
SR-BI/cells and/or inhibition of the SR-BI lipid transfer function
[67,93,96]. This led to the conclusion that SR-BI could mediate
HCV infection in the absence of lipoproteins, which would then
uncouple its HDL transport function from its ability to act as
a receptor for HCV. How HDL enhances HCV infectivity still
remains unclear. A role for apolipoprotein C1, a minor component
of HDL [47], has been suggested [100,102,103]. ApoC1 would
play a role through its association with the HCV particle, that
could subsequently enhance its membrane fusion properties.
The tetraspanin CD81
This tetraspanin was the first molecule described to interact
with a soluble form of HCV E2 [104], and therefore proposed
as an HCV receptor candidate. Ectopic expression of CD81
in CD81-negative hepatoma cells non-permissive to HCV infection restored permissiveness [36,105,106], or increased HCV
infectivity in permissive cell lines [107]. Recently, plasma
HCV entry into primary human hepatocytes was shown to be
inhibited by antibodies directed to CD81 or by CD81 gene
silencing [108]. Together these data established that CD81 is an
essential factor for HCV entry. CD81 belongs to the vast family
of tetraspanins, structurally related proteins with four membranespanning domains, and is an ubiquitous molecule present, e.g.,
on various epithelial cells, blood cells and myocytes [109]. The
reader is referred to reviews dedicated to their structure and
function [109–113]. These proteins are organized in the so-called
tetraspanin web, where tetraspanins interact with themselves,
other tetraspanins and/or integrins forming a net with a fine
mesh. The involvement of this web in downstream signalling
cascades by recruiting other protein partners intracellularly is
one of its key functions. CD81 has no known endogenous ligand
and does not possess any intracellular internalization motif [109].
This could explain its very slow endocytosis process [114]. In
the liver, CD81 is expressed on sinusoidal endothelium and on
hepatocytes, where its localization is mainly basolateral, although
some CD81-specific staining has been observed at the apical pole
[88]. CD81 ectodomain is formed by two extracellular loops, a
large (LEL) and a small (SEL) one. The LEL was shown to be
the reception platform for HCV [104], involving specific regions
of E2 [115–119]. A high density of cell-surface-exposed CD81
appeared to be crucial for productive HCV entry into human
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M. Perrault and E.-I. Pécheur
hepatoma cells [107]. However, the cascade of events triggered
after HCV interaction with CD81 remains unclear. Owing to the
slow internalization process of CD81, three hypotheses could be
raised that are not mutually exclusive: (i) HCV internalization
is mediated by other cell surface molecules; (ii) other surface
molecules promote CD81/HCV entry; (iii) HCV binding to CD81
induces a rapid internalization of the virus–receptor complex.
We have previously seen that other molecules play a role in
HCV entry. Recently, the idea emerged that HCV internalization
could involve a subtle interplay between CD81 and SR-BI.
Both molecules were found to act in a co-operative manner to
govern internalization of HCVcc in Huh-7 cells [120]. Further
investigation showed that HCVcc bound to cells non-permissive
for HCV infection only when SR-BI was expressed, but not CD81
[121]. Using antibodies against CD81 and SR-BI, Baumert and
co-workers showed that the kinetics of the inhibition of HCV
infection were comparable for both antibodies, suggesting that
HCV entry steps mediated by CD81 and SR-BI are linked to
each other [67]. Taken together, these data suggest the following
sequence of interaction: (i) SR-BI; (ii) CD81. However, further
investigation will be needed to determine the exact role of each
molecule in the early steps of HCV recognition at the molecular
level; it also remains to be determined whether one of these
molecules, both or none is(are) endocytosed along with HCV.
Claudin-1
Intriguingly, none of the ‘landing platform’ molecules for HCV
described above explains the hepatotropism of this virus. Indeed,
all of them are ubiquitous and involved in essential functions
for various tissues or cell types. A further level of complexity
came in 2007 with the astonishing discovery that claudin-1, a
protein of the cellular TJ, acted as a co-receptor for HCV entry
at a late post-binding step [121]. As described above, TJs in the
hepatocytes delimitate the apical membrane (Figure 3B), which is
not readily accessible for an element such as HCV coming from
the lumen of sinusoid capillaries. This discovery raises several
questions. (i) How does HCV find its way to the apical membrane
to encounter claudin-1? (ii) Why and how does HCV use such
a tedious pathway to enter hepatocytes? (iii) Is the polarity of
hepatocytes critical to explain HCV entry? Conversely, one can
wonder how specific claudin-1 localization is to TJs, how mobile
it is or could be, and whether HCV itself could trigger intracellular
signals leading to re- or mis-localization of claudin-1 to the
basolateral membrane.
TJs can be seen as seals between neighbouring cells, forming
discontinuous ridges in electron microscopy [122,123]. They are
essential in maintaining different lipid and protein compositions
on the apical and basolateral poles in polarized cells [124]. The
backbone of the TJ is organized as strands composed mainly of
claudins [125]. Claudins form a superfamily of proteins, all related
to functions in cell–cell contact, differentiation and proliferation.
They are integral membrane proteins with four transmembrane
passages and two extracellular loops, EL1 and EL2. Among
claudins, claudin-1 is expressed in the liver, but is not restricted to
this organ [126]. This molecule is therefore not the explanation
of HCV hepatotropism. In addition to claudins, TJs are composed of the cytosolic protein ZO-1 (zonula occludens-1) and
of the integral membrane protein occludin [124]. Owing to their
function, TJs are largely viewed as static structures under steadystate conditions. The group of Turner recently demonstrated that
this view could be revised at the light of their data obtained by
FRAP (fluorescence recovery after photobleaching) on TJs of
polarized kidney cells. Indeed, fluorescently tagged proteins
of the TJ displayed distinct behaviours towards FRAP, with
c The Authors Journal compilation c 2009 Biochemical Society
claudin-1 remaining stably localized at the TJ, occludin diffusing
rapidly within the TJ, and ZO-1 displaying a highly dynamic
behaviour and exchanging with intracellular pools [127]. It
therefore appears that binding interactions at the TJ are much
more dynamic than thought. However, claudin-1 and occludin
either remained localized at or moved within the TJ, suggesting a
somewhat constrained distribution.
Interestingly, in sections of normal liver, claudin-1 is found
at the apical/canalicular TJ region as well as at the basolateral/
sinusoidal membrane of hepatocytes [88]. Human hepatoma
HepG2 cells transfected with CD81 displayed a similar pattern,
and the pharmacological alteration of TJ integrity had only
minimal effects on cell polarity and HCV infection [128].
Altogether, these studies support the notion that a pool of claudins
present at the basolateral pole (and therefore non-junctional)
could be involved in HCV recruitment to the membrane. At the
basolateral membrane, claudin-1 co-localizes with SR-BI, but at
the apical pole it is located close to CD81 [129]. This would be
consistent with SR-BI acting before CD81 for HCV membrane
recruitment. A proposed sequence of events could then be
SR-BI, then CD81 and claudin-1, all present at the basolateral
pole. Interestingly, claudin-1 expression is enhanced at the
basolateral membrane in HCV-infected liver cells, where it
then co-localizes with CD81 [129]. Conversely, the pattern of
expression of SR-BI and CD81 remains unchanged [88]. This
suggests that HCV could induce re- or mis-localization of claudin1 to the basolateral membrane, thereby enhancing the physical
number of molecules available for interaction with the virus.
The hypothesis of an initial interaction between viral and cellular
partners at the basolateral pole does not contradict the view that
HCV could ‘travel’ from the basolateral to the apical membrane
to reach cell–cell contact zones enriched in TJ proteins, notably
claudin-1. Different forms of claudin-1 could be involved in HCV
entry [88,121]; the structure and localization of these forms are
unknown at present, and one could hypothesize that different
forms would be expressed in TJs and in non-junctional zones.
It also remains to be determined where effective internalization
occurs. Also, the nature of the interaction between claudin-1
and HCV is still a matter of debate, but increasing evidence
indicates that neither E1 nor E2 interact directly with any region
of claudin-1 [121,130]. Further studies are needed to clarify
these points, in particular in terms of binding and internalization
kinetics.
Occludin
A major breakthrough came most recently with the discovery
of occludin, a component of the TJ, as an HCV entry factor
[131,132]. Cells non-permissive to HCV infection became
permissive to HCVpp and HCVcc infection by expressing all
four molecules at their surface. These four molecules, SR-BI,
CD81, claudin-1 and occludin, present together on hepatocyte
membranes (but not necessarily on the same membranes nor
interacting together), seem therefore to be the ultimate quartet in
the finely tuned partnership HCV establishes with the hepatocyte
at the onset of infection. This again places hepatocyte polarity and
TJs at the center of the chessboard [133]. Occludin depletion did
not perturb claudin-1 expression or localization, suggesting that
both proteins function separately for HCV entry, but it severely
impaired HCV entry [131,134]. The initial binding steps of HCV
to the cell membrane were not affected by occludin gene silencing,
suggesting that occludin plays a role in a late entry event [134]. At
the TJ, occludin binds ZO-1 which in turn binds claudin-1 [126].
A close interaction between occludin and claudin-1 at the TJ
was suggested [135]. At present, a fine localization of occludin
Hepatitis C virus and its hepatic environment
309
precisely the absence of this molecule, to the ‘toxic game’ [136].
It was indeed demonstrated that HCV infection of Huh-7 cells
could be blocked by expressing the protein EWI-2wint (EWI-2
without its N-terminus) at the surface of these cells. EWI-2wint
is a cleavage product of EWI-2, which is known to be a partner
of the tetraspanin web [113], interacting with CD81 in a highly
stoichiometric manner [137,138]. It contributes to a variety of
CD81 functions seen in different cell types and tissues. EWI-2
was detected in human liver at the surface of hepatocytes, and is
associated with CD81 on freshly isolated hepatocytes [139]. EWI2wint is absent from hepatocytes [136], but present on other cells
harbouring EWI-2. It is able to inhibit the interaction between
CD81 and HCV glycoproteins. This is the first description of a
factor that could confer very specific properties to the membrane
of a cell, the hepatocyte, by being absent from that membrane.
This might therefore be the very first step toward the thorough
comprehension of the strict hepato-tropism of HCV.
EARLY STEPS OF HCV INTERNALIZATION
Figure 4 Proposed model for HCV interaction with its entry factors at the
surface of the hepatocyte
HCV would recognize SR-BI at the basolateral membrane (green), followed by its binding to
CD81 (blue). It would then migrate along the lateral face of the hepatocyte and encounter
claudin-1 (C) and occludin (O) at the TJ on the apical membrane. This could be followed
by clathrin-dependent endocytosis of the virus, engulfed into a clathrin-coated vesicle (clathrin
cage in brown), that would migrate along microtubules. Since HCV is found associated with
lipoproteins in the blood, this form is depicted as a virus with a yellow coating. It could recognize
the LDL-R at the basolateral membrane (orange, curved symbol) and SR-BI. The question mark
indicates that virus uptake by this pathway remains unclear. Entry factors are represented at the
membranes where they were reported to be present, and we hypothesize that SR-BI is the initial
contact molecule for HCV.
is not available yet; the question remains whether it could be
localized outside the TJ, e.g. at the basolateral pole in a similar
manner to what was observed for claudin-1. In the current context,
and considering the fact that claudin-1 and occludin are mainly
confined to the TJ [127], a model for HCV sequential recruitment
of these four molecules at the hepatocyte membrane is proposed
(Figure 4).
IN SEARCH FOR THE ‘HEPATO-TROPISM MOLECULE’ OR
EWI-2WINT, THE ABSENT PLAYER
At this point, we are still left with a major question: what
defines the propensity of HCV to infect hepatocytes and not
other cell types? None of the four molecules described above
is specific to the liver or to the hepatocyte. Is it a subtle (and
finely tuned) combination of each of these molecules, possibly
recruited by the virus itself that defines HCV hepatotropism?
Or a specific combination between these molecules in terms of
interaction kinetics? These questions remain entirely open at
present. Addressing the question in different terms, the group
of Dubuisson recently introduced another molecule, or more
After its interaction with several molecules, HCV is sent to intracellular compartments. Biochemical approaches using chemical
inhibitors of endosome acidification showed that HCV entered
cells via a pH-dependent pathway [36,39,140,141]. Strategies
blocking clathrin polymerization, silencing the clathrin heavy
chain gene or using dominant-negative mutants of proteins
involved in endocytosis resulted in the inhibition of HCVpp
or HCVcc entry into hepatoma cells [141–143]. This points to
a prominent role for clathrin-dependent endocytosis in HCV
internalization. However, it was suggested that additional low
pH-dependent events occurring after internalization would be
required for productive HCV entry [141]. The nature of these
events remains unclear. It is also unclear whether any of the
‘landing platform’ molecules, and in particular the quartet of
molecules used by HCV to enter cells, would be internalized
concomitantly and, if internalized, by which pathway. Several
viruses have developed various strategies to enter their target
cells (for a recent review, see [144]). Concerning HCV, not only
the involvement of receptor molecules in its endocytosis remains
to be determined, but also further investigation is clearly needed to
define whether other pathways of entry could exist. A recent
study demonstrates that intact and dynamic microtubules are
required for HCV entry into cells, and for post-fusion steps as well
[145]. Clathrin-coated vesicles transporting HCV may therefore
migrate along microtubules to ensure an efficient delivery of the
nucleocapsid at appropriate intracellular sites.
HCV MEMBRANE FUSION
On its pathway to productive infection, HCV has now arrived in
an intracellular compartment considered to be early endosomes.
The virus will have to get rid of its envelope, which will release the
viral nucleocapsid into the cytosol. This corresponds to the fusion
step, where the envelope of the virus will form one continuum
with the endosome membrane. Envelope proteins are thought to
play a key role in HCV membrane fusion.
Viral membrane fusion has been the subject of a plethora of
reviews, bringing out several common features from different
viruses and structurally unrelated fusion proteins (for recent
reviews, see [146–151]). Indeed, for viral fusion to occur, two
minimal partners are required: a fusion machinery and lipids.
These two partners must act in concert and in a co-operative
fashion so that the fusion process can be completed. Fusion machineries have therefore two things in common: (i) they interact
c The Authors Journal compilation c 2009 Biochemical Society
310
M. Perrault and E.-I. Pécheur
with lipids, so they possess hydrophobic segments (fusion
peptides or loops) or are able after rearrangements to enter into
hydrophobic interactions with membranes; (ii) they adopt specific
conformations related to the fusogenic and non-fusogenic states,
since fusion is limited in space and time. Dissimilar viruses could
therefore infect their host cells by very similar mechanisms at
the molecular level of proteins and lipids. Interestingly, similar
mechanisms can be brought about by structurally different fusion
machineries [146]. Concerning HCV, as already mentioned, no
three-dimensional structure of its envelope proteins is currently
available, which renders it difficult any study of the HCV fusion
mechanism at the molecular level. In this context, the first
questions to be addressed were the following. (i) Are HCV
envelope proteins involved in membrane fusion? (ii) Is fusion
pH-dependent? Does it depend on specific lipids? (iii) Which
protein is the fusion protein and where is the fusion peptide/loop?
A convenient way to assay viral membrane fusion directly is
to use artificial target bilayers or liposomes whose sizes can be
controlled, and in the membrane of which fluorescent lipid probes
have been incorporated. Fusion between virus and liposomes is
then measured as an increase in the fluorescence signal of the
probe [152]. Using this elegant in vitro fusion system, we could
demonstrate that HCV fusion relies on the presence of the functional envelope glycoproteins at its surface, for both HCVpp [153]
and HCVcc [154]. Indeed, viral particles devoid of the envelope
proteins did not display any fusion. This was corroborated in a
cell–cell fusion assay where the E1–E2 complex was expressed
at the surface of HEK-293T cells [human embryonic kidney 293
cells expressing the large T-antigen of SV40 (simian virus 40)]
[155]. In vitro fusion occurred only at low pH, but at a broad range
which would correspond to the pH of early to late endosomes
inside the cell [153–155]. This suggests that HCV could fuse
its envelope with the membrane of different endosomal compartments along the endocytic pathway. Cholesterol of the target
membrane was found to play a critical role in the fusion process.
Indeed, fusion was enhanced when cholesterol was incorporated
into liposomes [153]; conversely, cholesterol depletion from the
cell membrane inhibited HCV infection [120]. Cholesterol of
the viral membrane was also reported to play a key role in fusion,
since HCV depleted from its membrane cholesterol displayed
only minimal infectivity [156]. Interestingly, sphingomyelin had
a strong enhancing effect on HCV fusion when target membranes
also contained cholesterol [154], and sphingomyelin from the
viral membrane played a crucial role in HCV cell infectivity
[156]. This suggests that microdomains composed of cholesterol
and sphingomyelin, the so-called rafts, could play a role in HCV
entry, specially at the step of membrane fusion.
As mentioned above, HCV has the ability to associate with
β-lipoproteins and light fractions of HCVcc were found to be positive for apolipoproteins B and E. Indeed, we recently showed that
these fractions display the highest infectivity, which could be correlated with their higher fusogenicity as compared with fractions
of high density [154]. This is a strong indication that the lipids
associated with HCV, whatever this association may be [48], play
a key role in the process of HCV membrane fusion.
In the absence of structural data, several studies aimed at
determining which HCV glycoprotein is the fusion protein. These
studies were based upon indirect strategies analysing the effect
of point mutations in E1 or E2 on HCV infectivity and fusion.
From the postulate that a fusion protein contains (a) fusion determinant(s), being either a fusion peptide or a fusion loop, we
introduced carefully selected mutations at specific sites of E1 or
E2, expressed in the context of HCVpp. This strategy proved
useful, since it revealed the existence of three regions defined as
potential fusion peptide candidates [157]. One region is in E1,
c The Authors Journal compilation c 2009 Biochemical Society
the others in E2. Interestingly, three point mutations in a similar
region of E1 reduced HCVpp infectivity and severely perturbed
HCVcc production in hepatoma cells [158]. This region of E1
was also pointed out as a fusion peptide candidate in a study
by the group of Poumbourios [159]. Recently, we demonstrated
that one residue in E2 defined in the HCVpp context as essential
for infectivity and fusion also played a similar key role in the
HCVcc context [154]. This residue is a glycine at position 418 of
the sequence of E2, nearby a highly conserved glycosylation site
(Asn417 ). Therefore this region is probably not directly involved in
membrane destabilization, but it could act as a fusion ‘helper’ or
facilitator through (low pH-induced?) structural rearrangements
at the onset of fusion. These results also pointed to a crucial role
played by E2 in HCV membrane fusion. However, the question
of which protein is the fusion protein remains largely open at
present.
Recently, the group of Majano showed that occludin was
required for HCV glycoprotein-dependent cell–cell fusion [134].
This raises the elegant hypothesis that at least one protein shown
as a key factor for HCV entry could play a role in the fusion
mechanism. This would be consistent with the involvement of
occludin in late post-binding steps of HCV entry. However, we
are left with the question of how it would play this role. By
priming the fusion protein? Outside and/or inside the cell? In a
pH-dependent manner?
CONCLUSIONS AND PERSPECTIVES
Further studies are needed to understand the molecular aspects
of HCV entry and membrane fusion. Although HCV belongs to
the family of Flaviviridae, which includes viruses with fusion
proteins lying flat on the surface of their lipid envelope (class
II fusion proteins), HCV in several molecular details of its life
cycle does not behave as other members of this family. HCV is
a strict human pathogen, it is found associated with β-lipoproteins and displays a high heterogeneity in density (and therefore
probably in morphology). The ectodomains of HCV E1 and E2
are small compared with those of envelope proteins of other
viruses of the Flaviviridae family [18], and E1 or E2 share only
little sequence homology with these latter envelope proteins. This
structural difference probably translates into functional aspects
that are peculiar to HCV. However, the structure of the virus and
of its envelope proteins is currently unknown. HCV internalization
requires a quartet of receptors necessary and sufficient to allow
productive infection, but none of these molecules explains HCV
hepatotropism. Surprisingly, an explanation to this hepato-tropism
could come from the absence of a factor from the surface of
hepatocytes, which is the first description of a cellular property
conferred by the absence of a molecule.
In conclusion, the path to a thorough comprehension of HCV
infection is still long, and understanding HCV peculiarity is
essential for the development of an efficient and targeted pharmacological arsenal aimed at combating HCV toxicity.
ACKNOWLEDGEMENTS
E.-I. P. thanks Jean-Francis Michel for the expert drawing of a schematic HCV particle in
Figure 1, Agata Budkowska for critical reading, Elodie Teissier for thoughtful comments
and Dick Hoekstra for just being Dick Hoekstra.
FUNDING
This work was supported by the CNRS (Centre National de la Recherche Scientifique) and
by the Agence Nationale de Recherche contre le SIDA et les hépatites virales (ANRS) (to
E.-I. P.).
Hepatitis C virus and its hepatic environment
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