Hypervariable Region 1 Deletion and Required Adaptive Envelope Mutations Confer Decreased Dependency on Scavenger Receptor Class B Type I and Low-Density Lipoprotein Receptor for Hepatitis C Virus

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Hepatitis C virus (HCV) particles associate with lipoproteins and infect cells by using at least four cell entry factors. These factors include scavenger receptor class B type I (SR-BI), CD81, claudin 1 (CLDN1), and occludin (OCLN). Little is known about specific functions of individual host factors during HCV cell entry and viral domains that mediate interactions with these factors. Hypervariable region 1 (HVR1) within viral envelope protein 2 (E2) is involved in the usage of SR-BI and conceals the viral CD81 binding site. Moreover, deletion of this domain alters the density of virions. We compared lipoprotein interaction, surface attachment, receptor usage, and cell entry between wild-type HCV and a viral mutant lacking this domain. Deletion of HVR1 did not affect CD81, CLDN1, and OCLN usage. However, unlike wild-type HCV, HVR1-deleted viruses were not neutralized by antibodies and small molecules targeting SR-BI. Nevertheless, modulation of SR-BI cell surface expression altered the infection efficiencies of both viruses to similar levels. Analysis of affinity-purified virions revealed comparable levels of apolipoprotein E (ApoE) incorporation into viruses with or without HVR1. However, ApoE incorporated into these viruses was differentially recognized by ApoE-specific antibodies. Thus, SR-BI has at least two functions during cell entry. One of them can be neutralized by SR-BI-targeting molecules, and it is critical only for wild-type HCV. The other one is important for both viruses but apparently is not inactivated by the SR-BI binding antibodies and small molecules evaluated here. In addition, HVR1 modulates the conformation and/or epitope exposure of virus particle-associated ApoE.

Section: Discussionsupporting

confidence: 79%

Role of Hypervariable Region 1 for the Interplay of Hepatitis C Virus with Entry Factors and Lipoproteins

Bankwitz

Vièyres

Hueging

et al. 2014

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“…Transfection of CD81-deficient S29 cells (32) for determination of intra-and extracellular HCV Core titers and infectivity titers, respectively, was carried out as previously described (54,57,58). In brief, 400,000 S29 cells were plated in 6-well plates 24 h before transfection.…”

Section: Methodsmentioning

confidence: 99%

Adaptive Mutations Enhance Assembly and Cell-to-Cell Transmission of a High-Titer Hepatitis C Virus Genotype 5a Core-NS2 JFH1-Based Recombinant

Mathiesen

Prentoe

Meredith

et al. 2015

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Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1 C3405G,A3696G (termed SA13/JFH1 orig ), of the poorly characterized genotype 5a, to Huh7.5 cells, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log 10 focus-forming units (FFU)/ml. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1 orig mutant termed SA13/JFH1 Huh7-derived cells demonstrated that these changes did not affect replication but increased HCV assembly and specific infectivity as early as 24 h posttransfection. Infectious coculture assays in Huh7.5 cells showed a significant increase in cell-to-cell transmission for SA13/JFH1 Core-NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 hypervariable region 1 (HVR1) mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility of the CD81 binding site without affecting the usage of CD81 and SR-BI. We finally demonstrated that SA13/JFH1 orig and SA13/JFH1 Core-NS5B , with and without the E2 mutation T385P, displayed similar biophysical properties following iodixanol gradient ultracentrifugation. This study has implications for investigations requiring high virus concentrations, such as studies of HCV particle composition and development of whole-virus vaccine antigens. IMPORTANCEHepatitis C virus (HCV) is a major global health care burden, affecting more than 150 million people worldwide. These individuals are at high risk of developing severe end-stage liver diseases. No vaccine exists. While it is possible to produce HCV particles resembling isolates of all HCV genotypes in human hepatoma cells (HCVcc), production efficacy varies. Thus, for several important studies, including vaccine development, in vitro systems enabling high-titer production of diverse HCV strains would be advantageous. Our study offers important functional data on how cell culture-adaptive mutations identified in genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties. High-titer HCVcc like the one described in this study may be pivotal in future vaccine-related studies where large quantities of infectious HCV particles are necessary. Hepatitis C virus (HCV) is an important human pathogen with more than 150 million chronically infected individuals worldwide. These individuals are at high risk of developing severe end-stage liver diseases such as cirrhosis and hepatocellular carcinoma, making HCV the most frequent indication for liver transplantation in the United States and Europe (1, 2). HCV is an enveloped posi...

“…10C). We used a polyclonal anti-ApoE antibody because previous studies on viruses lacking HVR1 (⌬HVR1) have shown that polyclonal anti-ApoE antibodies differently neutralize ⌬HVR1 viruses, while monoclonal anti-ApoE antibodies do not (50,76,77). As shown in Fig.…”

Section: Selection Of a Monensin-resistant Virusmentioning

confidence: 99%

New Insights into the Understanding of Hepatitis C Virus Entry and Cell-to-Cell Transmission by Using the Ionophore Monensin A

Fénéant

Potel

François³

et al. 2015

In our study, we characterized the effect of monensin, an ionophore that is known to raise the intracellular pH, on the hepatitis C virus (HCV) life cycle. We showed that monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Using the selective pressure of monensin, we selected a monensin-resistant virus which has evolved to use a new entry route that is partially pH and clathrin independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, the Y297H mutation in E1 and the I399T mutation in hypervariable region 1 (HVR1) of E2, which confer resistance to monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces an N-glycosylation site within HVR1 and increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (anti-ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In summary, we identified a mutation in HVR1 that overcomes the vesicular pH dependence, modifies the biophysical properties of particles, and drastically reduces cellto-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation. IMPORTANCEAlthough several cell surface proteins have been identified as entry factors for hepatitis C virus (HCV), the precise mechanisms regulating its transmission to hepatic cells are still unclear. In our study, we used monensin A, an ionophore that is known to raise the intracellular pH, and demonstrated that cell-free and cell-to-cell transmission pathways are both pH-dependent processes. We generated monensin-resistant viruses that displayed different entry routes and biophysical properties. Thanks to these mutants, we highlighted the importance of hypervariable region 1 (HVR1) of the E2 envelope protein for the association of particles with apolipoprotein E, which in turn might control the pH dependency of cell-free and cell-to-cell transmission. H epatitis C virus (HCV) infection is a global public health problem affecting over 130 million individuals worldwide.Chronic HCV infection can result in liver cirrhosis and hepatocellular carcinoma (1). While previous interferon (IFN)-based therapies have been limited by drug resistance and marked toxicity (2), the recently clinically licensed direct-acting antivirals are expected to cure the large majority of infected patients without major adverse effects (3). Nevertheless, several challenges remain: high costs limit access to therapy ...