474 research outputs found

    Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I.

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    In the past several years, a number of cellular proteins have been identified as candidate entry receptors for hepatitis C virus (HCV) by using surrogate models of HCV infection. Among these, the tetraspanin CD81 and scavenger receptor B type I (SR-BI), both of which localize to specialized plasma membrane domains enriched in cholesterol, have been suggested to be key players in HCV entry. In the current study, we used a recently developed in vitro HCV infection system to demonstrate that both CD81 and SR-BI are required for authentic HCV infection in vitro, that they function cooperatively to initiate HCV infection, and that CD81-mediated HCV entry is, in part, dependent on membrane cholesterol

    Hepatoma cell density promotes claudin-1 and scavenger receptor BI expression and hepatitis C virus internalization.

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    Hepatitis C virus (HCV) entry occurs via a pH- and clathrin-dependent endocytic pathway and requires a number of cellular factors, including CD81, the tight-junction proteins claudin 1 (CLDN1) and occludin, and scavenger receptor class B member I (SR-BI). HCV tropism is restricted to the liver, where hepatocytes are tightly packed. Here, we demonstrate that SR-BI and CLDN1 expression is modulated in confluent human hepatoma cells, with both receptors being enriched at cell-cell junctions. Cellular contact increased HCV pseudoparticle (HCVpp) and HCV particle (HCVcc) infection and accelerated the internalization of cell-bound HCVcc, suggesting that the cell contact modulation of receptor levels may facilitate the assembly of receptor complexes required for virus internalization. CLDN1 overexpression in subconfluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced HCVpp entry and HCVcc internalization, demonstrating a rate-limiting role for SR-BI in HCV internalization

    Identification of a residue in hepatitis C virus E2 glycoprotein that determines scavenger receptor BI and CD81 receptor dependency and sensitivity to neutralizing antibodies.

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    Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses

    Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells.

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    Cell entry of hepatitis C virus, pseudoparticles (HCVpp) and cell culture grown virus (HCVcc), requires the interaction of viral glycoproteins with CD81 and other as yet unknown cellular factors. One of these is likely to be the scavenger receptor class B type I (SR-BI). To further understand the role of SR-BI, we examined the effect of SR-BI ligands on HCVpp and HCVcc infectivity. Oxidized low-density lipoprotein (oxLDL), but not native LDL, potently inhibited HCVpp and HCVcc cell entry. Pseudoparticles bearing unrelated viral glycoproteins or bovine viral diarrhea virus were not affected. A dose-dependent inhibition was observed for HCVpp bearing diverse viral glycoproteins with an approximate IC50 of 1.5 microg/mL apolipoprotein content, which is within the range of oxLDL reported to be present in human plasma. The ability of lipoprotein components to bind to target cells associated with their antiviral activity, suggesting a mechanism of action which targets a cell surface receptor critical for HCV infection of the host cell. However, binding of soluble E2 to SR-BI or CD81 was not affected by oxLDL, suggesting that oxLDL does not act as a simple receptor blocker. At the same time, oxLDL incubation altered the biophysical properties of HCVpp, suggesting a ternary interaction of oxLDL with both virus and target cells. In conclusion, the SR-BI ligand oxLDL is a potent cell entry inhibitor for a broad range of HCV strains in vitro. These findings suggest that SR-BI is an essential component of the cellular HCV receptor complex

    Persistent hepatitis C virus infection in vitro: coevolution of virus and host.

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    The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host

    Diverse CD81 proteins support hepatitis C virus infection.

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    Hepatitis C virus (HCV) entry is dependent on CD81. To investigate whether the CD81 sequence is a determinant of HCV host range, we expressed a panel of diverse CD81 proteins and tested their ability to interact with HCV. CD81 large extracellular loop (LEL) sequences were expressed as recombinant proteins; the human and, to a low level, the African green monkey sequences bound soluble HCV E2 (sE2) and inhibited infection by retrovirus pseudotype particles bearing HCV glycoproteins (HCVpp). In contrast, mouse or rat CD81 proteins failed to bind sE2 or to inhibit HCVpp infection. However, CD81 proteins from all species, when expressed in HepG2 cells, conferred susceptibility to infection by HCVpp and cell culture-grown HCV to various levels, with the rat sequence being the least efficient. Recombinant human CD81 LEL inhibited HCVpp infectivity only if present during the virus-cell incubation, consistent with a role for CD81 after virus attachment. Amino acid changes that abrogate sE2 binding (I182F, N184Y, and F186S, alone or in combination) were introduced into human CD81. All three amino acid changes in human CD81 resulted in a molecule that still supported HCVpp infection, albeit with reduced efficiency. In summary, there is a remarkable plasticity in the range of CD81 sequences that can support HCV entry, suggesting that CD81 polymorphism may contribute to, but alone does not define, the HCV susceptibility of a species. In addition, the capacity to support viral entry is only partially reflected by assays measuring sE2 interaction with recombinant or full-length CD81 proteins

    Hypoxia dampens innate immune signalling at early time points and increases Zika virus RNA levels in iPSC-derived macrophages

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    Type I interferons (IFNs) are the major host defence against viral infection and are induced following activation of cell surface or intracellular pattern recognition receptors, including retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs). All cellular processes are shaped by the microenvironment and one important factor is the local oxygen tension. The majority of published studies on IFN signalling are conducted under laboratory conditions of 18% oxygen (O2), that do not reflect the oxygen levels in most organs (1–5 % O2). We studied the effect of low oxygen on IFN induction and signalling in induced Pluripotent Stem Cell (iPSC)-derived macrophages as a model for tissue-resident macrophages and assessed the consequence for Zika virus (ZIKV) infection. Hypoxic conditions dampened the expression of interferon-stimulated genes (ISGs) following RLR stimulation or IFN treatment at early time points. RNA-sequencing and bio-informatic analysis uncovered several pathways including changes in transcription factor availability, the presence of HIF binding sites in promoter regions, and CpG content that may contribute to the reduced ISG expression. Hypoxic conditions increased the abundance of ZIKV RNA highlighting the importance of understanding how low oxygen conditions in the local microenvironment affect pathogen sensing and host defences

    Mutations in hepatitis C virus E2 located outside the CD81 binding sites lead to escape from broadly neutralizing antibodies but compromise virus infectivity.

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    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

    Polarization restricts hepatitis C virus entry into HepG2 hepatoma cells

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    The primary reservoir for hepatitis C virus (HCV) replication is believed to be hepatocytes, which are highly polarized with tight junctions (TJ) separating their basolateral and apical domains. HepG2 cells develop polarity over time, resulting in the formation and remodeling of bile canalicular (BC) structures. HepG2 cells expressing CD81 provide a model system to study the effects of hepatic polarity on HCV infection. We found an inverse association between HepG2-CD81 polarization and HCV pseudoparticle entry. As HepG2 cells polarize, discrete pools of claudin-1 (CLDN1) at the TJ and basal/lateral membranes develop, consistent with the pattern of receptor staining observed in liver tissue. The TJ and nonjunctional pools of CLDN1 show an altered association with CD81 and localization in response to the PKA antagonist Rp-8-Br-cyclic AMPs (cAMPs). Rp-8-Br-cAMPs reduced CLDN1 expression at the basal membrane and inhibited HCV infection, supporting a model where the nonjunctional pools of CLDN1 have a role in HCV entry. Treatment of HepG2 cells with proinflammatory cytokines, tumor necrosis factor alpha and gamma interferon, perturbed TJ integrity but had minimal effect(s) on cellular polarity and HCV infection, suggesting that TJ integrity does not limit HCV entry into polarized HepG2 cells. In contrast, activation of PKC with phorbol ester reduced TJ integrity, ablated HepG2 polarity, and stimulated HCV entry. Overall, these data show that complex hepatocyte-like polarity alters CLDN1 localization and limits HCV entry, suggesting that agents which disrupt hepatocyte polarity may promote HCV infection and transmission within the liver

    Type I interferon rapidly restricts infectious hepatitis C virus particle genesis

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    Interferon-alpha (IFNα) has been used to treat chronic hepatitis C virus (HCV) infection for over 20 years with varying efficacy, depending on the infecting viral genotype. The mechanism of action of IFNα is not fully understood, but is thought to target multiple stages of the HCV lifecycle, inhibiting viral transcription and translation leading to a degradation of viral RNA and protein expression in the infected cell. IFNα induces the expression of an array of interferon-stimulated genes within minutes of receptor engagement; however, the impact of these early responses on the viral lifecycle are unknown. We demonstrate that IFNα inhibits the genesis of infectious extracellular HCV particles within 2 hours of treating infected cells, with minimal effect on the intracellular viral burden. Importantly, this short duration of IFNα treatment of infected cells significantly reduced cell-free and cell-to-cell dissemination. The secreted viral particles showed no apparent change in protein content or density, demonstrating that IFNα inhibits particle infectivity but not secretion rates. To investigate whether particles released from IFNα-treated cells have a reduced capacity to establish infection we used HCV lentiviral pseudotypes (HCVpp) and demonstrated a defect in cell entry. Using a panel of monoclonal antibodies targeting the E2 glycoprotein, we demonstrate that IFNα alters glycoprotein conformation and receptor utilization. Conclusion: These observations show a previously unreported and rapid effect of IFNα on HCV particle infectivity that inhibits de novo infection events. Evasion of this response may be a contributing factor in whether a patient achieves early or rapid virological response, a key indicator of progression to sustained virological response or clearance of viral infection. (Hepatology 2014;60:1890–1900
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