EWI-2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry.
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ABSTRACT:
SUBMITTER: Potel J
PROVIDER: S-EPMC7162402 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.
| S-EPMC2898367 | biostudies-literature
Project description: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.
| S-EPMC7863897 | biostudies-literature
Project description:CD81 is a protein found in the cells of all vertebrates, including humans. CD81 performs an important role in the natural biology of a cell: it organises collections of proteins at the cell surface, allowing them to function together. Through this role, CD81 facilitates the activation of immune cells and the fusion of sperm and egg cells during fertilisation. As well these normal functions, CD81 is a receptor for hepatitis C virus, and malaria, to gain entry to liver cells. It was recently suggested that CD81 may be able to sense cholesterol at the cell surface, resulting in a switch-like movement in CD81, however, it is unknown whether this feature affects CD81 function. In this study, we generated variants of CD81 with disruptions in cholesterol sensing, and its associated switching mechanism. Our experiments suggest that HCV entry is dependent on the cholesterol-bound form of CD81, in which the switch is in a closed position. Our study provides evidence that cholesterol sensing is important for CD81 function; this work paves the way for future investigations into the role of CD81 in both health and disease.
2020-12-02 | PXD019260 | Pride
Project description:Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.
| S-EPMC2346731 | biostudies-literature
Project description:Tetraspanins are integral transmembrane proteins organized in microdomains displaying specific and direct interactions with other tetraspanins and molecular partners. Among them, CD81 has been implicated in a variety of physiological and pathological processes. CD81 also plays a crucial role in pathogen entry into host cells, including hepatitis C virus (HCV) entry into hepatocytes. HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV entry into hepatocytes is a complex process that requires the coordinated interaction of viral and host factors for the initiation of infection, including CD81, scavenger receptor BI, claudin-1, occludin, membrane-bound host cell kinases, Niemann-Pick C1 Like 1, Harvey rat sarcoma viral oncogene homolog (HRas), CD63 and transferrin receptor 1. Furthermore, recent data in HCV model systems have demonstrated that targeting critical components of tetraspanins and associated cell membrane proteins open new avenues to prevent and treat viral infection.
| S-EPMC3939486 | biostudies-literature
Project description:Hepatitis C virus (HCV) enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81). The tetraspanin hCD81 contains a large extracellular loop (LEL), which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop) is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81) functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F) do not and tetraspanins with intermediate homology (hCD9) show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors.
| S-EPMC5923501 | biostudies-literature
Project description: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.
| S-EPMC3457153 | biostudies-literature
Project description:Hepatitis C Virus (HCV) infection is a global public health problem affecting over 160 million individuals worldwide. Its symptoms include chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped RNA virus mainly targeting liver cells and for which the initiation of infection occurs through a complex multistep process involving a series of specific cellular entry factors. This process is likely mediated through the formation of a tightly orchestrated complex of HCV entry factors at the plasma membrane. Among HCV entry factors, the tetraspanin CD81 is one of the best characterized and it is undoubtedly a key player in the HCV lifecycle. In this review, we detail the current knowledge on the involvement of CD81 in the HCV lifecycle, as well as in the immune response to HCV infection.
| S-EPMC3939471 | biostudies-literature
Project description:UnlabelledIn spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies.ImportanceHepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins.
| S-EPMC4178871 | biostudies-literature
Project description:Interaction between the hepatitis C virus (HCV) envelope protein E2 and the host receptor CD81 is essential for HCV entry into target cells. The number of E2-CD81 complexes necessary for HCV entry has remained difficult to estimate experimentally. Using the recently developed cell culture systems that allow persistent HCV infection in vitro, the dependence of HCV entry and kinetics on CD81 expression has been measured. We reasoned that analysis of the latter experiments using a mathematical model of viral kinetics may yield estimates of the number of E2-CD81 complexes necessary for HCV entry. Here, we constructed a mathematical model of HCV viral kinetics in vitro, in which we accounted explicitly for the dependence of HCV entry on CD81 expression. Model predictions of viral kinetics are in quantitative agreement with experimental observations. Specifically, our model predicts triphasic viral kinetics in vitro, where the first phase is characterized by cell proliferation, the second by the infection of susceptible cells and the third by the growth of cells refractory to infection. By fitting model predictions to the above data, we were able to estimate the threshold number of E2-CD81 complexes necessary for HCV entry into human hepatoma-derived cells. We found that depending on the E2-CD81 binding affinity, between 1 and 13 E2-CD81 complexes are necessary for HCV entry. With this estimate, our model captured data from independent experiments that employed different HCV clones and cells with distinct CD81 expression levels, indicating that the estimate is robust. Our study thus quantifies the molecular requirements of HCV entry and suggests guidelines for intervention strategies that target the E2-CD81 interaction. Further, our model presents a framework for quantitative analyses of cell culture studies now extensively employed to investigate HCV infection.
| S-EPMC3234214 | biostudies-other