Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry

The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process.     

Critical role of leucine-valine change in distinct low pH requirements for membrane fusion between two related retrovirus envelopes

Many viruses use a pH-dependent pathway for fusion with host cell membrane, the mechanism of which is still poorly understood. Here we report that a subtle leucine (Leu)-valine (Val) change at position 501 in the envelope glycoproteins (Envs) of two related retroviruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), is responsible for their distinct low pH requirements for membrane fusion and infection. The Leu and Val residues are predicted to reside within the C-terminal heptad repeat (HR2) region of JSRV and ENTV Envs, particularly proximal to the hairpin turn of the putative six-helix bundle (6HB). Substitution of the JSRV Leu with a Val blocked the Env-mediated membrane fusion at pH 5.0, whereas replacement of the ENTV Val with a Leu rendered the ENTV Env capable of fusing at pH 5.0. A Leu-Val change has no apparent effect on the stability of native Env, but appears to stabilize an intermediate induced by receptor binding. These results are consistent with the existence of at least two metastable conformations of these viral glycoproteins, the native prefusion conformation and a receptor-induced metastable intermediate. Collectively, this work represents an interesting perhaps unique example whereby a simple Leu-Val change has critical impact on pH-dependent virus fusion and entry.     

Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor

Several cell surface molecules have been proposed as receptor candidates, mediating cell entry of hepatitis C virus (HCV) on the basis of their physical association with virions or with soluble HCV E2 glycoproteins. However, due to the lack of infectious HCV particles, evidence that these receptor candidates support infection was missing. Using our recently described infectious HCV pseudotype particles (HCVpp) that display functional E1E2 glycoprotein complexes, here we show that HCV is a pH-dependent virus, implying that its receptor component(s) mediate virion internalization by endocytosis. Expression of the CD81 tetraspanin in non-permissive CD81-negative hepato-carcinoma cells was sufficient to restore susceptibility to HCVpp infection, confirming its critical role as a cell attachment factor. As a cell surface molecule likely to mediate endosomal trafficking, we demonstrate that the human scavenger receptor class B type 1 (SR-B1), a high-density lipoprotein-internalization molecule that we previously proposed as a novel HCV receptor candidate due to its affinity with E2 glycoproteins, is required for infection of CD81-expressing hepatic cells. By receptor competition assays, we found that SR-B1 antibodies that blocked binding of soluble E2 could prevent HCVpp infectivity. Furthermore, we establish that the hyper-variable region 1 of the HCV E2 glycoprotein is a critical determinant mediating entry in SR-B1-positive cells. Finally, by correlating expression of HCV receptors and infectivity, we suggest that, besides CD81 and SR-B1, additional hepatocyte-specific co-factor(s) are necessary for HCV entry.     

Identification of interactions in the E1E2 heterodimer of hepatitis C virus important for cell entry

Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry.     

Distinct roles in folding, CD81 receptor binding and viral entry for conserved histidine residues of hepatitis C virus glycoprotein E1 and E2

The protonation of histidine in acidic environments underpins its role in regulating the function of pH-sensitive proteins. For pH-sensitive viral fusion proteins, histidine protonation in the endosome leads to the activation of their membrane fusion function. The HCV (hepatitis C virus) glycoprotein E1-E2 heterodimer mediates membrane fusion within the endosome, but the roles of conserved histidine residues in the formation of a functional heterodimer and in sensing pH changes is unknown. We examined the functional roles of conserved histidine residues located within E1 and E2. The E1 mutations, H222A/R, H298R and H352A, disrupted E1-E2 heterodimerization and reduced virus entry. A total of five out of six histidine residues located within the E2 RBD (receptor-binding domain) were important for the E2 fold, and their substitution with arginine or alanine caused aberrant heterodimerization and/or CD81 binding. Distinct roles in E1-E2 heterodimerization and in virus entry were identified for His691 and His693 respectively within the membrane-proximal stem region. Viral entry and cell-cell fusion at neutral and low pH values were enhanced with H445R, indicating that the protonation state of His445 is a key regulator of HCV fusion. However, H445R did not overcome the block to virus entry induced by bafilomycin A1, indicating a requirement for an endosomal activation trigger in addition to acidic pH.     

Different domains of CD81 mediate distinct stages of hepatitis C virus pseudoparticle entry

The CD81 tetraspanin was first identified as a hepatitis C virus (HCV) receptor by its ability to bind the soluble ectodomain of envelope glycoprotein E2 (sE2). More recently, it has been suggested that CD81 is necessary but not sufficient for HCV entry into target cells. Here we present further evidence that putative human hepatocyte-specific factors act in concert with CD81 to mediate sE2 binding and HCV pseudoparticle (HCVpp) entry. Moreover, we show that CD81-mediated HCVpp entry entails E2 binding to residues in the large extracellular loop as well as molecular events mediated by the transmembrane and intracellular domains of CD81. The concept that CD81 receptor function progresses in stages is further supported by our finding that anti-CD81 monoclonal antibodies inhibit HCVpp entry by different mechanisms. The half-life of CD81-HCVpp binding was determined to be approximately 17 min, and we propose that binding is followed by CD81 oligomerization, partitioning into cholesterol-rich membrane domains, or other, lateral protein-protein interactions. This results in the formation of a receptor-virus complex that undergoes endocytosis and pH-dependent membrane fusion.     

The Roles of Histidines and Charged Residues as Potential Triggers of a Conformational Change in the Fusion Loop of Ebola Virus Glycoprotein

Ebola virus (EBOV) enters cells from late endosomes/lysosomes under mildly acidic conditions. Entry by fusion with the endosomal membrane requires the fusion loop (FL, residues 507–560) of the EBOV surface glycoprotein to undergo a pH-dependent conformational change. To find the pH trigger for this reaction we mutated multiple conserved histidines and charged and uncharged hydrophilic residues in the FL and measured their activity by liposome fusion and cell entry of virus-like particles. The FL location in the membrane was assessed by NMR using soluble and lipid-bound paramagnetic relaxation agents. While we could not identify a single residue to be alone responsible for pH triggering, we propose that a distributed pH effect over multiple residues induces the conformational change that enhances membrane insertion and triggers the fusion activity of the EBOV FL.     

Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry

Hepatitis C virus (HCV) is an important human pathogen associated with chronic liver disease. Recently, based on a genotype 2a isolate, tissue culture systems supporting complete replication and infectious virus production have been developed. In this study, we used cell culture-produced infectious HCV to analyze the viral entry pathway into Huh-7.5 cells. Bafilomycin A1 and concanamycin A, inhibitors of vacuolar ATPases, prevented HCV entry when they were present prior to infection and had minimal effect on downstream replication events. HCV entry therefore appears to be pH dependent, requiring an acidified intracellular compartment. For many other enveloped viruses, acidic pH triggers an irreversible conformational change, which promotes virion-endosomal membrane fusion. Such viruses are often inactivated by low pH. In the case of HCV, exposure of virions to acidic pH followed by return to neutral pH did not affect their infectivity. This parallels the observation made for the related pestivirus bovine viral diarrhea virus. Low pH could activate the entry of cell surface-bound HCV but only after prolonged incubation at 37 degrees C. This suggests that there are rate-limiting, postbinding events that are needed to render HCV competent for low-pH-triggered entry. Such events may involve interaction with a cellular coreceptor or other factors but do not require cathepsins B and L, late endosomal proteases that activate Ebola virus and reovirus for entry.     

Morphological Characterization and Fusion Properties of Triglyceride-rich Lipoproteins Obtained from Cells Transduced with Hepatitis C Virus Glycoproteins

The density of hepatitis C virus (HCV) particles circulating in the blood of chronically infected patients and of cell-culture produced HCV is heterogeneous. Specific infectivity and fusion of low density particles are higher than those of high density particles. We recently characterized hybrid particles produced by Caco-2 colon or Huh-7.5 liver cells transduced with HCV E1 and E2 envelope glycoproteins. Caco-2-derived particles, called empty lipo-viral particles (eLVP), are composed of triglyceride-rich lipoproteins positive for apolipoproteins B (i.e. apoB100 and apoB48) and contain HCV E1 and E2. Here we aimed at characterizing the morphology and in vitro fusion properties of eLVP using electron microscopy and fluorescence spectroscopy. They displayed the aspect of β-lipoproteins, and immunogold labeling confirmed the presence of apoB and HCV E1 and E2 at their surface. These particles are able to fuse with lipid bilayers (liposomes) in a fusion process leading to the coalescence of internal contents of triglyceride-rich lipoproteins particles and liposomes. Fusion was pH-dependent and could be inhibited by either Z-fFG, a peptide known to inhibit viral fusion, or by monoclonal antibodies directed against HCV E2 or the apolipoprotein moiety of the hybrid particle. Interestingly, particles derived from Huh-7.5 cells failed to display equivalent efficient fusion. Optimal fusion activity is, thus, observed when HCV envelope proteins are associated to apoB-positive hybrid particles. Our results, therefore, point to a crucial role of the E1 and E2 proteins in HCV fusion with a subtle interplay with the apolipoprotein part of eLVP.     

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells.     

Herpesvirus Entry into Host Cells Mediated by Endosomal Low pH

Herpesviral pathogenesis stems from infection of multiple cell types including the site of latency and cells that support lytic replication. Herpesviruses utilize distinct cellular pathways, including low pH endocytic pathways, to enter different pathophysiologically relevant target cells. This review details the impact of the mildly acidic milieu of endosomes on the entry of herpesviruses, with particular emphasis on herpes simplex virus 1 (HSV‐1). Epithelial cells, the portal of primary HSV‐1 infection, support entry via low pH endocytosis mechanisms. Mildly acidic pH triggers reversible conformational changes in the HSV‐1 class III fusion protein glycoprotein B (gB). In vitro treatment of herpes simplex virions with a similar pH range inactivates infectivity, likely by prematurely activating the viral entry machinery in the absence of a target membrane. How a given herpesvirus mediates both low pH and pH‐independent entry events is a key unresolved question.      

Human immunodeficiency virus infection of CD4-bearing cells occurs by a pH-independent mechanism.

The effect of weak bases (NH4Cl and amantadine) and carboxylic ionophores (monensin) on the infection of CD4 (T4) positive human cell lines by HIV-1 is examined. These reagents, which raise the pH of acidic intracellular organelles, fail to inhibit HIV-1 entry and the events leading to viral protein synthesis at concentrations inhibitory for low pH-dependent fusogenic enveloped viruses. The infectivity of VSV (HIV-1) pseudotypes is unaffected by weak bases at concentrations causing 95% plaque reduction of VSV in its own envelope. HIV-1 dependent cell--cell fusion (syncytium formation) occurs in medium maintained at pH 7.4-7.6, and virions are not irreversibly inactivated by incubation in acid medium. Our results show that HIV-1 entry and membrane fusion do not require exposure to low pH. The production of infectious HIV-1 particles, however, is inhibited in cells treated with NH4Cl.     

Transient Activation of the PI3K-AKT Pathway by Hepatitis C Virus to Enhance Viral Entry

The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed against CD81 and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction between HCV E2 and its two co-receptors CD81 and claudin-1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibited HCV infection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3K-AKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients.     

Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus

Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH.     

Inhibition of Ebola virus entry by a C-peptide targeted to endosomes

Ebola virus (EboV) and Marburg virus (MarV) (filoviruses) are the causative agents of severe hemorrhagic fever. Infection begins with uptake of particles into cellular endosomes, where the viral envelope glycoprotein (GP) catalyzes fusion between the viral and host cell membranes. This fusion event is thought to involve conformational rearrangements of the transmembrane subunit (GP2) of the envelope spike that ultimately result in formation of a six-helix bundle by the N- and C-terminal heptad repeat (NHR and CHR, respectively) regions of GP2. Infection by other viruses employing similar viral entry mechanisms (such as HIV-1 and severe acute respiratory syndrome coronavirus) can be inhibited with synthetic peptides corresponding to the native CHR sequence ("C-peptides"). However, previously reported EboV C-peptides have shown weak or insignificant antiviral activity. To determine whether the activity of a C-peptide could be improved by increasing its intracellular concentration, we prepared an EboV C-peptide conjugated to the arginine-rich sequence from HIV-1 Tat, which is known to accumulate in endosomes. We found that this peptide specifically inhibited viral entry mediated by filovirus GP proteins and infection by authentic filoviruses. We determined that antiviral activity was dependent on both the Tat sequence and the native EboV CHR sequence. Mechanistic studies suggested that the peptide acts by blocking a membrane fusion intermediate.     

The final conformation of the complete ectodomain of the HA2 subunit of influenza hemagglutinin can by itself drive low pH-dependent fusion

One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1–185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.     

Endocytic entry of HIV-1

Enveloped viruses enter target cells by membrane fusion or endocytosis. In the latter case, fusion of the viral envelope is induced by the acidic pH of the endocytic vesicle [1]. As with most other retroviruses, entry of the human immunodeficiency virus (HIV) is thought to be exclusively by pH-independent membrane fusion after interaction of its envelope with CD4 and a chemokine co-receptor on the target cell [2,3]. Expression of CD4 on the virus-producing cell impairs the release and infectivity of HIV-1(NL4-3) particles [4-6]. In sharp contrast, we found that the infectivity of another HIV isolate, HIV-1SF2, was enhanced by expression of CD4 on the producer cells, which correlated with significantly increased amounts of viral proteins in the vesicular fraction of target cells. Endocytic inhibitors decreased infectivity of HIV-1SF2 but enhanced that of HIV-1 NL4-3. Expression of CD4 in the producer cell did not remove gp41 from HIV-1SF2 virions. With these cells, the formation of syncytia could be induced by acidic medium. Thus, HIV-1SF2 can enter the cytoplasm by an endocytic route after activation of gp41 by the acidic pH of endocytic vesicles. Endocytic entry might expand the range of cells that HIV could infect and should be considered in antiviral strategies against AIDS.     

Functional Analysis of Cell Surface-Expressed Hepatitis C Virus E2 Glycoprotein

Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT. The E2661-HATMCT chimeric glycoprotein was able to bind a number of conformation-dependent monoclonal antibodies and a recombinant soluble form of CD81, suggesting that it was folded in a manner comparable to "native" E2. Furthermore, cell surface-expressed E2661-HATMCT demonstrated pH-dependent changes in antigen conformation, consistent with an acid-mediated fusion mechanism. However, E2661-HATMCT was unable to induce cell fusion of CD81-positive HEK cells after neutral- or low-pH treatment. We propose that a stretch of conserved, hydrophobic amino acids within the E1 glycoprotein, displaying similarities to flavivirus and paramyxovirus fusion peptides, may constitute the HCV fusion peptide. We demonstrate that influenza virus can incorporate E2661-HATMCT into particles and discuss experiments to address the relevance of the E2-CD81 interaction for HCV attachment and entry.     

Adaptation of Hepatitis C Virus to Mouse CD81 Permits Infection of Mouse Cells in the Absence of Human Entry Factors

Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.     

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.