Antiviral and Vaccine Targets - American Chemical Society

Review pubs.acs.org/journal/aidcbc

Structure and Function of the Hepatitis C Virus Envelope Glycoproteins E1 and E2: Antiviral and Vaccine Targets Holly Freedman,* Michael R. Logan, John Lok Man Law, and Michael Houghton* Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada ABSTRACT: The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are critical in viral attachment and cell fusion, and studies of these proteins may provide valuable insights into their potential uses in vaccines and antiviral strategies. Progress has included elucidating the crystal structures of portions of their ectodomains, as well as many other studies of hypervariable regions, stem regions, glycosylation sites, and the participation of E1/E2 in viral fusion with the endosomal membrane. The available structural data have shed light on the binding sites of cross-neutralizing antibodies. A large amount of information has been discovered concerning heterodimerization, including the roles of transmembrane domains, disulfide bonding, and heptad repeat regions. The possible organization of higher order oligomers within the HCV virion has also been evaluated on the basis of experimental data. In this review, E1/E2 structure and function is discussed, and some important issues requiring further study are highlighted. KEYWORDS: hepatitis C virus, E1/E2 heterodimer, envelope glycoprotein, viral fusion protein, hypervariable region, neutralizing antibody

1. INTRODUCTION

heterodimerization interface of the two proteins as deletion of these domains abolishes formation of the heterodimer.7 E1 and E2 are highly glycosylated, with glycans accounting for almost 50% of the mass of the heterodimer.8 The addition of sugar groups and the formation of disulfide linkages occur during the folding of the two proteins, which is assisted by host chaperone proteins such as calnexin9,10 and BiP.11 In the virion envelope, the glycoproteins are associated with serum lipoproteins, leading to a much lower buoyant density for HCV than for other viruses. 12,13 These serum lipoproteins (composed of apolipoproteins, phospholipids, triglycerides, and cholesterol) interact with HCV to form hybrid lipoviral particles14 and facilitate viral entry and secretion. In addition, it has been documented that HCV-associated lipoproteins may partially mask the virion from the action of neutralizing antibodies.15−17 During viral entry, HCV first binds through its associated lipoproteins to the cellular receptor SR-BI on hepatocytes in the liver. This is followed by an essential lipid transfer action by SR-BI, possibly involving an alteration in the association of HCV with lipoproteins.1 After the initial interaction with SR-BI, E2 is exposed, allowing binding to the cellular tetraspanin receptor, CD81.18 The binding of CD81 is E1 independent1 and triggers association with tight-junction proteins claudin 119−21 and occludin,22 Upon these sequential interactions of

The hepatitis C virus (HCV) has a positive-stranded RNA genome, encoding a polyprotein that is processed into seven nonstructural and three structural proteins. The virion is made of a nucleocapsid composed of the core protein and viral RNA enclosed within a host-derived lipid envelope embedded with glycoproteins E1 and E2. These glycoproteins form a heterodimer, and this structure is crucial for viral entry, fusion, and secretion.1 Because it is at the surface of the virion, the heterodimer E1/E2 is the major target of the humoral response during HCV infection, giving rise to a range of virusneutralizing antibodies.2,3 The immunogenicity of E1/E2 has led to various ongoing attempts to develop one or both envelope glycoproteins into a vaccine.4 Because of this practical aspect, a large number of studies in the literature have been devoted to the determination of the structures of E1 and E2, how these two proteins interact, and how the E1/E2 heterodimer is arranged within the envelope of the HCV virion. The two glycoproteins E1 and E2 comprise residues 192− 383 and 384−746, respectively, of the genotype 1a H77 strain.5 Following translation of the HCV polyprotein, each of E1 and E2’s C-terminal transmembrane domains (TMDs) forms a hairpin of antiparallel α-helices, that is, a pair of α-helices folded over on one another so as to span the membrane twice.6,7 Upon cleavage by signal peptide peptidase at the endoplasmic reticulum, the transmembrane helices of E1 and E2 each span the viral membrane in a single long, straight α-helix.6 The TMDs are thought to comprise the most important © XXXX American Chemical Society

Special Issue: Host-Pathogen Interactions Received: June 19, 2016

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DOI: 10.1021/acsinfecdis.6b00110 ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

Review

sheet formed from two sets of three strands, again from two different monomers, and is stabilized by two disulfide bridges shown in Figure 1. Interestingly, in the search for the best match among all known protein structures for structural homology, this homodimeric six-stranded β-sheet structure was determined to have a structure similar to that of phosphatidylcholine transfer protein.28 This suggests that the two types of interfaces formed in the dimer may be replaced by an interdomain interface in the monomer in which the β-hairpin folds back, interacting with β5.28 As a result, E1 may be more compact instead of taking on an extended, open structure as in the crystal structure. Indeed, the fact that the correct folding of E1 is known to require the cotranslation of E231,32 suggests that the 4UOI structure may potentially have deficiencies. 2.2. E2. The structure of the central E2 ectodomain was solved by two independent groups in 201326 and 2014.27 To crystallize E2, Kong et al.26 expressed 41 different recombinant E2 proteins from HCV genotype 1a (strain H77) in mammalian cells. One of these provided a crystal structure, 4MWF,26 of a peptide corresponding to the central core region of E2, namely, residues 384−645, in complex with the neutralizing anti-E2 antibody, AR3C.33 This structure omitted some flexible regions including the hypervariable 1 (HVR1) domain (384−411) and the conserved region neighboring it (412−420), residues belonging to HVR2 (453−491), and some of the residues (574−577) belonging to the hypervariable region IgVR (inter-genotype variable region). On the other hand, Khan et al.27enzymatically removed glycosyl groups and determined a crystal structure, 4WEB (Figure 2), of the E2 ectodomain from HCV genotype 2a (strain J6) in complex with the non-neutralizing Fab 2A12. With the exception of the disulfide bonding networks, these E2 structures were very similar to each other. On the basis of the crystal structures, the E2 core structure has been described as consisting of a more basic front layer and a more hydrophobic back layer (Figure 2).8 The front layer contains an Ig-fold, a motif consisting of a central β-sandwich, which is typically found in domains I, II, and III of class II viral fusion proteins (especially the latter), and the back layer contains a second β-sheet with a novel fold roughly perpendicular to the plane of the central β-sandwich. The CD81 binding site is at the interface of these two layers.26,27 Negative stain cryo-EM by Kong et al.26 showed residues 384−717 of E2 bound to the non-neutralizing antibody AR2A33 and to CD81. This work and a previous mutational study34 demonstrated that binding to CD81 is mediated by the loop connecting the inner and outer sheets of the β-sandwich designated the CD81 binding loop (residues Y527, W529, G530, and D535), along with a few residues on the N-terminus of 4MWF (W420, L427, and N428) and F442 belonging to HVR3. 2.2.1. Hypervariable Regions. E2 contains several hypervariable regions, that is, regions of high sequence diversity, namely, hypervariable regions HVR1 (384−411),35 HVR2 (461−481),36 HVR3 (431−466),37 and IgVR (570−580).38 This diversity has been partially driven by immune evasion.39 A few functions have been proposed for HVR1. These include involvement in binding to key HCV cell entry receptors40,41 and acting as an immune decoy to prevent effective Ab neutralization.42 One assigned function for the E2 HVR1 domain is during viral attachment and binding to the HCV cell entry receptor, SR-BI. A direct or indirect interaction between HVR1 and SR-

receptors, the virus is internalized via clathrin-mediated endocytosis.1 The E1/E2 heterodimer mediates fusion of the viral lipid envelope with the host cell endosomal membrane in a pH-dependent process.1 However, the molecular details of the HCV fusion mechanism have not yet been elucidated. On the basis of sequence similarities with flaviviruses, glycoproteins of HCV were presumed to be class II fusion proteins.23,24 Homologies between E1 and E2 and class II fusion proteins have been used to create models both of the E2 structure23,24 and of the E1/E2 complex structure.25 More recently, crystal structures have been solved for the N-terminal domain of E1 and the central domain of E226−28 and demonstrated more globular rather than elongated structures. These findings have ruled out that either E1 or E2 is a class II fusion protein. However, because E1 and E2 are highly glycosylated and membrane-bound, crystallization efforts and structural resolution of the full-length glycoproteins have not yet been successful.

2. STRUCTURES OF E1 AND E2 2.1. E1. In 2014, a crystal structure, 4UOI (Figure 1), was solved for the N-terminal domain of E1 (in the absence of E2)

Figure 1. 4UOI crystal structure, with each of the four E1 monomers colored differently (gray, yellow, red, and blue). (Inset) Intradimer and interdimer symmetry axes, in cyan and magenta, respectively. Green balls represent side chains of disulfide-linked Cys residues, and glycans are shown in stick mode. Reprinted with permission from ref 28. Copyright 2014 Nature Publishing Group.

consisting of residues 192−270, with the flexible region from 245 to 259 omitted. This molecule lacked N-glycosylation due to the presence of an N-glycosylation inhibitor in the tissue culture media.11 In this crystal structure, the N-terminus forms a β-hairpin, which extends out in a perpendicular manner from the 16-residue α-helix that follows it. This α-helix is flanked by a three-stranded β-sheet; a flexible region that could not be assigned is situated between the last two strands, β4 and β5, of this β-sheet. A protease site was engineered at the C-terminus of the protein construct in place of the putative fusion peptide. Hydrophobic residues making up this site pack against β5, near a region where it is proposed to bind to apolipoproteins ApoB and ApoE.28,29 The structure is a dimer of dimers, and the oligomeric arrangement in the crystal structure displays two types of dimer interfaces. In the first of these, the two N-terminal β-hairpins from two monomers are arranged into an antiparallel β-sheet, linked noncovalently,28 in what is most likely a domainswapped formation.30 The second interface is a six-stranded βB



Review

Figure 2. 4WEB crystal structure, colored by electrostatic potential (a, d) with blue being basic, red being acidic, and white being neutral, colored to represent residues with high sequence identity of HCV variants in green (b, e), and in ribbon representation (c, f) with N-glycosylation sites labeled and in stick mode. Reprinted with permission from ref 27. Copyright 2014 Nature Publishing Group.

particle types. It has been suggested, therefore, that ApoE conformation or epitope exposure may differ between wild-type and ΔHVR1 viral particles, perhaps due to differences in their lipid content.41 In addition to its role in mediating binding to cellular receptors, HVR1 may also serve to modulate antibody neutralization. In a recent study, mice transplanted with human hepatocytes infected with ΔHVR1 HCV (genotype 2a) particles, but not wild-type particles, were shown to be fully protected by passive immunization with IgG from a chronically infected human HCV (genotype 1a) patient.47 In HCVinfected patients, neutralizing Abs directed to regions of HVR1 are present, and it is known that HVR1 residues give rise to viral mutation and escape.48 It has been shown that anti-HVR1 antibodies can block the binding of certain broadly neutralizing Abs to HCV.49 In contrast, HVR2 and IgVR do not seem to be the targets for neutralizing Abs.50 HVR1 and HVR2 are missing from the E2 crystal structures, but their general positions were observed in a cryo-EM density map positioned on opposite sides of the central core domain; the loop corresponding to the part of IgVR that is missing from the crystal structure is positioned close to HVR2.26 Deletion of HVR1 in HCVpp does not affect folding, CD81 binding, or heterodimerization, but can lead to diminished virion infectivity.51 In addition, infectivity correlates with the number of basic residues in HVR1.52 Deletion of HVR2 or IgVR abolishes the formation of E1E2 heterodimers, reduces viral entry, and disrupts folding and CD81 binding in the context of HCVpp.51 In the context of E2 alone, deletion of HVR1, HVR2, or IgVR alone does not affect CD81 binding, nor does the simultaneous deletion of all three regions. Interestingly, deletion of HVR1 and HVR2 reduced the binding to CD81 by a factor of about half.38,53 Alhammad et al.54 characterized the antibodies raised to the cytosolic portion of E2 (E2661) or to E2661 lacking the three hypervariable regions, Δ123 (where IgVR is represented by the

BI has been suggested, possibly mediated through ApoA lipoproteins.43 It has been reported that neutralizing Abs that bind to the C-terminal portion of HVR1 inhibit the binding of HCV pseudoparticles to SR-BI.44 In a separate study it was observed that anti-SR-BI antibodies could neutralize wild-type HCV particles, but exhibited reduced efficacy toward particles that expressed E1E2 lacking HVR1.40 A plausible mechanism for the HCV neutralization observed by anti-SR-BI antibodies is that this may interfere with interactions between SR-BI and HVR1, which are required for exposure of E2’s CD81 binding site8 and which involve changing the position of HVR1 so that it does not shield the CD81 binding site. Thus, for wild-type HCV particles bound by these SR-BI-specific Abs, shielding of the CD81 binding site by HVR1 may take place, but not for ΔHVR1 particles and, hence, no neutralization occurs. Nevertheless, SR-BI has been demonstrated to be equally important for infectivity by ΔHVR1 particles and wild-type particles because modulation of SR-BI by overexpression or gene silencing has similar effects on both particle types.41 This implies that HCV has a second dependency on SR-BI for infectivity, independent of HVR1, which may involve the lipid transfer activity of SR-BI during viral attachment.1 E2 HVR1 is also important for interaction with the lowdensity lipoprotein receptor (LDL-R), which, like SR-BI, mediates lipid transfer41 and may have a redundant role in HCV entry with SR-BI.45 For example, wild-type HCV particles were observed to be more effectual at binding parental CHO cells (which lack the SR-BI receptor but express LDL-R) compared to ΔHVR1 particles. The interaction of HVR1 with LDL-R may be mediated, in part, by ApoE (which has been implicated in the interaction of HCV with LDL-R46) because ApoE-specific Abs neutralize wild-type HCV particles more effectively than ΔHVR1 particles.41 The buoyant density of ΔHVR1 particles was also found to be higher than that of wildtype particles, suggesting a reduced level of lipid association. The levels of viral-associated ApoE are, however, similar in both C



Review

N8, and N10) have been shown to be involved in correct folding and heterodimerization.59,60 N7 has a genotype-specific role in viral entry.60 Glycans also contribute to immune evasion because mutation of N1, N2, N4, N6, or N11 strongly increases sensitivity to antibody-mediated neutralization, and mutating the first four of these leads to improved binding affinity of E1/ E2 to the large extracellular loop (LEL) of CD81.60

number 3). They identified two neutralizing Abs directed to E2661 that required HVR1 for optimal binding to E2 (one of which bound directly to a peptide corresponding to HVR1). Binding of these antibodies was reduced if IgVR was deleted, but not if both IgVR and HVR2 were deleted. On the other hand, several Abs raised to Δ123 were more reactive to E2 and better able to reduce E2−CD81 interactions when any one of the hypervariable regions was deleted from E2. One of these was found to be broadly neutralizing against all seven HCV genotypes and to recognize epitope I, and for this antibody, deleting two or more of the hypervariable regions of E2 increased the ability of this Ab to inhibit E2−CD81 interactions still further.54 In summary, the presence of HVR1, HVR2, and/ or IgVR can block the formation of some Abs that neutralize CD81 binding, and HVR2 and IgVR appear to be able to decrease and increase, respectively, the exposure of the binding sites of HVR1-specific Abs. As mentioned before, however, Abs to HVR1 can neutralize important viral interactions with the cellular receptor SR-BI, and whether Abs to the hypervariable regions can affect interactions of HCV with other cellular receptors such as claudin 1 and occludin remains an open question. 2.3. Glycosylation. Depending on the viral genotype, E1 has five or six potential N-linked glycosylation sites (Asn residues in the sequence Asn-X-Ser/Thr, where X denotes any residue but Pro), which are N196, N209, N234, 250, N305, and N325 in the H77 strain.55 The glycosylation sequon at N250 is specific to genotypes 1b and 6, and the third glycosylation sequon is shifted in position by one residue in genotype 2b and can occur at both the shifted and the more common position simultaneously in genotype 1b.56 The site at N325 does not appear to be glycosylated because the large hydrophobic amino acids Trp 326 and Pro 328 interfere with glycosylation.57 Mutation of the Asn residues at 196 or 305 results in loss of E1/E2 heterodimerization, suggesting that glycosylation on these residues is essential for E1−E2 interaction. The glycosylation of N305 precludes the involvement in disulfide linkage of the Cys residue at position 306, and it has been suggested that the formation of aggregates observed in the N305 mutant may result from an aberrant disulfide bond network through dysregulation at C306.57 Mutations of Nglycosylation sites may also affect the interaction of E1 with calnexin,52 which has previously been shown to correlate with the number of glycans on E1;57 in the same way it is possible that genotypic variations in glycosylation may affect chaperone interactions with E1. There are 9−11 N-linked glycosylation sites belonging to E2, again with variations due to genotypic differences.55 In the H77 reference strain, these are N1 (417), N2 (423), N3 (430), N4 (448, HVR3), N5 (476, HVR2), N6 (532, CD81-binding loop), N7 (540), N8 (556), N9 (576, IgVR), N10 (623), and N11 (645). With a few exceptions, these N-linked glycosylation sites are highly conserved.58 Specifically, N7 is not present in genotype 3 or 6, and N5 is not present in genotype 5, can be absent in genotype 1b, and is often shifted in position by one or two residues.55,56 Most of these residues are observed in the 4MWF crystal structure,26 with the exceptions of N4 (mutated to Asp), N9 (deleted along with neighboring residues in IgVR to facilitate crystallization), and N1 and N5, both of which are absent in the crystal structure. Four of these residues, N7, N8, N10, and N11, were also observed in the 4WEB crystal structure,27 each of which was located on E2’s basic surface (Figure 2). Some of the N-linked glycosylation sites on E2 (N1,

3. FUSION Before the solved structures of E1 and E2, both proteins were predicted to be class II fusion proteins on the basis of their homology to the envelope protein of tickborne encephalitis virus (TBEV).24,61 Thus, an early model of E2 was created by threading E2 onto a class II template in a manner to match locations of β-strands, assisted by the identification of disulfide bridges.24 On the basis of the model, regions of E2 have been termed Domain I, Domain II, Domain III, following the terminology for class II fusion proteins. A typical class II fusion protein comprises three domains, composed mostly of β-sheets. Prior to fusion, the class II fusion protein is elongated and parallel to the lipid bilayer and in a dimeric state. Domain I, containing the protein’s N-terminus, is positioned in the center between the other two domains and has a compact, barrel-like structure. On one side, Domain I is flanked by the elongated Domain II, which contains the fusion loop, a segment composed of between 16 and 26 hydrophobic residues that inserts into the target membrane. It is linked to Domain I by two long insertions connecting two β-strands from Domain I. On the other side of Domain I is Domain III, which has an Ig-fold motif and is anchored in the viral membrane and connected to Domain I by a flexible linker (assigned as IgVR in the model of E2 based on class II fusion proteins24). The fusion protein is synthesized together with a companion, or chaperone, protein, which covers and protects the fusion loop until the companion protein is proteolytically processed in the endosome, priming the virus for fusion.62,63 The exposure of the fusion loop allows for its insertion into the endosomal membrane by a mechanism that has been determined to be cholesterol-dependent in many viruses.62,63 At the same time, the protein transitions from dimeric to trimeric, and the change in pH in the endosome causes Domain III to undergo a conformational change and then fold over Domain I, bringing the viral membrane close to the cell membrane and resulting in the hairpin conformation that facilitates fusion.62,63 Cooperativity within and between oligomers contributes during the fusion process by concentrating force on the viral membrane.62,63 The structures of the N-terminal ectodomains of E1 and E2 were unexpected because each was too compact to be a class II fusion protein. It has been proposed that by acting cooperatively, HCV E1 and E2 together might function as a class II fusion protein.64 The same suggestion has likewise been made about the E1 and E2 proteins from the bovine viral diarrhea virus (BVDV), a pestivirus with an unknown fusion mechanism, following crystallization of its E2 ectodomain.64,65 Besides being heterodimeric, E1/E2 also differs in other regards from typical class II fusion proteins. For one thing, neither E1 nor E2 appears to be proteolytically cleaved in the endosome prior to fusion, and no other companion protein that is proteolytically cleaved has yet been reported. Instead, it seems likely that the low-pH environment in the endosome may disrupt disulfide bonds within E1/E2 in a protease-independent manner.66 Moreover, unlike for typical class II fusion proteins, D



Review

E2, demonstrating a helical conformation for residues 687−703 and a fraying helix formed by C-terminal residues 706−714.74 During formation of the postfusion hairpin structure, the short helical regions in the E1/E2 stem domains may interact with and destabilize the endosomal membrane and in this way facilitate its fusion with the viral membrane.75 Direct evidence for the involvement of the stem regions of E1 and E2 in fusion was provided by experiments in which insertions at residues 341 of E1 and 682 of E2 were observed to specifically disrupt fusion;76 moreover, the H693A/R E2 mutations disrupt viral entry but not CD81 binding, most likely by destabilizing the postfusion hairpin.77 In another study the peptide consisting of 671−705 of E2 was found to block fusion,78 potentially by trapping E1/E2 in the prefusion state by competitive inhibition of interactions between E1 and the E2 stem domain formed in a prefusion intermediate, similar to previous attempts to inhibit fusogenic conformational changes in other viruses.63 Interestingly, Ala 360 and Leu 725 within the TMDs have also been shown to be critical for fusion.79 The protonation of His residues upon internalization within the endosome often acts as a trigger of viral fusion. In HCV, the mutation of E2 residue His 445 to Arg enhances fusion and infectivity, and in fact H445R and H445 K are naturally occurring polymorphisms that must alter the threshold of fusion activation.77 A recent study found that the drug flunarizine inhibits fusion.80 Escape mutations selected in E1/E2 by flunarizine exposure are M267V and Q289H in E1 and M405T in E2. The first E1 mutation occurs just N-terminal to the β-strands in the 4UOI crystal structure, the second belongs to the putative fusion peptide, and the E2 mutation is located in HVR1. It is not clear if flunarizine binds directly to E1/E2. It may be, rather, that flunarizine is membrane-embedded or functions as an ion-channel inhibitor, and the escape mutations could be involved in enhancing fusion in the presence of a change in membrane or ionic properties under flunarizine treatment. For instance, the mutations may allow lipids to bind better to E1/ E2 or may stabilize the pre-hairpin intermediate. On the other hand, these mutations may be located at the binding site of flunarizine.80 Interestingly, the sequence positions of two of these mutations are close to two mutations, I262L and N415D, in E1 and E2, respectively, that arise in ΔHVR1 deletions and restore infectivity, partially in the case of the former mutation and completely in the case of the latter.51

lipoproteins are essential for HCV viral entry and fusion. In summary, whereas E2 displays some similarity to Domain III from class II fusion proteins, it is likely that E1/E2 represents a new class of fusion proteins. Currently, most indications support that a highly conserved region between residues 264 and 290 of E1 contains the fusion peptide. This was suggested by bioinformatics analysis using the alignments of the corresponding peptides from multiple HCV genotypes with flavivirus fusion peptides.67 The presence of a highly conserved acidic residue (D279), as is commonly found in low-pH activated fusion peptides, was noted in multiple HCV genotypes in a similar position as in several flavivirus’ fusion peptides. The sequence similarity also included two highly conserved Cys residues (C272 and C281) and two Gly residues. A set of Gly residues in this E1 sequence is also seen with a similar spacing in the fusion peptides of paramyxoviruses, where they are thought to be crucial structural elements.67 Experiments have established the hydrophobicity/interfaciality of a peptide corresponding to HCV E1 residues 265−296 and demonstrated that it could mediate high levels of fusion.68 Moreover, when residues 276−286 were mutated individually, one of theseF285Awas shown to abolish viral entry.69 Finally, deletion of residues 262−290 in an E1 construct truncated after residue 340 is required for secretion of the protein, indicating that these residues can serve as a transmembrane anchor of truncated E1.67 Two sequences belonging to E2 were also identified as having sequence homology to fusion peptides, 502−52024 and 429−45270 (the latter with proven membranotropic properties68). These peptides were located within the central β-sheet and the CD81 binding domain, respectively, on the basis of recent E2 structures.26,27,70 Although this makes it unlikely that either is the fusion-loop per se, it is still possible that one or both of these sequences may embed in the target membrane, assisting in the fusion process. E2 may serve other roles in viral fusion, and indeed mutations in the TMDs of E1 and E2 that correlate with a loss of E1/E2 heterodimerization also correlate with a loss of fusion activity.71 First, E2 may contribute to shielding the fusion peptide in E1 from premature fusion at earlier stages of viral maturation; at a later stage, the low pH in the endosome may disrupt E1/E2 interactions. Second, the stem region of E2 including its heptad repeat sequence could be involved in costabilizing the postfusion hairpin by doubling over and helping to pull the viral membrane closer to the endosomal membrane. The stem regions of E1 (309−349) and E2 (662−717) connect the ectodomains to the TMDs. In class II fusion proteins, when Domain III folds over Domain I to bridge the space between the viral and host membranes, the stem region at the C-terminal end of Domain III plays an essential role by packing against Domain III, stabilizing it and drawing the TMD closer to the cellular membrane. A similar involvement of the stem regions in fusion may be expected in the case of HCV.72 Within the stem regions of both proteins E1 and E2, there are sequences of heptad repeats, that is, seven consecutive hydrophobic, charged, and polar residues arranged in a specific order, characteristically forming coiled-coil motifs. Both are believed to contain two short α-helices.66 In the case of E1, an α-helical structure was determined by NMR for a peptide corresponding to residues 314−342 in a low-polarity solvent used to mimic the interfacial region where the stem domain is most likely located.73 An NMR spectra was likewise obtained for the peptide composed of residues 684−719 belonging to

4. NEUTRALIZING ANTIBODIES Whereas neutralizing antibodies have been mapped to E1 and to epitopes involving both E1 and E2 sequences, the majority of neutralizing Abs do not require E1 for binding and target the binding site on E2 of the cellular receptor protein CD81; Abs to the CD81 binding site can also be non-neutralizing if this region is incorrectly structured.81 The antigenic regions of E2 are illustrated in Figure 3, which also shows the CD81 binding site. Among the antigenic regions overlapping the CD81 binding site, Antigenic Region 3 (AR3)33/Domain B82 is naturally immunogenic and is bound by broadly neutralizing Abs. It is characterized by binding to residues 523−535 in the CD81 binding loop,3 and other residues can also be important for binding, as is observed in the 4MWF26 crystal structure of the E2 ectodomain bound by the AR3C Ab.33 In particular, AR3 overlaps with two other neutralizing domains corresponding to Abs directed to the CD81 binding site, namely, epitope E



Review

There are also some E1-specific neutralizing Abs.90 These include the weakly neutralizing Ab H-111, binding to residues 192−202 at the N-terminus of E1,91 and the broadly neutralizing Abs IGH505 and IGH526, which bind to stem residues 313−328.92 The latter of these two Abs has been crystallized in a peptide-bound structure with the peptide taking on a helical conformation.90 The AR4 and AR5 Abs bind to epitopes present in the E1/ E2 heterodimer, but not in either protein alone. These Abs were discovered by an exhaustive panning strategy after preblocking E2 with known Abs to the CD81 binding site.93 AR4A is a particularly potent Ab, which was shown to have exceptionally broad neutralizing activity. By using mutational analysis, three regions were identified that are important for binding of both AR4A and AR5A antibodies: 201−206 on E1, 657−659 on E2, and 692 on E2. That is, AR4A and AR5A recognize residues in the N-terminal region of E1 and in the stem region of E2. In addition, two extremely conserved residues are needed for binding by AR4A and AR5A, respectively, namely, D698 and R639, the latter of which appears in both crystal structures 4MWF and 4WEB. Despite having overlapping binding regions, the two Abs do not compete with each other, and in fact AR5A (but not AR4A) competes93 with CBH-7,82 one of the antibodies that bind to AR2 at E2 sites 541−549. 4.1. Clinical Impact of E1/E2−Ab Interactions. Although the role of virus-specific CD4+ and CD8+ T cells in eradication of acute hepatitis C virus (HCV) infection has been directly demonstrated,94,95 recent data also indicate an important role for neutralizing antibodies in eradication of acute infection. Many studies have shown the temporal association of recovery from acute or chronic infection with the development of neutralizing antibodies,96−99 and a protective role for such antibodies has been directly demonstrated in mouse100,101 and chimpanzee102−104 models of HCV infection. Moreover, a vaccine comprising a recombinant 1a E1/E2 envelope glycoprotein heterodimer, derived from a single 1a strain, has been shown to significantly reduce the carrier state in vaccinated chimpanzees that were challenged with either homologous or heterologous 1a virus, which is the most common genotype in the United States and Canada.4 This remains the only HCV vaccine candidate shown to reduce the carrier rate in the reliable chimpanzee model. Such vaccinated chimpanzees elicit broadly cross-neutralizing antibodies against all HCV genotypes.107 This vaccine adjuvated with MF59 is well-tolerated in humans108 and elicits broadly cross-neutralizing antibodies targeting many different B-cell epitopes in E1/E2.105,106 In addition, strong CD4+ T cell lymphoproliferative responses to the E1/E2 vaccine were observed in vaccinated humans.108 When combined with clinical data showing that hypo-γ-globulinemics experience more severe disease,109 an HCV vaccine that primes both broad cross-neutralizing antibodies and cross-reactive T cells may provide optimal protection against this highly heterogeneous virus.

Figure 3. Antigenic surface of the 4MWF crystal structure. The neutralizing face (red) is in a lateral position in the left-hand structure and is rotated to face the viewer in the structure on the right. Hypervariable regions and Abs binding to AR1, AR2/Domain C, and AR3/Domain B are labeled. (Here VR3 represents IgVR.) The CD81 binding site is outlined by a cyan-colored dotted line. A white-dotted line surrounds helix α1 and corresponds to Domain D/epitope II. The red dashed line connecting HVR1 to the crystallized construct represents Domain E/epitope I. Reprinted with permission from ref 26. Copyright 2013 American Association for the Advancement of Science.

I/Domain E and epitope II/Domain D, both shown in Figure 3. Epitope I3/Domain E84−86 precedes the start of the crystal structure 4MWF at residue 421 immediately downstream of HVR1. These Abs recognize overlapping epitopes with different neutralizing potentials, particularly within the region 412−423.3 Abs binding in this region block interaction with CD81 via contact with W420, a residue important in binding CD81. However, binding by these Abs leads to selection of escape mutations. Moreover, seroprevalence is low in this region, meaning that only a very small percentage of Abs raised in HCV-infected patients compete for binding to these epitopes and suggesting that this region may be masked.3 One factor leading to immune evasion is the flexibility of this region, because there are two conformations (a β-hairpin87 and an extended conformation) recognized by different Abs.87,88 The other region mentioned above as overlapping with AR3/ Domain B is Domain D89/ epitope II.3 These Abs bind to 434−446,3 residues located within HVR3another region where binding can in some cases lead to neutralization of CD81 binding. There is also some overlap between AR3 and AR233/Domain C,82 an epitope separate from the CD81 binding site (Figure 3). AR2/Domain C Abs have restricted neutralizing capabilities and bind to a site centered on N540.3 Amino acids tolerant to change are often targets of these Abs.3 One of these, AR2A,33 was used by Kong et al.26 for EM imaging. AR2 is proximal to another epitope, AR1,33 non-neutralizing or weakly neutralizing and encompassing residues 538−540.3 Another set of Abs that are not directed to the CD81 binding site are those targeting HVR1. The mechanisms of these Abs can involve inhibition of either postattachment entry or infectivity.48 They generally bind to the C-terminal region of HVR1, are isolate-specific and neutralizing, and may be immunodominant in natural infection.53 Abs binding to the N-terminal portion of HVR1 all are non-neutralizing.3,48 For the sake of completeness in our discussion of Abs binding to E2, we last mention that Domain A82,83 refers to a set of non-neutralizing Abs.2

5. HETERODIMERIZATION 5.1. Transmembrane Domains. Heterodimerization of E1/E2 is controlled by the TMDs (350−383 in E1 and 718− 742 in E2). Both TMDs are believed to consist of a single αhelix. This was concluded for E2 because its TMD consists of two hydrophobic stretches (718−727 and 731−742), each of F



Review

Intramolecular disulfide bonding in the noncovalent heterodimer E1/E2 has been investigated by mutation of cysteine residues in each protein. It was found that the disulfide linkages within the E1 protein are flexible, in the sense that mutations of cysteine residues only reduce viral infectivity,114 whereas in E2 such mutations severely affect E1/E2 heterodimerization.115 Interestingly, all E1 mutations of cysteine residues induce better accessibility of CD81 to its binding site on E2,114 demonstrating the influence of E1 on E2 structure or possibly indicating a change in the oligomeric state. Heterodimerization of E1/E2 does not appear to be affected by mutation of E1 cysteine residues to Ala or C226A−C229A,114 but all E2 Cys to Ala mutations greatly decreased or eliminated heterodimerization with E1.115 Thus, all E2 cysteine residues have structural significance to heterodimerization with E1, in the sense of maintaining E2 in the correct conformation to heterodimerize. It is likely that many of these E2 cysteines exert this influence by participating in intramolecular disulfide bonds. However, this is likely not the case for all E2 cysteines because the study by Vieyres et al.48 provided evidence that some of the Cys residues in the E1/E2 heterodimer are involved in covalent oligomeric interactions of heterodimers. In the 4MWF crystal structure, all of the cysteines are involved in intramolecular disulfide bonds.26 The remaining cysteines not seen in the 4MWF crystal structure could possibly form two more pairs of disulfide bonds, that is, C459−C486 and C652−C677 (of which the former pair of cysteines but not the latter was included in the crystallized construct but was too flexible to be assigned).26 The disulfide bonding network differs in the second crystal structure, 4WEB.27 Another study attempted to deduce disulfide linkages in E2 from Ab reactivity and CD81 binding patterns against Cys to Ala mutants.116 Residue pairs were assigned as disulfide-linked if Ab reactivity and CD81 binding patterns decreased for mutations of either residue or, in the case of C652−C677, mutation of either residue did not affect Ab reactivity or binding to CD81-LEL, but led to loss of heterodimerization.116 The disulfide linkages determined from the two crystal structures and from the Ab binding patterns are shown in Table 1. Another set of disulfide linkages has been determined by trypsin digestion of the E2 ectodomain under reducing and nonreducing conditions to identify peptides susceptible to reduction, followed by high-performance liquid chromatog-

which is too small to span the membrane as a helix. In the case of E1, NMR and circular dichroism experiments have verified that residues 350−370 form a conformation that is mainly αhelical, although the N-terminal residues (350−353) are unstructured; the remaining C-terminal residues of the E1 TMD are expected to extend this helix.110 Single membranespanning topologies for E1 and E2 have also been confirmed by epitope tagging and fluorescent imaging of the proteins’ Ctermini.6 The TMDs consist of mostly hydrophobic residues but also contain charged residues in their centers that act as ER retention signals111 and are directly involved in heterodimerization, namely, Lys 370 in E1 and Asp 728 and Arg 730 in E2. Mutational analysis demonstrated large reductions in heterodimerization when Ala is inserted at the centers of the TMDs of E1 or E2,110 Asp 728 and Arg 730 are mutated to Ala,111 or Lys 370 or Asp 728 is mutated to Trp.79 Of the two charged residues in the E2 TMD, on the basis of the reduction of heterodimerization upon mutations to Leu, Asp 728 is more important in heterodimerization than Arg 730 (reduction to 12%, compared to 50%).71 These data suggest that Asp 728 and Lys 370 might form an ion pair linking the two TMDs; however, this is unconfirmed, and in fact replacement of Asp 728 by Lys did not seem to change heterodimerization.71 Two GXXXG motifs (intramembrane protein−protein interaction motifs) belonging to the N-terminus of E1’s TMD also contain residues important for heterodimerization,110 especially the two Gly residues in the G354XXXG motif.79 5.2. Disulfide Bonds. E1 and E2 have 8 and 18 conserved cysteine residues, respectively. Recombinant E1/E2 isolated as a noncovalently linked heterodimer9,112 has been postulated to represent the native state, whereas a disulfide-linked aggregate form has been thought to represent misfolded E1E2.112 The correct folding of E1 appears to require E2, particularly the E2 TMD residues.32 Fully oxidized, and presumably disulfidebonded, E1 is formed only in the presence of E2,31 and its formation coincides with that of the noncovalently linked form of E1/E2.9 A recent study has called into question the native state of E1/E2 in the context of assembled viral particles. Vieyres et al.48 observed that the majority of E1/E2 found in the virion was in disulfide-linked complexes with molecular weights of at least 440 kDa (cf. roughly 30/70 kDa for the glycosylated E1/E2 heterodimer.) The existence of these complexes in a native state was verified by testing for binding to antibodies and to CD81. On the other hand, the same study found that most intracellular E1/E2 exists as a noncovalently linked heterodimer. It is unclear when and how E1 and E2 are disulfide-linked to one another in the heterodimer within the viral envelope. It may be that the disulfide linkage observed by Vieyres et al.48 takes place only between different E1/E2 heterodimers in the virion. It seems reasonable that the localization of disulfide-linked complexes to the viral envelope may simply be due to the scaffold afforded by the other components of the viral envelope providing the right environment and organization necessary for oligomerization of E1/E2 to take place. Alternatively, as suggested by Vieyres et al.,48 a mechanism may be in place for selectively incorporating disulfide-linked E1/E2 into secreted virions, and E1−E2 interactions might play an active role in secretion. In this regard, the recently reported structural homology between the E1 N-terminal region28 and the phosphatidylcholine transfer protein113 could indicate a potential role of E1 in lipid transfer during viral secretion.

Table 1. Disulfide Bonds in E2 Predicted from Crystal Structures and Ab Reactivity experimental source 4MWF 429−503 452−620 459−620 486−585 486−620 494−564 508−552 569−581 585−597 597−569 607−644 652−677 a

G

+ + − − − + + + + − +

a

4WEBb

Ab reactivityc

− − − + + + − − + +

+ − + + − + + + − − + +

Reference 26. bReference 27. cReference 116. DOI: 10.1021/acsinfecdis.6b00110 ACS Infect. Dis. XXXX, XXX, XXX−XXX


Review

(measured by ability to bind CD81). However, similar to E2, all helix-breaking mutations led to loss of entry, as did three of five helix-stabilizing mutations.69 His 352 in the E1 stem region (Cterminal to the heptad repeat sequence) has been shown to be important for E1 structure and heterodimerization.77 5.4. Other Residues Contributing to Heterodimerization. Yi et al.121 used far-Western blotting with E2 as a probe to demonstrate that a recombinant protein corresponding to residues 192−238 and comprising the N-terminal region of E1 and, less so, one corresponding to residues 239−340 further on in the E1 sequence bind E2. In the same study, it was found that the N-terminal part of E2 containing residues 415−500 interacts with E1. In particular, the highly conserved residues from 484 to 491 containing the “W489HY” sequence and flanking HVR2, were found to be essential for heterodimerization on the basis of mutational analysis.121 Rychlowska et al. performed another important study that gave information about residues in E1/E2 involved in heterodimerization.76 Linker scanning mutagenesis was used to generate 5aa insertions at 34 random positions within the E1/E2 sequence. None of the viable insertions within E1 affected heterodimerization, whereas this experiment pointed to several residues in E2 important for heterodimerization. In particular, residues 587−597, a flexible region on E2 absent from both crystal structures, as well as 692−727 belonging to the pretransmembrane and TM region of E2 were shown to be critical specifically for heterodimerization, because inserts at these regions affected heterodimerization without causing a global change in E2 structure.76 A study by Douam et al.122 used domain complementation in dual-strain chimeric E1/E2 heterodimers from strains H77 and A40 to investigate which residues of E1 and E2 are involved in cross talk between the two proteins. Residues and domains in E1 and E2, respectively, were identified that when mutated to match the strain corresponding to the heterodimeric partner (E2 or E1, respectively) could restore HCVpp infectivity. Although structural data now prove otherwise, at the time the study was performed, E2 was believed to have the architecture of a class II fusion protein, and thus regions matching class II domains were swapped between strains. The study pointed to the significance of certain residues belonging to the E1 stem region, namely, M308I, T330A, and L345M, because each single mutation slightly improved cell entry, and together the three E1 mutations led to a 7-fold increase in cell entry. Likewise, residues belonging to Domain III of E2 are important for optimal interactions with E1, because substituting Domain III in E1A40/E2H77 with that of E2A40 restored HCVpp entry to the same level as A40. The residues differing in Domain III of E2 between the two different strains are L603I, L608M, N610D, and I629 V. The crosstalk between E1 and E2 was shown to simultaneously involve both the three residues in E1 and E2’s Domain III. Moreover, this crosstalk was shown to modulate binding to both SR-BI and CD81 and to be influential in fusion. Complementation by HVR1, Domain I, Domain II, or IgVR did not lead to improved heterodimer functionality.122

raphy and sequencing of peptides’ N-termini; paired Cys residues are 429−552, 452−459, 486−494, 503−508, 564− 569, 581−585, 607−644, and 652−677, of which only the last two pairs match the other data sets.24 In E1, the Cys residues are C207, C226, C229, C238, C272, C281, C304, and C306, of which C229 and C238 are disulfide linked in the 4UOI crystal structure.28 The disagreement among the experimental data for E2 disulfide bonding, discussed above, makes it very difficult to form conclusions about the disulfide-bonding pattern in E1/E2. This is further complicated by indications that these linkages isomerize following virion incorporation and/or cellular attachment. Fraser et al.117 showed that free thiol groups in both E1 and E2 are needed for cellular attachment, based on the elimination of viral entry by addition of a sulfhydryl alkylating agent, and suggested that free Cys residues could be responsible for catalyzing disulfide isomerization. This type of requirement is already known in some leukemia viruses, where the prefusion complex has a labile disulfide bond that is reduced by a Cys residue belonging to a CXXC motif, activating fusion.118,119 In HCV, fusion does not occur immediately in the endosome, but instead, takes place only after lengthy incubation at 37 °C, indicating a kinetically activated process.120 Thus, the isomerization of disulfide bonds upon attachment must precede further pH-induced rearrangements within the endosome. The connection between attachment and isomerization is unclear as the alkylating agent does not affect the ability to bind to CD81 and, conversely, soluble CD81 does not affect the overall oxidation state of the glycoproteins. In HCV, E1 contains a highly conserved C226(V/L)PC protein disulfide isomerase motif, suggesting that C226 may be one of the free cysteines required for cell entry. This suggests that in the endosome, C226 might form a new disulfide bridge with C229 and catalyze the dissociation of the disulfide linkage between C229 and C238. However, if this happens, then the effect of C226 in catalyzing the cleavage of this disulfide linkage seems to be kinetic only, on the basis of experiments demonstrating that, although the C226A mutation completely eliminates infectivity 48 h postelectroporation, it has a similar effect as the double-mutant C226A/C229A after 72 h.114 5.3. Heptad Repeat Regions. Drummer and co-workers have investigated how mutating residues in the heptad repeat regions of E1 and E2 affect cell entry and heterodimerization.69,72 When Ala and Pro mutations, representing helixstabilizing and helix-breaking residues, respectively, were introduced at residues in the a and d positions of the heptad repeat region on E2 (675−699), all but one (S687A) eliminated the ability of HCVpp to infect Huh7 cells. Moreover, the four residues Leu 675, Ser 678, Leu 689, and Leu 692 were all found to be determinants of heterodimerization, in that heterodimerization is partially blocked in HCVcc and completely blocked in HCVpp by all mutations to Pro and by all mutations to Ala except for S678A.72 Ala substitutions were reported to be less severe, emphasizing the importance of helical structure in this region. His 691 in this region is also essential for heterodimerization.77 The helix-stabilizing mutations P676A and P683A were also introduced; both allow for heterodimerization, although P683A eliminates Huh7 cell-entry competence.72 Helix-breaking and helix-forming mutations were also made in E1 at residues within the heptad repeat sequence (330−347) at the a and d positions. Unlike E2, mutations in E1 had no effect on heterodimerization or folding

6. OLIGOMERIZATION Work by Falson et al.123 indicates that the oligomeric structure of E1/E2 is a trimer of heterodimers (see Figure 4). When E1 is expressed in HCVcc or HCVpp, analysis by SDS-PAGE at 37 °C under reducing conditions reveals non-disulfide-linked E1 trimers, and the formation of these trimers in HCVpp is H



Review

Although three-dimensional imaging of the HCV virion has so far been hindered by difficulties in growing sufficient quantities of HCV in cell culture, Yu et al.124 succeeded in using cryo-electron microscopy (EM) to reconstruct HCV-like particles based on a baculovirus system at a 30 Å resolution (Figure 5). Cryo-EM images of HCV virions purified from cell culture show spherical particles with a smooth outer surface, all with a diameter of about 500 Å, and the HCV-like particles appear similar, but particle sizes are somewhat more variable. Visualization of these particles reveals an arrangement of E1/E2 with 2-, 3-, and 5-fold symmetry axes. According to the authors’ reconstruction, E1/E2 is arranged in dimers of heterodimers in a head-to-tail configuration, similarly as in dengue virus, based on fitting the structure of dengue virus E protein into the density map.124 However, this dimeric arrangement should now be re-evaluated in consideration of the trimeric arrangement proposed by Falson et al.123 because the class II fusion protein E from dengue is no longer believed to be an appropriate model for E1/E2. Visualization by cryo-EM along with cryo-electron tomography has also been used to show that HCVcc particles are coated with an outer layer containing ApoE, ApoA, and ApoB lipoproteins. The attached apolipoproteins lead to more variable sizes for HCVcc lipoviral particles, ranging from 400 to 1000 Å.43

Figure 4. Model proposed by Falson et al. in which E1/E2 is arranged as a trimer of heterodimers, with E1 components interacting with one another at the center of the trimer through their TMDs. K370 is shown in stick and is assumed to interact with the TMD of the E2 monomer within the same heterodimer. Reprinted with permission from ref 123. Copyright 2015 American Society for Microbiology.

dependent upon the coexpression of E2. This oligomeric structure is heat-labile because when the experiments were performed at 95 °C, mainly monomers and dimers of E1 were observed. Mutating residues in the G354XXXG motif found in the N-terminal region of E1’s TMD led to complete loss of infectivity along with loss of trimerization; this points to the importance of E1/E2 trimerization to infectivity and to the importance of this motif in E1 TMDs to trimerization.123 We note that the absence of E2 from the observed trimer indicates that there was not a stable noncovalent E1/E2 heterodimer in the virion during these experiments.

7. CONCLUSIONS The HCV virion demonstrates a capacity for obscuring its envelope proteins, E1 and E2, from the immune system. Some of the tactics it uses to shield E1/E2 are glycosylation,

Figure 5. Surface (a, c) and density slice (b, d) images of 3D reconstruction of HCV lipoviral pseudoparticles. The particles shown in panels c and d are labeled with anti-E1 Abs. Panels a and c are color-coded according to radius, and 2-, 3-, and 5-fold symmetry axes are visible and labeled on the figure. The red arrowheads in panels b and d indicate the lipid bilayer, and the black arrows point to E1/E2. In panel b, the blue arrow points to the capsid, and in panel d, the thick red arrow points to an outer layer believed to correspond to Ab density. Adapted with permission from ref 124. I



Review

association with lipoproteins, the flexibility and variability of the hypervariable regions on E2, and its structural versatility that entails shape alterations during cell entry and fusion by the reisomerization of disulfide bonds. The neutralizing antibody response of the host strives to target conserved epitopes on E1/ E2 that are key to viral functions including CD81 binding of E2 during cell entry, interaction of HVR1 with cellular receptors, and involvement of the interfacial/stem regions of both E1 and E2 in fusion. A structural picture of E1/E2 is gradually emerging; both proteins have ectodomains with globular structures composed mainly of β-sheets, stem regions containing two α-helices, and single-domain α-helical C-terminal TMDs. Many structural details of the E1/E2 heterodimer are, however, still incomplete, such as the complete binding sites of the potent AR4A and AR5A cross-neutralizing antibodies (that target the E1/E2 interface), the spatial positions of the E1/E2 glycosyl groups, and hypervariable regions, as well as the nature and time course of lipoprotein association and re-isomerization of disulfide bridges. HCV appears to have a fusion mechanism different from those previously observed in other viruses, and a detailed understanding of this mechanism may facilitate the design of peptides or small molecules to interfere with this process and hence facilitate an important new antiviral.

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(7) Vieyres, G., Dubuisson, J., and Pietschmann, T. (2014) Incorporation of hepatitis C virus E1 and E2 glycoproteins: the keystones on a peculiar virion. Viruses 6, 1149−1187. (8) Douam, F., Lavillette, D., and Cosset, F. L. (2015) The mechanism of HCV entry into host cells. Prog. Mol. Biol. Transl. Sci. 129, 63−107. (9) Dubuisson, J., and Rice, C. M. (1996) Hepatitis C virus glycoprotein folding: disulfide bond formation and association with calnexin. J. Virol. 70, 778−786. (10) Choukhi, A., Ung, S., Wychowski, C., and Dubuisson, J. (1998) Involvement of endoplasmic reticulum chaperones in the folding of hepatitis C virus glycoproteins. J. Virol. 72, 3851−3858. (11) Liberman, E., Fong, Y. L., Selby, M. J., Choo, Q. L., Cousens, L., Houghton, M., and Yen, T. S. (1999) Activation of the grp78 and grp94 promoters by hepatitis C virus E2 envelope protein. J. Virol. 73, 3718−3722. (12) Thomssen, R., Bonk, S., Propfe, C., Heermann, K. H., Kochel, H. G., and Uy, A. (1992) Association of hepatitis C virus in human sera with β-lipoprotein. Med. Microbiol. Immunol. 181, 293−300. (13) Thomssen, R., Bonk, S., and Thiele, A. (1993) Density heterogeneities of hepatitis C virus in human sera due to the binding of β-lipoproteins and immunoglobulins. Med. Microbiol. Immunol. 182, 329−334. (14) Andre, P., Komurian-Pradel, F., Deforges, S., Perret, M., Berland, J. L., Sodoyer, M., Pol, S., Brechot, C., Paranhos-Baccala, G., and Lotteau, V. (2002) Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J. Virol. 76, 6919−6928. (15) Meunier, J. C., Engle, R. E., Faulk, K., Zhao, M., Bartosch, B., Alter, H., Emerson, S. U., Cosset, F. L., Purcell, R. H., and Bukh, J. (2005) Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1. Proc. Natl. Acad. Sci. U. S. A. 102, 4560−4565. (16) Dreux, M., Pietschmann, T., Granier, C., Voisset, C., RicardBlum, S., Mangeot, P. E., Keck, Z., Foung, S., Vu-Dac, N., Dubuisson, J., Bartenschlager, R., Lavillette, D., and Cosset, F. L. (2006) High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of the scavenger receptor BI. J. Biol. Chem. 281, 18285−18295. (17) Voisset, C., Op de Beeck, A., Horellou, P., Dreux, M., Gustot, T., Duverlie, G., Cosset, F. L., Vu-Dac, N., and Dubuisson, J. (2006) High-density lipoproteins reduce the neutralizing effect of hepatitis C virus (HCV)-infected patient antibodies by promoting HCV entry. J. Gen. Virol. 87, 2577−2581. (18) Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner, A. J., Houghton, M., Rosa, D., Grandi, G., and Abrignani, S. (1998) Binding of hepatitis C virus to CD81. Science 282, 938−941. (19) Farquhar, M. J., Hu, K., Harris, H. J., Davis, C., Brimacombe, C. L., Fletcher, S. J., Baumert, T. F., Rappoport, J. Z., Balfe, P., and McKeating, J. A. (2012) Hepatitis C virus induces CD81 and claudin-1 endocytosis. J. Virol. 86, 4305−4316. (20) Harris, H. J., Davis, C., Mullins, J. G., Hu, K., Goodall, M., Farquhar, M. J., Mee, C. J., McCaffrey, K., Young, S., Drummer, H., Balfe, P., and McKeating, J. A. (2010) Claudin association with CD81 defines hepatitis C virus entry. J. Biol. Chem. 285, 21092−21102. (21) Evans, M. J., von Hahn, T., Tscherne, D. M., Syder, A. J., Panis, M., Wolk, B., Hatziioannou, T., McKeating, J. A., Bieniasz, P. D., and Rice, C. M. (2007) Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446, 801−805. (22) Ploss, A., Evans, M. J., Gaysinskaya, V. A., Panis, M., You, H., de Jong, Y. P., and Rice, C. M. (2009) Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457, 882−886. (23) Yagnik, A. T., Lahm, A., Meola, A., Roccasecca, R. M., Ercole, B. B., Nicosia, A., and Tramontano, A. (2000) A model for the hepatitis C virus envelope glycoprotein E2. Proteins: Struct., Funct., Genet. 40, 355−366. (24) Krey, T., d’Alayer, J., Kikuti, C. M., Saulnier, A., Damier-Piolle, L., Petitpas, I., Johansson, D. X., Tawar, R. G., Baron, B., Robert, B.,

AUTHOR INFORMATION

Corresponding Authors

*(H.F.) E-mail: [email protected]. *(M.H.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.F. thanks Jason Wong for helpful discussions. We are grateful for funding support from the Canadian Excellence Research Chair (CERC) grant held by M.H.

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ABBREVIATIONS Ab, antibody; AR, antigenic region; EM, electron microscopy; ER, endoplasmic reticulum; HCV, hepatitis C virus; HCVcc, HCV cell culture system; HCVpp, HCV pseudoparticle; HVR, hypervariable region; IgVR, intergenomic variable region; TMD, transmembrane domain

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REFERENCES

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