Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease worldwide. With 180 million persistently infected people, chronic hepatitis C infection, which induces end-stage liver disease such as liver cirrhosis and hepatocellular carcinoma, represents a major public health problem of high socio-economic impact (Ref. Reference Shepard, Finelli and Alter1). However, treatment options for chronic hepatitis C are limited and a vaccine is not available.
HCV is an enveloped, positive-stranded RNA virus of the hepacivirus genus of the Flaviviridae family (Ref. Reference Lindenbach, Rice, Knipe and Howley2). The size of its genome is approximately 10 kb, comprising a large open reading frame encoding a precursor polyprotein of approximately 3000 amino acids. Cleavage of this polyprotein occurs at translation and afterwards to generate at least 11 viral proteins, including a core protein, two glycoproteins, E1 and E2, which are exposed at the surface of HCV particles, and several nonstructural proteins (Ref. Reference Penin3). The transmembrane domains of both glycoproteins contain heterodimerisation and endoplasmic reticulum (ER) retention sequences (Refs Reference Cocquerel4, Reference Cocquerel5, Reference Cocquerel6). Consistent with the predominant localisation of these glycoproteins in the ER, HCV is thought to assemble intracellularly, which is a common trait of the Flaviviridae (reviewed in Ref. Reference Murray, Jones and Rice7).
Another characteristic feature of HCV is the heterogeneity of the viral forms in vivo (Refs Reference Andre8, Reference Bradley9, Reference Hijikata10, Reference Nielsen11, Reference Thomssen12, Reference Felmlee13). HCV RNA is usually detected with diverse buoyant density ranging from <1.06 to >1.25 g/ml, in the blood of infected patients, and this structural heterogeneity results from virion binding to or association with different serum components such as immunoglobulins and lipoproteins as well as from ‘naked’, envelope-free nucleocapsids (reviewed in Refs Reference Burlone and Budkowska14, Reference Andre15). The topology and the assembly process of the low-density, lipoprotein-associated particles are still being debated. HCV particles might directly bind to lipoproteins or incorporate lipoprotein components, such as lipids and apolipoproteins, either in trans (through their interaction within the blood of infected patients) or in cis (through their interaction in virus producer cells). The low-density HCV particles, also called lipoviro particles (LVPs), have been purified from the plasma of chronically infected patients and partially characterised. Models of LVP structure consisting of an HCV core protein as well as viral RNA and E1 and E2 glycoproteins encrusted into lipoprotein-like structures enriched in phospholipids, neutral lipids (e.g. cholesteryl esters and triacyglycerols) and several apolipoproteins (ApoB, ApoC and ApoE) have been proposed (Refs Reference Andre8, Reference Nielsen11, Reference Felmlee13, Reference Andre15, Reference Icard16, Reference Bartenschlager17, Reference Meunier18). The remarkable property of HCV to associate with β-lipoprotein might modulate receptor usage and entry routes and might protect HCV from anti-HCV or anti-E2 antibodies, by the shielding of epitopes on viral particles (Refs Reference Andre8, Reference Nielsen11, Reference Maillard19, Reference Prince20, Reference Agnello21, Reference Molina22).
So far, the investigation of natural HCV and its different lipoprotein- or immunoglobulin-associated forms has been laborious because the infectivity of HCV-containing sera ex vivo, in either primary hepatocytes or hepatoma cell lines, is very low (Refs Reference Rumin23, Reference Fournier24). Furthermore, the mechanisms by which LVPs mediate cell penetration, leading to release of HCV genetic material and replication, have not yet been characterised functionally.
To overcome these severe limitations of the molecular characterisation of HCV infection, several surrogate assays or tools have been developed (Table 1). Two relevant and complementary infection assays consist of cultured genuine HCV (HCVcc) derived from a fulminant hepatitis C, JFH-1 and JFH-1-derived recombinant genomes (Refs Reference Lindenbach25, Reference Wakita26, Reference Zhong27), and of HCV pseudo particles (HCVpp) harbouring authentic E1E2 glycoproteins, which are particularly amenable to mutagenesis analysis (Refs Reference Bartosch, Dubuisson and Cosset28, Reference Drummer, Maerz and Poumbourios29, Reference Hsu30). Similarly to HCV derived from plasma, HCVcc RNA displays a broad density profile with high specific infectivity associated with low-density fractions, which might reproduce some features of HCV association with lipoprotein compounds (Refs Reference Lindenbach25, Reference Podevin31, Reference Yi32, Reference Gastaminza, Kapadia and Chisari33, Reference Bankwitz34, Reference Grove35, Reference Lindenbach36). In a recent study, the lipid composition of HCVcc particles was determined and found to resemble that of low-density lipoproteins (LDLs) and very low-density lipoproteins (VLDLs) (Ref. Reference Merz37). Importantly, highly purified HCVcc particles contain several ApoE molecules on their surface as well as ApoC-I and, to some extent, ApoB, in line with the finding that HCV formation and secretion depend on the VLDL assembly and secretion pathway (Refs Reference Meunier18, Reference Merz37, Reference Gastaminza38, Reference Chang39, Reference Huang40, Reference Jiang and Luo41, Reference Cun, Jiang and Luo42, Reference Hishiki43, Reference Owen44, Reference Benga45). Although HCVccs, which are produced in human hepatocarcinoma cells (typically Huh-7.5 cells), further permit investigation of the late steps of infection, HCVpps offer a particularly flexible platform to study the structure–function relationship of HCV glycoproteins, both in cell culture and in vitro, in liposome membrane-fusion assays (Refs Reference Lavillette46, Reference Lavillette47). Of note, because they are produced from 293T kidney cells, HCVpp particles are not associated with lipoproteins (Ref. Reference Flint48), which therefore allows the investigation of cell entry events specifically linked to the function of E1E2 glycoproteins and reconstitution of some of the interactions of HCV with lipoproteins or apoliproteins (Refs Reference Bartosch49, Reference Dreux50, Reference Dreux51, Reference Voisset52, Reference Meunier53). Thus, both HCVcc and HCVpp infection assays reproduce several cell entry characteristics of native HCV and together allow precise dissection of the cellular and viral factors involved in the early events of HCV infection.
Table 1. Major assays and tools for the study of HCV entry
Viral attachment and entry, which represent the first encounter of the virus with the host cell (Fig. 1), are major targets of the adaptive immune response. The detailed understanding of the HCV entry process should offer interesting opportunities and targets for the development of novel therapeutic strategies to prevent or cure HCV infection. HCV entry is thought to be a multistep process (reviewed in Refs Reference Burlone and Budkowska14, Reference Cocquerel, Voisset and Dubuisson54, Reference Bartosch and Cosset55, Reference Dubuisson, Helle and Cocquerel56, Reference von Hahn and Rice57). The interactions of envelope glycoproteins or apolipoproteins incorporated on the viral surface with heparan sulfate proteoglycans (HSPGs) might contribute to the primary binding of virus particles to host cells and capture from the bloodstream (Ref. Reference Barth58). Because of the association of HCV with VLDL and LDL and/or the viral incorporation of lipoprotein components such as ApoE and LDL receptor (LDLR), might provide alternative docking sites (Refs Reference Agnello21, Reference Molina22, Reference Owen44). Following this initial engagement, a minimal set of four cell-entry factors comprising the scavenger receptor class B type I (SR-BI/SCARB1) (Ref. Reference Scarselli59), the CD81 tetraspanin (Ref. Reference Pileri60), and the tight junction proteins Claudin-1 (CLDN1) (Ref. Reference Evans61) and occludin (OCLN) (Refs Reference Ploss62, Reference Liu63) contribute to binding, uptake and internalisation of HCV in a clathrin-dependent manner (Refs Reference Blanchard64, Reference Meertens, Bertaux and Dragic65). The polarity of hepatic cells and the cellular distribution of these HCV entry factors might have a role in viral entry or propagation (Refs Reference Mee66, Reference Mee67). SR-BI, similarly to CD81, is expressed at the sinusoidal surface of hepatocytes within normal tissue (Ref. Reference Reynolds68), which is consistent with physiological routes of viral access. That HCV entry depends on tight junction proteins suggests that it exploits such intercellular structures for transmission, although evidence is limited, owing to the lack of suitable polarised cell culture models (Ref. Reference Mee66). Nevertheless, there is evidence for cell–cell transmission of HCV, which, compared with cell-free transmission, might favour HCV dissemination into its host by optimising viral particle delivery and by limiting the impact of neutralising antibodies (Refs Reference Timpe69, Reference Witteveldt70, Reference Brimacombe71).
Figure 1. Cell entry of HCV. Native HCV appears under heterogeneous forms, associated or not with lipoprotein components, complexed with immunoglobulins or free of envelope. For the sake of clarity, only two representations of HCV are shown here, with and without association with lipoprotein. (a) HCV entry is thought to be a multistep process. SR-BI, along with other attachment factors such as the LDLR and HSPGs, might contribute to the primary binding of viral particles to host cells as a result of its association with lipoprotein components. Following this initial engagement, SR-BI might then mediate virion binding, concomitantly with CD81, initiating downstream events such as uptake and cellular internalisation, possibly involving the other HCV-entry factors CLDN1 and OCLN. (b) SR-BI-mediated lipid uptake of HDL CE leads to an increase in the cholesterol content of the target cell membrane (1). By creating membrane microdomains, enriched in cholesterol, this function might favour the association of the four entry factors to form a receptor complex. The formation of this complex possibly activates distinct signalling pathways that might provide different beneficial roles for HCV entry, during internalisation or fusion processes. SR-BI might also contribute to the release of ApoC-I, an exchangeable apolipoprotein, and subsequent recruitment on the HCV particle surface would favour fusion of viral and cell membranes (2). (c) The HCV receptor complex mediates the internalisation of HCV particles by clathrin-dependent endocytosis. The identity of the HCV-entry factors directly involved in this process remains elusive. (d) SR-BI-mediated local cholesterol enrichment of target membranes might favour pH-dependent viral fusion processes, which result in the release and delivery of viral RNA to the cytoplasm. The role and localisation of the HCV-entry factors during such fusion is so far unknown. Abbreviations: CE, cholesteryl ester; CLDN1, Claudin-1; HCV, hepatitis C virus; HDL, high-density lipoprotein; HSPG, heparan sulfate proteoglycan; LDLR, LDL-receptor; OCLN, occludin; SR-BI, scavenger receptor class B type I; TAG, triacylglycerol.
Using HCVpp and HCVcc infection assays as well as in vitro membrane fusion assays, HCV entry was shown to occur in a pH-dependent manner (Refs Reference Hsu30, Reference Lavillette46, Reference Bartosch72, Reference Lavillette73, Reference Tscherne74), through endocytosis of the viral particles (Refs Reference Blanchard64, Reference Meertens, Bertaux and Dragic65). Similarly to other Flaviviridae (Ref. Reference Kielian and Rey75), the low endosomal pH might induce conformational rearrangement of HCV glycoproteins, leading to fusion of the viral membrane with that of the endosome. Important progress has been made in recent years to decipher the function of these HCV-entry cofactors, particularly SR-BI, which will be discussed here, and how they permit HCV attachment, internalisation and membrane fusion.
SR-BI is a 509 amino acid glycoprotein (57 kDa) with two C- and N-terminal cytoplasmic domains separated by a large extracellular domain that is involved in lipid metabolism. SR-BI was originally called CLA-1 and was identified as a class B scavenger receptor (Ref. Reference Calvo and Vega76) in a family that includes CD36, LIMPII and SR-BII, an SR-BI isoform with a different cytoplasmic tail (Ref. Reference Webb77). SR-BI is a cell surface receptor that is highly expressed in liver and steroidogenic tissues. It plays a key role in high-density lipoprotein (HDL) cholesterol metabolism and that regulate the supply of cholesterol to steroidogenic tissues. SR-BI also mediates binding and lipid transfer from different classes of lipoprotein (Ref. Reference Krieger78) and facilitates the metabolism of LDL, VLDL, and oxidised or acetylated lipoproteins (Ref. Reference Van Eck79). This accounts for the multiple functions of SR-BI in cholesterol metabolism, such as removal of peripheral unesterified cholesterol, steroidogenesis, and bile acid synthesis and secretion. Other essential physiological roles of SR-BI include uptake of HDL into the adrenal gland (Ref. Reference Hoekstra80), interaction with LPS (Refs Reference Cai81, Reference Vishnyakova82) and the modulation of vascular tone via endothelial nitric oxide synthase (NOS) activity in response to HDL (reviewed in Ref. Reference Saddar, Mineo and Shaul83). These functions clearly have implications for the development of therapeutics targeting SR-BI.
SR-BI stimulates the bidirectional flux of free cholesterol between cells and lipoproteins, an activity that might be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of free cholesterol from plasma HDL. In hepatic cells, SR-BI also mediates the selective uptake of cholesteryl ester from HDL, a process by which HDL cholesteryl ester is incorporated into the plasma membrane without degradation of the HDL particle (Ref. Reference Silver84). SR-BI-mediated uptake of HDL cholesteryl ester is a two-step process that requires high-affinity binding of HDL followed by incorporation of cholesteryl ester to the plasma membrane pool and subsequent transfer of the lipid to an inaccessible pool. Cholesteryl ester uptake is followed by hydrolysis to free cholesterol by a neutral cholesteryl ester hydrolase. SR-BI-mediated lipid uptake leads to increase in cholesterol content of the target cell membrane (Refs Reference de la Llera-Moya85, Reference Kellner-Weibel86, Reference Parathath87) and activates distinct signalling pathways (Refs Reference Saddar, Mineo and Shaul83, Reference Zhang88).
Similarly to CD81, SR-BI was identified as an HCV receptor through its capacity to mediate binding of soluble recombinant HCV E2 (sE2; Table 1) to human hepatic cells (Ref. Reference Scarselli59). Interactions between sE2 and SR-BI were found to be selective; neither mouse SR-BI nor the closely related human scavenger receptor CD36 was able to bind sE2. SR-BI binding to sE2 could be specifically outcompeted with polyclonal sera and monoclonal antibodies against SR-BI (Refs Reference Bartosch, Dubuisson and Cosset28, Reference Scarselli59, Reference Barth89, Reference Catanese90).
Functions of SR-BI in HCV entry
The functional role of SR-BI in HCV entry into cells was demonstrated using HCVpp and antibodies against SR-BI. Indeed, similarly to their neutralising activity in sE2 binding assays, polyclonal anti-SR-BI antibodies inhibited HCVpp entry specifically by up to fourfold (Refs Reference Bartosch72, Reference Lavillette73). Likewise, in Huh-7 human hepatoma cells in which SR-BI expression was downregulated to less than 5% of endogenous levels, entry of HCVpp was reduced three- to tenfold (Ref. Reference Lavillette73). This applied, albeit with significant variation, to all major HCV genotypes and subtypes. These early findings were confirmed in several different studies that also included the HCVcc from major genotypes and subtypes, HCV recovered from HCVcc-infected chimpanzees and/or human primary hepatocytes (Refs Reference Grove35, Reference Dreux51, Reference Catanese90, Reference Dreux91, Reference Grove92, Reference Kapadia93, Reference Zeisel94, Reference Gottwein95, Reference Catanese96, Reference Regeard97, Reference Dhillon98).
In contrast to CD81, CLDN1 and OCLN, whose involvement in HCV entry has been unambiguously demonstrated by mutation analysis in cells that were rendered susceptible to HCV entry upon their ectopic expression (Refs Reference Hsu30, Reference Evans61, Reference Ploss62, Reference Bartosch72), the broadness of SR-BI expression made the investigation of the functional properties of this molecule in HCV infection difficult, owing to the paucity of SR-BI-negative human cell lines. Furthermore, efficient or stable SR-BI downregulation in HCV-susceptible cell types, such as Huh-7 hepatoma cells, is difficult to achieve without compromising cell viability and has produced differing results (Refs Reference Bartosch49, Reference Voisset52, Reference Lavillette73, Reference Zeisel94, Reference Catanese96, Reference Randall99). Remarkably, cells of rodent and human origin with undetectable or negligible to low SR-BI expression have been generated upon ectopic expression of SR-BI and other entry cofactors, which subsequently allowed efficient HCV entry (Refs Reference Ploss62, Reference Dreux91, Reference Catanese96), hence unambiguously demonstrating that SR-BI is an essential HCV entry factor. As discussed below, through the design of SR-BI molecules mutated in the extracellular and the cytoplasmic domains, important features of SR-BI functions regarding its involvement during HCV entry have been uncovered.
SR-BI binding: direct and indirect interactions with HCV particles
Owing to the association of HCV with VLDL and LDL, and more particularly, given that ApoE, a ligand of SR-BI in its lipoprotein-bound form (Refs Reference Chroni100, Reference Li101, Reference Thuahnai102), is constitutively and abundantly expressed on the surface of HCV particles (Refs Reference Merz37, Reference Chang39, Reference Jiang and Luo41, Reference Cun, Jiang and Luo42, Reference Hishiki43, Reference Owen44), SR-BI might bind the HCV virions independently of the viral surface glycoproteins themselves. Indeed, purified VLDL, as well as antibodies directed against β-lipoproteins, particularly ApoE antibodies, efficiently inhibited SR-BI-mediated binding and uptake of HCV LVPs (Ref. Reference Maillard19). Because this primary interaction is not thought to be sufficient to induce further steps of HCV entry such as membrane fusion, it is believed that the E1E2 glycoprotein heterodimer, which is expressed on HCV particles or LVPs, mediates direct interaction with SR-BI or with other HCV-entry factors (Fig. 1), through mechanisms that remain poorly defined. As discussed above, SR-BI mediates binding of sE2 (Ref. Reference Scarselli59) through an interaction that involves the hypervariable region-1 (HVR1), a 27 amino acid peptide located at the N-terminus of the HCV E2 glycoprotein. Indeed, recombinant forms of sE2 deleted from HVR1 are unable to bind SR-BI and antibodies that are specific for HVR1 inhibit sE2 binding to SR-BI (Ref. Reference Scarselli59).
HCV particles harbouring HVR1 deletion (Refs Reference Bankwitz34, Reference Bartosch72, Reference Prentoe103, Reference Forns104) or virions incubated with anti-HVR1 antibodies (Refs Reference Dreux51, Reference Bartosch72, Reference Voisset105, Reference Farci106) display reduced infectivity in cell culture and in vivo, in agreement with a role of HVR1 for SR-BI binding. However, in infection assays with both HCVpp (which express ApoE-free viral particles) and HCVcc, deletion of HVR1 does not completely abrogate HCV entry and yields a significant residual infectivity, which varies between isolates or subtypes (Refs Reference Bankwitz34, Reference Bartosch72, Reference Prentoe103). This indicates that other viral surface determinants in addition to HVR1 modulate SR-BI binding, or alternatively that E2 and SR-BI binding is not the sole function of SR-BI during HCV entry. Along these lines, small compounds targeting SR-BI [BLTs, ‘blockers of lipid transfer’ (Ref. Reference Nieland107)], which completely prevent sE2 and SR-BI binding (Ref. Reference Dreux91), did not significantly inhibit HCV entry (Refs Reference Bartosch49, Reference Voisset52). Hence, because SR-BI was found to be required in receptor complementation assays (Refs Reference Ploss62, Reference Dreux91), along with the other three HCV-entry cofactors (CD81, CLDN1 and OCLN), these data indicate that alternative HVR1- and sE2-binding-independent SR-BI functions are at play during HCV entry. Consistently, the murine SR-BI orthologue, which does not bind HCV-E2 (Refs Reference Scarselli59, Reference Catanese96), could fully substitute its human counterpart in receptor complementation assays for HCVpp and HCVcc entry (Refs Reference Ploss62, Reference Catanese96).
It is therefore important to address the role of SR-BI together with the other HCV-entry factors. As discussed before, four cell-surface cofactors have been shown to be essential for HCV entry in receptor complementation assays and the inter-relationships are progressively being unravelled (Refs Reference Krieger108, Reference Harris109). Expressed individually, CD81, CLDN1 and OCLN do not appear to mediate binding of HCVcc particles to the cell surface, in contrast to SR-BI (Ref. Reference Evans61). These observations imply that a first contact with SR-BI, or with an as yet undiscovered factor, is necessary before the viral particle can interact with CD81 or the other HCV-entry factors. Furthermore, the possibility that SR-BI mediates entry functions that are independent of HCV binding (Refs Reference Ploss62, Reference Catanese96) is intriguing and could be linked to its capacity, along with the other HCV-entry factors, to modulate the formation of a receptor complex composed of the different HCV-entry cofactors, which initiates virus uptake and mediates its internalisation. Several determinants and functions of SR-BI could account for this and are discussed below (Fig. 1).
Internalisation of HCV particles
SR-BI may induce HCV endocytosis by itself, as suggested by its capacity to mediate internalisation of its natural ligands (Refs Reference Silver84, Reference Harder110, Reference Wustner111), in a manner determined by its C-terminal cytoplasmic tail. Indeed, deletion or substitution of the SR-BI cytoplasmic tail reduced its capacity to mediate HCV entry (Ref. Reference Dreux91). However, expression of SR-BII, an mRNA splice variant that differs from SR-BI at the C-terminus (Ref. Reference Calvo and Vega76), which confers intracellular localisation of SR-BII and rapid internalisation of HDL (Refs Reference Webb77, Reference Eckhardt112), induced reduced HCVpp and HCVcc entry compared with SR-BI (Refs Reference Dreux91, Reference Grove92). Although this indicated that SR-BI, rather than SR-BII, is a preferred receptor for HCV entry, these results suggest that determinants on the SR-BI cytoplasmic tail other than those controlling its endocytosis may regulate HCV entry. Moreover, functional restoration of the SR-BII dileucine endocytic motif in the SR-BI C-terminal cytoplasmic tail, which induces rapid internalisation of SR-BI–HDL complexes (Ref. Reference Eckhardt113), did not increase HCV entry but rather reduced it (Ref. Reference Dreux91). Altogether, these results indicate that if SR-BI initiates or promotes HCV endocytosis, it could be through its interaction with other HCV-entry factors in an appropriate timing rather than through a classical binary virus–receptor interaction. In line with this notion, a recent study suggested that modulation of receptor levels by cell–cell contacts might facilitate the assembly of receptor complexes required for virus internalisation and demonstrated a rate-limiting role for SR-BI in HCV internalisation (Ref. Reference Schwarz114).
Membrane targeting and organisation of the HCV receptor complex
Alternative determinants of the C-terminal cytoplasmic tail of SR-BI might contribute to HCV-entry functions through modulation of its intracellular trafficking and membrane localisation. Interestingly, expression of an SR-BI mutant that abrogates interaction with PDZK1 (Na+/H+ exchange regulatory cofactor NHE-RF3), an adaptor molecule that modulates its intracellular transport, localisation, assembly and scaffolding (Refs Reference Ikemoto115, Reference Komori116), did not affect HCV entry in receptor complementation assays (Ref. Reference Dreux91), whereas PDZK1 downregulation in Huh-7 or HepG2 human hepatoma cells moderately inhibited HCVpp and HCVcc infection two- to fourfold (Refs Reference Dreux91, Reference Eyre, Drummer and Beard117). By contrast, alternative mutations in the SR-BI C-terminal cytoplasmic tail that prevent palmitoylation (Ref. Reference Gu118) and thus its potential association to lipid raft microdomains indicated that localisation of SR-BI in specific micro-environments could have a role in HCV entry (Ref. Reference Dreux91). Indeed, subcellular fractionation experiments showed that SR-BI localises in plasma membrane lipid rafts (Ref. Reference Rhainds119) and caveolae (Ref. Reference Babitt120), which may have a crucial role in SR-BI-mediated transfer of lipids between HDL and cells (Refs Reference de la Llera-Moya85, Reference Subbaiah, Gesquiere and Wang121) and, possibly, HCV entry. Such low-density membrane microdomains are enriched in cholesterol and glycolipids, and are involved in several transport and signalling events that could be important for virus endocytosis and intracellular transport (Ref. Reference Marsh and Helenius122).
HDL stimulation of HCV entry
Several reports have demonstrated that HDL enhances the infectivity of HCVpp and HCVcc (Refs Reference Bartosch49, Reference Dreux51, Reference Voisset52, Reference Meunier53, Reference Catanese90, Reference Voisset105). This original mechanism is controlled by the HCV glycoproteins, and more particularly by conserved residues of HVR1 (Refs Reference Bartosch49, Reference Voisset52). HDL-mediated enhancement of infection clearly involves SR-BI, as shown using SR-BI lipid transfer inhibitors (Refs Reference Bartosch49, Reference Voisset52), SR-BI blockade or downregulation (Refs Reference Bartosch49, Reference Voisset52), and mutagenesis of SR-BI residues that control lipid transfer from HDL (Ref. Reference Dreux91). Yet, this occurs neither through direct binding of HDL to HCV particles, nor through an increase in HCV binding to SR-BI (Refs Reference Bartosch49, Reference Dreux51, Reference Voisset52). As discussed above, through lipid uptake, SR-BI increases cellular cholesterol mass, alters cholesterol distribution in plasma membrane domains (Refs Reference Kellner-Weibel86, Reference Parathath87) and activates distinct signalling pathways (Refs Reference Saddar, Mineo and Shaul83, Reference Zhang88). These events might provide different beneficial roles for HCV entry.
First, using liposome-based in vitro fusion assays, HCVpp and HCVcc membrane fusion is facilitated when cholesterol is present in the target membrane (Refs Reference Lavillette46, Reference Haid, Pietschmann and Pecheur123). By analogy with fusion processes of Flaviviruses and Alphaviruses that have been widely studied (Ref. Reference Stiasny and Heinz124), cholesterol enrichment of target membranes might induce specific curvatures that could positively influence the early interactions of HCV fusion proteins. Alternatively, local cholesterol enrichment might facilitate binding (Ref. Reference Umashankar125) and conformational changes (Ref. Reference Rawat126) within the HCV glycoproteins that are required for membrane fusion processes.
Second, HCVpp internalisation was shown to be specifically accelerated by HDL (Ref. Reference Dreux51). As discussed above, this effect is likely to be indirect. Indeed, the internalisation rate of HCVpp has a half-life that is much longer than that of HDL internalisation (Refs Reference Harder110, Reference Wustner127). Furthermore, HDL added during the initial stage of infection suppresses a 1 h time lag during which cell-bound virions are not internalised (Ref. Reference Dreux51). This could reflect the time interval required to assemble a functional HCV receptor complex, which remains poorly defined, but might comprise the different HCV-entry cofactors. This time lag might be reduced on SR-BI activation through modifications of the cell membrane. One possibility is that interaction between HDL and SR-BI augments the rate of CD81 recruitment at virion-binding sites and internalisation of HCV–CD81 complexes through a cholesterol-dependent pathway. In agreement with this assumption, SR-BII or SR-BI mutants that increase HDL internalisation are less effective than wild-type SR-BI at mediating HCV entry (Refs Reference Dreux91, Reference Grove92), which suggests that HCV internalisation is probably driven by interaction of SR-BI with other HCV-entry factors rather than through SR-BI alone. In this respect, it is interesting that CD81 and SR-BI function cooperatively to initiate HCV infection (Refs Reference Kapadia93, Reference Zeisel94), that CD81-mediated HCV entry seems dependent on membrane cholesterol (Ref. Reference Kapadia93) and that SR-BI–HDL-mediated HCV entry enhancement still requires CD81 (Refs Reference Bartosch49, Reference Dreux51). In addition, CD81, through several interactions with other tetraspanin proteins, forms a multiprotein tetraspanin web, which is regulated by cholesterol and might modulate their function (Ref. Reference Charrin128). Recently, both SR-BI and CD81 have been proposed to be cell factors allowing Plasmodium sporozoite invasion or intracellular parasite development in mouse (Ref. Reference Yalaoui129) and human (Ref. Reference Rodrigues130) hepatocytes, perhaps through SR-BI-induced regulation of the organisation of CD81 at the plasma membrane by mediating an arrangement that is permissive to penetration by sporozoites (Ref. Reference Yalaoui129). Alternatively, homo-oligomerisation of SR-BI seems to be associated with functional expression of the selective HDL cholesteryl ester uptake pathway (Ref. Reference Reaven131) and might contribute to the formation of an HCV receptor complex.
ApoC-I is an exchangeable apolipoprotein that stimulates HCV fusogenicity
An essential component of HDL that seems to be responsible for infection enhancement is ApoC-I (Refs Reference Dreux50, Reference Meunier53). ApoC-I is a small plasma protein (57 amino acids) composed of two amphipathic α-helices and is the smallest of the exchangeable apolipoproteins. It circulates in the bloodstream mainly associated with HDL and with VLDL and chylomicron particles (Refs Reference Cohn132, Reference Malmendier133). HCVcc or authentic HCV from infected chimpanzees are efficiently immunoprecipitated and neutralised by anti-ApoC-I antibodies, demonstrating that ApoC-I is a component of HCV (Refs Reference Meunier18, Reference Merz37, Reference Dreux50). Using HCVpp and HCVcc assays, recent data suggested that in cell culture, ApoC-I could be transferred from HDL to the HCV particle membrane during SR-BI-mediated lipid transfer (Ref. Reference Dreux50).
Interestingly, recruitment of ApoC-I to the surface of the viral particle predisposes the HCV envelope for fusion with a target membrane (Ref. Reference Dreux50), presumably via alterations of its outer phospholipid layer (Refs Reference Gillotte134, Reference Segrest135), in a process that could depend on the α-helical amphipathic structure of ApoC-I and its affinity to phospholipid surfaces and that is modulated by HVR1 (Ref. Reference Dreux50). Such a process of membrane fusion priming is reminiscent of analogous mechanisms adopted by surface glycoproteins of different enveloped viruses to modulate the extent of membrane fusion (Refs Reference Munoz-Barroso136, Reference Salzwedel, West and Hunter137, Reference Schibli, Montelaro and Vogel138, Reference Saez-Cirion139).
The unexpected role of a serum protein in promoting fusion enhancement is another remarkable feature of the ability of HCV to hijack blood and lipoprotein components to facilitate its replication. As ApoC-I in vivo is present in mainly HDL and in VLDL to which HCV is associated, but not as a lipoprotein-free form (Ref. Reference Cohn132), it could be released from either lipoprotein type following their interaction with SR-BI to allow subsequent recruitment on the HCV particle surface also bound to SR-BI (Ref. Reference Dreux50). Alternatively, similarly to ApoE, ApoC-I could be associated intracellularly as a cellular lipoprotein component with the surface of HCV particles during viral morphogenesis through the VLDL assembly pathway (Refs Reference Meunier18, Reference Merz37).
A combined role of SR-BI and HVR1 in avoiding HCV neutralisation
It is remarkable that several aspects of the HCV–SR-BI interaction depend on HVR1 despite its intense variability. The importance of this determinant is suggested in vivo by the attenuated phenotype of HVR1-deleted HCV in chimpanzees and by the abrogation of infectivity by HVR1 antibodies (Refs Reference Forns104, Reference Farci106, Reference Shimizu140). The analysis of HVR1 sequences from different HCV strains indicates that its variability is not random (Ref. Reference Penin141), because the chemicophysical properties and conformation of this basic domain are well conserved. This suggests the involvement of this region during cell entry (Ref. Reference Penin141), and indeed, the presence of basic residues in HVR1 was found to facilitate virus entry (Ref. Reference Callens142). As discussed above, HVR1 also modulates the interaction of HCV-E2 with SR-BI (Refs Reference Scarselli59, Reference Bartosch72), the infectivity of HCVpp and HCVcc particles (Refs Reference Bankwitz34, Reference Bartosch72, Reference Prentoe103), and enhancement of HCV infection by HDL (Refs Reference Bartosch49, Reference Voisset52). In addition, HVR1 is not the sole E2 determinant that governs interaction with SR-BI. Indeed, in a manner similar to HVR1 deletion or modification, several mutations immediately downstream of HVR1 (Refs Reference Dhillon98, Reference Tao143), as well as the more downstream G451R mutation (Ref. Reference Grove35), rendered JFH1 HCVcc insensitive or less sensitive to SR-BI blocking antibodies and to HDL-mediated enhancement.
HVR1 is a viral determinant targeted by the host humoral response
Because of its antigenicity and its important functions, it is not surprising that HVR1 is primarily targeted by the neutralising immune response during acute and chronic infection. HVR1 contains at least one neutralising epitope that is recognised by antibodies from patients or from HCV-inoculated chimpanzees (Refs Reference Bartosch, Dubuisson and Cosset28, Reference Bartosch72, Reference Farci106, Reference von Hahn144, Reference Bartosch145, Reference Vieyres, Dubuisson and Patel146). Sequence changes that appear in response to immune pressure occur almost exclusively within HVR1 (Ref. Reference Farci147), indicating that a strong humoral response drives its evolution and might allow HCV to adapt to its host and to escape from neutralising antibodies (Refs Reference Kato148, Reference Liu149). The early development of such HCV quasispecies and, particularly, the variation of HVR1 have been suggested to correlate with persistent infection, whereas reduction of genetic diversity, leading to increasingly homogeneous virus populations, is a consistent feature associated with viral clearance in sustained responders (Ref. Reference Farci147). Furthermore, the emergence of antibodies against HVR1 in inoculated chimpanzees was linked to variations in HVR1 sequence, whereas no variation was detected in the absence of detectable HVR1 antibodies (Ref. Reference van Doorn150). Finally, HVR1 evolution is reduced in agammaglobulinaemic patients (Ref. Reference Booth151). This evidence in support of HVR1 selection by the humoral response (Ref. Reference Ray152) led to the notion that HVR1 functions as an ‘immunological decoy’, which stimulates a strong immune response causing variant selection, but which is ineffective for viral clearance (Ref. Reference Mondelli153).
HVR1 determines the density of HCV
The distribution of HCV in its heterogeneous forms in density gradients depends on the stage of disease. In sera from chronically infected patients, HCV is often present in higher-density forms as a complex with immunoglobulins in addition to its low-density forms (Refs Reference Hijikata10, Reference Thomssen154, Reference Choo155, Reference Thomssen, Bonk and Thiele156). By contrast, in immunodeficient and immunosuppressed patients, as well as in acute infection before the onset of immune responses, the virus is more prevalent in low-density forms (Refs Reference Hijikata10, Reference Nielsen11). Importantly, mutations within HVR1 lead to the accumulation of immunoglobulin-free virus particles of low density (Ref. Reference Choo155), suggesting that in patients with chronic infection, the presence or increase of low-density virus, possibly as lipoprotein-associated forms, is a result of immune escape.
In cell culture, HVR1 also seems to modulate the distribution and infectivity of viral particles in the HCV populations isolated by density gradients from HCVcc-containing supernatants (Refs Reference Bankwitz34, Reference Prentoe103). Indeed, deletion of HVR1 from HCVcc resulted in a moderate to strong decrease in the number of low-density virions (1.00–1.10 g/ml), in a manner that varies between HCV isolates or subtypes, suggesting that HVR1 influences the interplay between HCV and lipids, apolipoproteins or lipoproteins during assembly or subsequently. Furthermore, in contrast to the high specific infectivity of parental HCVcc in the low-density fractions (Refs Reference Lindenbach25, Reference Podevin31, Reference Yi32, Reference Gastaminza, Kapadia and Chisari33, Reference Bankwitz34, Reference Grove35) and irrespective of the virus genotype, HVR1-deleted HCVcc low-density particles were not at all or were poorly infectious, whereas infectivity was greater in the intermediate-density fractions (1.11–1.14 g/ml) (Refs Reference Bankwitz34, Reference Prentoe103). These results indicated that the dependence of HCVcc on HVR1 is different between HCV particles of low density compared with those of intermediate density, with HVR1 having particular importance in the infectivity of particles of low density (Refs Reference Bankwitz34, Reference Prentoe103). This perhaps reflects the importance of HVR1 in recruiting or interacting with lipoprotein factors that can support infection during assembly of viral particles or during virus entry into cells.
HVR1–SR-BI interaction and modulation of neutralisation
It is interesting that HVR1, a determinant that controls escape from neutralising antibodies through its variability, also determines the specific infectivity of low-density HCV forms. Indeed, low-density HCV particles are themselves more resistant to neutralisation by E1E2 antibodies than those of intermediate density (Refs Reference Nielsen11, Reference Maillard19, Reference Prince20, Reference Grove35), as a possible result of virus masking within β-lipoproteins.
Although this suggests that direct interactions between HCV surface and lipoprotein components are mediated by HVR1 and induce shielding of neutralising epitopes, there are alternative possibilities that could involve an effect of HVR1 in the conformation of the E1E2 glycoprotein heterodimer itself and hence its sensitivity to neutralisation. In fact, in cell culture, for both HCVpp and HCVcc, HVR1 attenuates the effect of neutralising antibodies and of crossneutralising antibodies present in the sera of acutely or chronically infected patients (Refs Reference Bankwitz34, Reference Bartosch49, Reference Prentoe103). Indeed, through the use of HVR1-deleted HCV particles, a relatively strong neutralisation response against both autologous and heterologous HCV sequences was shown in acute-phase patients who seemingly did not show neutralising antibodies against the autologous virus (Refs Reference Bartosch49, Reference Lavillette157). Results indicated that these patients had successfully mounted crossneutralising antibody responses against epitopes outside HVR1 but that these antibodies were relatively ineffective against viruses possessing HVR1, possibly because these crossneutralising epitopes were poorly accessible or were masked. Moreover, sera from patients with chronic infection showed a strongly increased capacity to neutralise HVR1-deleted HCVpp or HCVcc particles compared with unmodified HCV particles (Refs Reference Bankwitz34, Reference Bartosch49, Reference Prentoe103). Finally, most mouse and human monoclonal antibodies targeted against E2 and, more specifically, those against crossneutralising epitopes representing areas of CD81–E2 interaction were much more efficient at neutralising HVR1-deleted HCVpp and HCVcc particles (Refs Reference Bankwitz34, Reference Bartosch49, Reference Dreux51). Importantly, similar results of neutralisation attenuation were obtained whether HCVcc particles were produced in cell culture or in vivo from human hepatocytes in human liver chimeric mice (Ref. Reference Prentoe103). That HVR1 represents a primary viral factor involved in HCV escape from crossneutralising antibodies suggested that, as a determinant that modulates interaction with SR-BI, this latter HCV receptor could also be involved in neutralisation attenuation. This assumption proved to be correct because, in further studies, downregulation of SR-BI or blockade of its lipid transfer function was found to efficiently restore crossneutralisation of both HCVcc and HCVpp particles by monoclonal antibodies and patient-derived polyclonal antibodies (Refs Reference Dreux51, Reference Voisset105, Reference Dreux and Cosset158).
Although these different results, which were initially obtained using the HCVpp assay (Refs Reference Bartosch49, Reference Dreux51, Reference Voisset105, Reference Dreux and Cosset158), were largely confirmed when HCVcc particles became available (Refs Reference Bankwitz34, Reference Prentoe103), interesting qualitative differences between HCVpp and HCVcc could be detected. Indeed, whereas both HCVcc and HCVpp particles required a functional interaction with the SR-BI receptor in target cells to attenuate neutralisation (Refs Reference Dreux51, Reference Voisset105, Reference Dreux and Cosset158), HCVpp additionally required the presence of human serum or HDL (Refs Reference Bartosch49, Reference Dreux51). This difference probably indicated that HVR1 induces the masking of crossneutralising epitopes only when HCV is produced in a cellular environment providing some lipoprotein components, such as HCVcc from Huh-7.5 hepatocarcinoma cells, and that such properties of HCVcc can be reproduced to some extent upon incubation of HCVpp produced from 293T kidney cells with HS or HDL in the presence of SR-BI.
Overall, these data suggest that HVR1 masks or induces masking of the viral CD81-binding site that most crossneutralising antibodies target. This hypothesis was indeed confirmed by the findings that soluble CD81 (CD81-LEL) inhibited and precipitated HVR1-deleted HCVcc more efficiently than wild-type particles (Ref. Reference Bankwitz34). Consistently, as mentioned earlier, HCVcc particles do not bind efficiently to cells individually expressing CD81, in contrast to SR-BI (Ref. Reference Ploss62), and deletion of HVR1 increases sE2 binding to CD81 (Refs Reference Scarselli59, Reference Roccasecca159). This suggests that the CD81-binding site in wild-type particles is not accessible until HCV has previously interacted with a host factor that induces a conformational change in the E1E2 complex, making the CD81-binding site accessible for interaction with this crucial entry factor. This parallels human immunodeficiency virus type (HIV-1) for which a previous interaction with CD4 induces a conformational rearrangement that allows subsequent interaction with either the CXCR4 or CCR5 chemokine receptors (Ref. Reference Wyatt and Sodroski160). Interestingly, some mutations in regions downstream of HVR1 that rendered HCV less dependent on SR-BI (Refs Reference Grove35, Reference Dhillon98, Reference Tao143) also modulated accessibility to the CD81-binding site, resulting in viral particles that were more sensitive to neutralisation by polyclonal antibodies from chronically infected patients and by monoclonal antibodies that target the E2–CD81 interaction.
HVR1, SR-BI and quasispecies selection
Because HVR1 might protect HCV from neutralisation in vivo, preservation of its functions should be essential for viral persistence. This could be achieved by maintaining its capacity to mediate binding of HCV glycoproteins to SR-BI because HVR1–SR-BI interaction attenuates crossneutralisation, as discussed above. Preservation of this interaction could explain why HCV has evolved, within a conserved framework of residues, a hypervariable cluster of residues that are targeted by the humoral response. Indeed, under immune pressure, the variants escape from HVR1 antibodies by antigenic drift, leading to the formation of quasispecies. The emerging HVR1 variants that maintain viral ‘fitness’, by preserving the SR-BI–HDL interaction, escape the immune response, maintain HVR1 immune modulatory or decoy functions and gain a considerable advantage over their parental viral species. They might spread better and replace the parental sequences to become the new dominant viral species, until they themselves become targets of the host's immune system. This is consistent with the notion that, despite its high degree of genetic variability, highly conserved amino acids are found throughout HVR1 and that, even at some less conserved residues, the physicochemical properties of amino acids are maintained (Ref. Reference Penin141). Indeed, in cell culture, non-conservative substitution of such conserved amino acids had a dramatic effect on SR-BI interaction, infection enhancement and neutralisation attenuation (Refs Reference Bartosch49, Reference Dreux50), suggesting that genetic diversification of HVR1 compromises immune escape and enhancement of cell entry. Although the conserved residues might be responsible for maintaining HVR1 in a conformation that allows interplay with lipoprotein and SR-BI, the truly variable positions are probably involved in HVR1 antigenicity: a type of organisation similar to that of immunoglobulin and T-cell receptor variable domains that show variable sequences but conserved conformations. The importance of this HCV determinant during viral replication in vivo has previously been suggested by the attenuated phenotype of HVR1-deleted virus in chimpanzees and by abrogation of wild-type virus infectivity by HVR1 antibodies (Refs Reference Forns104, Reference Farci106).
Conclusions and clinical implications
SR-BI is an intriguing receptor because it is essential for HCV replication in cell culture and because the virus seems to exploit some aspects of its physiological functions during cell entry. Many questions regarding SR-BI usage remain, such as what is the molecular basis of the HCV–SR-BI interaction, because the initial binding of serum-derived HCV to SR-BI does not seem to be mediated by HVR1 or by alternative regions of the E2 glycoprotein. Instead, the association of VLDL with virus particles seems to have a critical role in the primary interaction with SR-BI (Ref. Reference Maillard19). In addition, although SR-BI mediates HCV entry in cell culture independently of its binding capacity, it is clear that direct HCV binding to SR-BI facilitates infection, allows HDL-mediated HCV entry enhancement and attenuates crossneutralisation (Ref. Reference Dreux51). Other important aspects linked to SR-BI usage in vivo that remain to be defined are its involvement in neutralisation attenuation and the characterisation of the HCV and host determinants that mediate this process. Importantly, inhibition of the HCV–SR-BI interaction might result in inhibition of HCV propagation, as well as in stimulation of cross-genotype virus neutralisation.
SR-BI is therefore an important target for therapeutic intervention, and recent data suggest that it should be possible to define inhibitors of HCV–SR-BI interaction that do not compromise its important and varied physiological functions related to lipid transfer, or at least, in a manner that does not increase the risk of atherosclerosis, as shown in a mouse model for ITX5061 (Ref. Reference Masson161), a clinical stage compound that targets SR-BI to inhibit HCV entry (Ref. Reference Syder162). As discussed in this review, SR-BI seems to be involved in different steps of HCV transmission. As a determinant that mediates direct HCV binding by E2 interaction, or by indirect binding through lipoprotein-associated virions, SR-BI could be responsible for the initial recognition of viral particles. Furthermore, SR-BI is also involved at a post-binding step, and polyclonal antibodies as well as mouse and human monoclonal antibodies have been derived that inhibit both binding and post-binding steps of HCV-entry and HDL-mediated HCV entry enhancement (Refs Reference Bartosch49, Reference Catanese90, Reference Zeisel94, Reference Catanese96). Such antibodies and, more generally, SR-BI binding blockers (Refs Reference Dreux91, Reference Syder162) could prove excellent tools for passive (immuno-) therapy against chronic HCV infection, provided they do not inhibit crucial physiological functions of SR-BI. Recent evidence argues for this. Indeed SR-BI mutants that do not mediate HCV binding or HDL-mediated enhancement and that do not impair receptor oligomerisation and lipid transfer (HDL binding and cholesterol ester efflux or uptake) have been described (Ref. Reference Catanese96). In addition, blockers of the HCV–SR-BI interaction have been shown to fully restore the potency of monoclonal antibodies targeting E2–CD81 interaction and of polyclonal antibodies induced in HCV patients that are attenuated by HDL–SR-BI (Refs Reference Dreux51, Reference Voisset105, Reference Dreux and Cosset158). Thus, blocking HCV–SR-BI interaction should have a dual benefit for the patient in clinical settings, both by reducing HCV entry and by stimulating the neutralising activity of antibodies inoculated in patients or naturally induced by HCV. That antibodies or small compounds targeting E2–SR-BI interaction inhibit cell–cell transmission in addition to cell-free transmission (Ref. Reference Brimacombe71) further identifies SR-BI as a suitable therapeutic target.
There are alternative opportunities to develop safe agents that selectively inhibit HCV–SR-BI interaction with no repercussion on HDL metabolism. Natural inhibitors of the HCV–SR-BI interaction have been discovered, such as oxLDL, another natural ligand of SR-BI, which potently inhibits both HCVpp and HCVcc entry (Ref. Reference vonHahn163). Recently, SR-BI has also been shown to bind and internalise serum amyloid protein (SAA) (Refs Reference Baranova164, Reference Cai165). SAA is an acute-phase protein that is produced mainly by the liver immediately after infection, tissue damage or inflammation (reviewed in Ref. Reference Uhlar and Whitehead166) and that might have a beneficial role in host defence during the acute phase of HCV infection. The effect of SAA on HCV entry has recently been tested (Refs Reference Cai167, Reference Lavie168) and was found to inhibit HCV infection in a dose-dependent manner by affecting an early step of the virus life cycle, by interacting with the viral particle.
However, more knowledge is required to fully characterise how HCV surface glycoproteins, such as HVR1, a most important determinant of HCV–SR-BI interaction, interact with SR-BI, and also with other regions in E2. As discussed in this review, mutations of such determinants can reduce the infectivity of low-density HCV particles (Refs Reference Bankwitz34, Reference Grove35, Reference Prentoe103, Reference Tao143) and increase the accessibility of the CD81-binding site, leading to more efficient HCV neutralisation by human or mouse monoclonal antibodies and patient-derived sera (Refs Reference Bankwitz34, Reference Grove35, Reference Bartosch49, Reference Dreux51, Reference Dhillon98, Reference Prentoe103, Reference Voisset105, Reference Tao143, Reference Dreux and Cosset158). Interestingly, HVR1-targeted antibodies are not attenuated by HDL (Refs Reference Bartosch49, Reference Dreux51, Reference Voisset105). Furthermore, the chemicophysical properties of HVR1 seem to be highly conserved, suggesting that despite the high level of amino acid variability, HVR1 might adopt only one or a restricted range of related conformations (Refs Reference Callens142, Reference Puntoriero169). Conserved amino acid positions have been suggested to maintain a global conformation of HVR1 and this might be used to design crossreactive antibodies or small compounds that target HVR1 or other E2 determinants (Ref. Reference Zucchelli170).
Acknowledgements and funding
We thank the referees for their constructive comments.
Our work is supported by the European Research Council (ERC-2008-AdG-233130-HEPCENT) and the ‘Agence Nationale pour la Recherche contre le SIDA et les Hépatites Virales’ (ANRS). V.L.D.T. is supported by a pre-doctoral fellowship from Région Rhône-Alpes. M.D. and F.-L.C. are supported by Inserm and CNRS, respectively.
