The CD81 partner EWI-2wint inhibits hepatitis C virus entry

Two to three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Here, we have identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2wint in a hepatoma cell line susceptible to HCV infection blocked viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. This finding suggests that, in addition to the presence of specific entry factors in the hepatocytes, the lack of a specific inhibitor can contribute to the hepatotropism of HCV. This is the first example of a pathogen gaining entry into host cells that lack a specific inhibitory factor.     

Hepatitis C virus entry and the tetraspanin CD81

CD81, a member of the tetraspanin integral membrane protein family, has been identified as an essential receptor for HCV (hepatitis C virus). The present review highlights recent published data on the role that CD81 plays in HCV entry, including the importance of actin-dependent lateral diffusion of CD81 within the cell membrane, CD81 endocytosis and the CD81-Claudin-1 receptor complex in HCV internalization. Additional functions for CD81 in the viral life cycle and the role of HCV-CD81 interactions in HCV-induced B-cell and CNS (central nervous system) abnormalities are discussed.     

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.     

Hepatitis C virus envelope glycoprotein E2 glycans modulate entry, CD81 binding, and neutralization

Hepatitis C virus (HCV) is a major human pathogen that causes serious liver disease, including cirrhosis and hepatocellular carcinoma. The primary target cells of HCV are hepatocytes, and entry is restricted by interactions of the envelope glycoproteins, E1 and E2, with cellular receptors. E1 and E2 form noncovalently linked heterodimers and are heavily glycosylated. Glycans contribute to protein folding and transport as well as protein function. In addition, glycans associated with viral envelopes mask important functional domains from the immune system and attenuate viral immunogenicity. Here, we explored the role of N- and O-linked glycans on E2, which is the receptor binding subunit of the HCV envelope. We identified a number of glycans that are critical for viral entry. Importantly, we showed that the removal of several glycans significantly increased the inhibition of entry by sera from HCV-positive individuals. Only some of the glycans that affected entry and neutralization were also important for CD81 binding. Our results show that HCV envelope-associated glycans play a crucial role in masking functionally important regions of E2 and suggest a new strategy for eliciting highly neutralizing antibodies against this virus.     

Loss of surface EWI‐2 on CD9 null oocytes

CD9, a member of the tetraspanin family, associates with a variety of other proteins to form the tetraspanin web. CD9 forms direct and relatively stable associations with the immunoglobulin superfamily proteins EWI‐2 and EWI‐F. Deletion of the Cd9 gene results in female infertility since Cd9 null mice produce oocytes that fail to fuse. It is thought that the absence of CD9 causes the inability of the oocytes to fuse. In this study, we report that the expression level of EWI‐2 on the Cd9^?/? oocyte surface is <10% of the wild‐type level. Hence, the severe reduction in EWI‐2 activity may be responsible for the loss of fusion ability. An entirely different mutant of CD9, not a deletion but a depalmitoylated construct, does not affect in vivo female fertility suggesting that the palmitate modification of CD9 is not essential for its putative fusion function. Additionally, the level of EWI‐2 on the surface of the oocytes from these females was comparable to the EWI‐2 level on wild‐type oocytes. We also found that soluble, recombinant EWI‐2 binds preferentially to acrosome‐reacted sperm but the bound EWI‐2 does not inhibit sperm–oocyte fusion. Overall, the results indicate that deletion of CD9, which is known to have multiple associations, may have pleiotropic effects on function that will require further dissection. Mol. Reprod. Dev. 76: 629–636, 2009. © 2008 Wiley‐Liss, Inc.     

Characterization of Hepatitis C Virus E2 Glycoprotein Interaction with a Putative Cellular Receptor, CD81

A truncated soluble form of the hepatitis C virus E2 glycoprotein, E2661, binds specifically to the surface of cells expressing human CD81 (hCD81) but not other members of the tetraspanin family (CD9, CD63, and CD151). No differences were noted between the level of E2661 binding to hCD81 expressed on the surface of rat RBL or KM3 cells compared to Daudi and Molt-4 cells, suggesting that additional human-cell-specific factors are not required for the primary interaction of E2 with the cell surface. E2 did not interact with African green monkey (AGM) CD81 on the surface of COS cells, which differs from the hCD81 sequence at four residues within the second extracellular region (EC2) (amino acids [aa] 163, 186, 188, and 196), suggesting that one or more of these residues defines the site of interaction with E2. Various recombinant forms of CD81 EC2 show differences in the ability to bind E2, suggesting that CD81 conformation is important for E2 recognition. Regions of E2 involved in the CD81 interaction were analyzed, and our data suggest that the binding site is of a conformational nature involving aa 480 to 493 and 544 to 551 within the E2 glycoprotein. Finally, we demonstrate that ligation of CD81 by E2661 induced aggregation of lymphoid cells and inhibited B-cell proliferation, demonstrating that E2 interaction with CD81 can modulate cell function.     

A Neutralization Epitope in the Hepatitis C Virus E2 Glycoprotein Interacts with Host Entry Factor CD81

The identification of a specific immunogenic candidate that will effectively activate the appropriate pathway for neutralizing antibody production is fundamental for vaccine design. By using a monoclonal antibody (1H8) that neutralizes HCV in vitro, we have demonstrated here that 1H8 recognized an epitope mapped between residues A524 and W529 of the E2 protein. We also found that the epitope residues A524, P525, Y527 and W529 were crucial for antibody binding, while the residues T526, Y527 and W529 within the same epitope engaged in the interaction with the host entry factor CD81. Furthermore, we detected “1H8-like” antibodies, defined as those with amino acid-specificity similar to 1H8, in the plasma of patients with chronic HCV infection. The time course study of plasma samples from Patient H, a well-characterized case of post-transfusion hepatitis C, showed that “1H8-like” antibodies could be detected in a sample collected almost two years after the initial infection, thus confirming the immunogenicity of this epitope in vivo. The characterization of this neutralization epitope with a function in host entry factor CD81 interaction should enhance our understanding of antibody-mediated neutralization of HCV infections.     

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation.     

Hepatitis C virus induces CD81 and claudin-1 endocytosis

Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.     

Mutations in Hepatitis C Virus E2 Located outside the CD81 Binding Sites Lead to Escape from Broadly Neutralizing Antibodies but Compromise Virus Infectivity

Broadly neutralizing antibodies are commonly present in the sera of patients with chronic hepatitis C virus (HCV) infection. To elucidate possible mechanisms of virus escape from these antibodies, retrovirus particles pseudotyped with HCV glycoproteins (HCVpp) isolated from sequential samples collected over a 26-year period from a chronically infected patient, H, were used to characterize the neutralization potential and binding affinity of a panel of anti-HCV E2 human monoclonal antibodies (HMAbs). Moreover, AP33, a neutralizing murine monoclonal antibody (MAb) to a linear epitope in E2, was also tested against selected variants. The HMAbs used were previously shown to broadly neutralize HCV and to recognize a cluster of highly immunogenic overlapping epitopes, designated domain B, containing residues that are also critical for binding of viral E2 glycoprotein to CD81, a receptor essential for virus entry. Escape variants were observed at different time points with some of the HMAbs. Other HMAbs neutralized all variants except for the isolate 02.E10, obtained in 2002, which was also resistant to MAb AP33. The 02.E10 HCVpp that have reduced binding affinities for all antibodies and for CD81 also showed reduced infectivity. Comparison of the 02.E10 nucleotide sequence with that of the strain H-derived consensus variant, H77c, revealed the former to have two mutations in E2, S501N and V506A, located outside the known CD81 binding sites. Substitution A506V in 02.E10 HCVpp restored binding to CD81, but its antibody neutralization sensitivity was only partially restored. Double substitutions comprising N501S and A506V synergistically restored 02.E10 HCVpp infectivity. Other mutations that are not part of the antibody binding epitope in the context of N501S and A506V were able to completely restore neutralization sensitivity. These findings showed that some nonlinear overlapping epitopes are more essential than others for viral fitness and consequently are more invariant during earlier years of chronic infection. Further, the ability of the 02.E10 consensus variant to escape neutralization by the tested antibodies could be a new mechanism of virus escape from immune containment. Mutations that are outside receptor binding sites resulted in structural changes leading to complete escape from domain B neutralizing antibodies, while simultaneously compromising viral fitness by reducing binding to CD81.Over 170 million people worldwide are infected with hepatitis C virus (HCV). While acute infection is usually silent, the majority of infected individuals develop persistent infections. Approximately 30% of acute infections are spontaneously resolved. Cellular immunity is clearly necessary, as robust and sustained CD4⁺ and CD8⁺ T-cell responses are temporally associated with virus clearance leading to disease resolution (7). Persistent infection is associated with an inability to sustain a vigorous CD4⁺ response. The role of antibodies in disease resolution is increasingly recognized but less understood. Clinical trials with gamma globulin administration prior to the discovery of HCV achieved prophylactic effects on transfusion-associated non-A, non-B hepatitis cases, most of which were subsequently shown to be HCV related (28, 46). Animal studies showed that gamma globulin therapy delayed the onset of acute HCV infection (29). Preincubation of the infectious inoculum with pooled gamma globulin from HCV-positive donors prevented infection in challenged chimpanzees (55). The protection afforded by gamma globulin preparations correlated with antibody titers blocking infection of target cells with retroviral pseudotype particles expressing HCV E1E2 glycoproteins (HCVpp) (4). In addition, chimpanzees vaccinated with recombinant HCV E2 glycoproteins were protected against infection in a manner that correlated with serum antibody titers inhibiting binding of E2 to CD81 (19, 40, 41), a receptor required for entry by both HCVpp and cell culture infectious HCV (HCVcc) (5, 17, 33, 53, 56). Two recent studies observed that patients with strong and progressive neutralizing antibody responses demonstrated decreasing viremia and control of viral replication (31, 39). A third study, however, reported the lack of neutralizing antibodies to heterologous HCVpp isolates in the sera of patients who eventually controlled their viremia during acute HCV infection (21). Furthermore, 10⁴ to 10⁶ virions per milliliter of serum are usually detected during chronic infection in the presence of high titers of serum neutralizing antibodies.A driver of persistent viremia is a high degree of viral variants, or “quasispecies.” Owing to a high viral replication rate (10¹² copies per day) and an error-prone viral RNA-dependent polymerase, the estimated mutation rate is 2.0 × 10⁻³ base substitutions per genome per year (9, 34). This high rate of quasispecies formation contributes to the emergence of escape viral variants from immune surveillance. Mutations within major histocompatibility complex class I-restricted HCV epitopes lead to escape from cytotoxic T-cell responses (7). Mutations leading to escape from humoral immunity, particularly in E2 hypervariable region 1 (HVR1), known to be the target of host neutralizing antibodies, are also documented (10, 22, 30, 45). Protection in chimpanzees is achieved following challenge with an inoculum that had been preincubated with antibodies to autologous HVR1 (10). Yet over time, these isolate-specific antibodies drive the emergence of new viral variants that the concurrent immune response poorly recognizes. A study of sequential HCV isolates obtained from a patient, H, who was meticulously followed for a 26-year period starting 3 weeks after exposure to the virus, showed that the serial HCV variants were poorly neutralized by the concurrent serum antibodies (52). Escape was associated in part with mutations in HVR1 leading to decreased binding and neutralization by monoclonal antibodies (MAbs) to HVR1 that were produced against the first isolate obtained from this patient.Broadly neutralizing antibodies are usually directed against conformational epitopes within E2 (2, 8, 13, 14, 44). We previously described a panel of neutralizing and nonneutralizing human MAbs (HMAbs) to conformational epitopes on HCV E2 that were derived from peripheral B cells of individuals infected with either genotype 1a or 1b HCV. Cross-competition analyses delineated at least three immunogenic clusters of overlapping epitopes with distinct functions and properties (23-25). All nonneutralizing antibodies fell within one cluster, designated domain A (24). Neutralizing HMAbs segregated into two clusters, designated domains B and C, with domain B HMAbs having greater potency than domain C HMAbs in blocking infection with the strain JFH1 genotype 2a HCVcc (23, 25).The epitopes of increasing numbers of anti-HCV E2 neutralizing antibodies include residues that are also critical for binding of E2 to CD81. All of our domain B HMAbs inhibit binding of E2 to CD81. Alanine scanning mutagenesis of E2 regions implicated in binding to CD81 identified two highly conserved residues, G530 and D535, that are needed for all domain B antibodies, with a subset also requiring W529 (25, 26, 36). Other laboratories have isolated similar neutralizing antibodies to epitopes containing these residues (20, 32, 38). A similar panel of E2 mutants was previously used to identify five amino acid residues, W420, Y527, W529, G530, and D535, that are essential for interaction with CD81 (37, 42). These findings show that domain B antibodies exert their potent neutralization of HCV infectivity by directly competing with CD81 for binding to E2. It also explains the breadth of neutralization against different HCV genotypes and subtypes for many of these antibodies, since any changes in their epitopes could affect CD81 binding and virus entry. The conserved nature of this cluster of overlapping epitopes makes them of interest for vaccine and immunotherapeutic development. A critical question involves the likelihood that immune selection could lead to escape from neutralization by domain B HMAbs. The series of sequential HCVpp variants derived from patient H over a span of 26 years (52) provide a unique resource for studying the extent and mechanisms of virus escape from broadly neutralizing antibodies. This report describes evidence of escape from immune containment of some but not other domain B HMAbs. Interestingly, a single H variant with reduced HCVpp infectivity and diminished CD81 binding was resistant to neutralization by all domain B antibodies as well as MAb AP33, recognizing a highly conserved linear epitope spanning residues 413 to 420 (35, 47). Sequence analysis revealed multiple mutations on E2 at a considerable distance from CD81 binding residues that could account for the immune escape, although it is unlikely that they are part of the domain B HMAb or the AP33 epitopes. Site-directed substitutions at these mutations restored neutralization sensitivity to all antibodies and CD81 dependency.     

Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be induced by antibody mediated cross-linking of CD81. In addition, EGFR ligands that enhance the kinetics of HCV entry induce EGFR internalization and colocalization with CD81. While EGFR kinase inhibitors inhibit HCV infection primarily by preventing EGFR endocytosis, antibodies that block EGFR ligand binding or inhibitors of EGFR downstream signaling have no effect on HCV entry. These data demonstrate that EGFR internalization is critical for HCV entry and identify a hitherto-unknown association between CD81 and EGFR.     

Cholesterol sensing by CD81 is important for hepatitis C virus entry

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81''s function as a molecular scaffold; these insights are relevant to CD81''s varied roles in both health and disease.     

Discovery of highly potent small molecule Hepatitis C Virus entry inhibitors

Novel, highly potent small molecule HCV entry inhibitors are reported. The SAR exploration of a hit molecule identified from screening of a compound library led to the identification of highly potent compounds with IC(50) values of <5 nM in the tissue culture HCV infectious assay.     

Hepatitis C virus infection propagates through interactions between Syndecan‐1 and CD81 and impacts the hepatocyte glycocalyx

The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan‐1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan‐1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan‐1 or Xylosyltransferase 2, a key enzyme of Syndecan‐1 biosynthesis. Simultaneous knockdown of Syndecan‐1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan‐1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan‐1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan‐1 and Xylosyltransferase 2 was altered within days post‐infection, and the remaining Syndecan‐1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation.     

Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.