Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein

Wahid, Ahmed; Helle, François; Descamps, Véronique; Duverlie, Gilles; Pénin, François; Dubuisson, Jean

doi:10.1128/jvi.02659-12

Cited by 51 publications

(63 citation statements)

References 65 publications

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“…The roles of C272 and C281 were previously investigated by Wahid et al using an HCVcc system in which E1 is expressed in cis from the viral genome (41). Those researchers found that an alanine mutation at these two positions significantly reduced but did not completely abolish HCV infectivity (41). In contrast, we found that mutations at these two positions completely abolished HCV infectivity in our study.…”

Section: Discussioncontrasting

confidence: 56%

“…These results suggest that the role of C272 and D279 in the virus life cycle may be more toward HCV morphogenesis rather than virus entry. The roles of C272 and C281 were previously investigated by Wahid et al using an HCVcc system in which E1 is expressed in cis from the viral genome (41). Those researchers found that an alanine mutation at these two positions significantly reduced but did not completely abolish HCV infectivity (41).…”

Section: Discussionmentioning

confidence: 99%

“…It is possible that this might be due to the different adaptive mutations in the virus genomes used in these two studies. While we used a JFH1 genome that contains an adaptive mutation in NS5A (EEDDT deletion), Wahid et al used a JFH1 genome that contains two adaptive mutations in the core protein (F172C and P173S) as well as A4 epitope swapping in E1 to aid in the detection of E1 (41). More studies will be needed to address this question.…”

Section: Discussionmentioning

confidence: 99%

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Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans -Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis

Tong

Chi

Yang

et al. 2017

J Virol

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Hepatitis C virus (HCV) is an enveloped RNA virus belonging to the Flaviviridae family. It infects mainly human hepatocytes and causes chronic liver diseases, including cirrhosis and cancer. HCV encodes two envelope proteins, E1 and E2, that form a heterodimer and mediate virus entry. While E2 has been extensively studied, less has been done so for E1, and its role in the HCV life cycle still needs to be elucidated. Here we developed a new cell culture model for HCV infection based on the trans-complementation of E1. Virus production of the HCV genome lacking the E1-encoding sequence can be efficiently rescued by the ectopic expression of E1 in trans. The resulting virus, designated HCVΔE1, can propagate in packaging cells expressing E1 but results in only single-cycle infection in naive cells. By using the HCVΔE1 system, we explored the role of a putative fusion peptide (FP) of E1 in HCV infection. Interestingly, we found that the FP not only contributes to HCV entry, as previously reported, but also may be involved in virus morphogenesis. Finally, we identified amino acid residues in FP that are critical for biological functions of E1. In summary, our work not only provides a new cell culture model for studying HCV but also provides some insights into understanding the role of E1 in the HCV life cycle. IMPORTANCE Hepatitis C virus (HCV), an enveloped RNA virus, encodes two envelope proteins, E1 and E2, that form a heterodimeric complex to mediate virus entry. Compared to E2, the biological functions of E1 in the virus life cycle are not adequately investigated. Here we developed a new cell culture model for single-cycle HCV infection based on the trans-complementation of E1. The HCV genome lacking the E1-encoding sequence can be efficiently rescued for virus production by the ectopic expression of E1 in trans. This new model renders a unique system to dissect functional domains and motifs in E1. Using this system, we found that a putative fusion peptide in E1 is a multifunctional structural element contributing to both HCV entry and morphogenesis. Our work has provided a new cell culture model to study HCV and provides insights into understanding the biological roles of E1 in the HCV life cycle. KEYWORDS E1, envelope proteins, fusion peptide, hepatitis C virus, transcomplementation, virus entry, virus morphogenesis H epatitis C virus (HCV) is a major human pathogen that infects about 170 million people worldwide and can lead to chronic hepatitis and more severe liver diseases such as cirrhosis and hepatocellular carcinoma (1-3). For a long time, standard therapy for chronic HCV infection consisted of the administration of pegylated interferon alpha Functional analysis of hepatitis C virus (HCV) envelope protein E1 using a transcomplementation system reveals a dual role of a putative fusion peptide of E1 in both HCV entry and morphogenesis. J Virol 91:e02468-16.

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Section: Discussioncontrasting

confidence: 56%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans -Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis

Tong

Chi

Yang

et al. 2017

J Virol

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“…Importantly, authentic virions bearing S327T mutations were rendered highly susceptible to 1:7 neutralization. Recently, mutations in E1 were shown to render viruses more sensitive to inhibition by soluble CD81 (36). Similarly, our data suggest that conservative S-to-T mutations in E1 can render HCV highly susceptible to antibodies targeting the discontinuous CD81 binding regions in E2.…”

Section: Resultsmentioning

confidence: 54%

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

Brown

Koutsoudakis

Urbanowicz

et al. 2014

J Virol

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Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codonswitching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic "fossil record" of historical purifying selection against E1E2 intermediate phenotypes. Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus and causes a significant disease burden worldwide (1). A chronic infection ensues in around 80% of cases, predisposing carriers to an increased risk of cirrhosis and hepatocellular carcinoma (2). HCV exhibits substantial genetic diversity globally, with six well-sampled genotypes and a multitude of subtypes having been described (3). This observed genetic heterogeneity results from a high mutation/replicative rate in vivo (4, 5) and extends to the intrahost level, where the virus exists as an ensemble of genetically related yet distinct variants (6, 7). However, this genetic heterogeneity is not evenly distributed throughout the viral genome. The greatest levels of diversity are observed in the E1E2 genes, encoding the envelope glycoproteins E1 and E2, heterodimers of which are distributed over the surface of the viral lipid envelope. Genotype-specific positive selection, mediated by humoral and cellular targeting, contributes to global E1E2 evolution (8). Progressive substitutional evolution of the E1E2 genes, in par...

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“…6 indicate that both recombinant glycoproteins bind to CD81-LEL in a concentration dependent manner, with almost identical reactivity. Although the interaction of CD81 is specific for E2, recent studies using E1-cysteine mutants in cell-cultured HCV (HCVcc) indicated that E1 glycoprotein has a modulating effect in the binding of E2 to CD81 [22]. However, this effect must be restricted to the context of the E1E2 heterodimer, since the isolated E2 ectodomain has the same CD81-binding capacity when it folds either independently (E2 661 ) or in the presence of E1 (E2 661 p).…”

Section: Antigenic Characterization Of E2 661 P a Panel Of Six Hcv-pmentioning

confidence: 99%

Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein

Ortega-Atienza

Lombana

Gómez-Gutiérrez

et al. 2014

Protein Expression and Purification

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Hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, are involved in the first steps of virus infection. The E2 ectodomain can be produced as an isolated form (E2 661 ). However, there is some concern about its proper conformation and the role that E1 can play as a chaperone for the folding of E2. In order to verify this fact we have expressed a chimeric protein (E1tmbE2) based on the fulllength E1 sequence followed by the E2 ectodomain using the baculovirus-insect cells system. The E2 ectodomain is folded in the presence of the E1, proteolytically processed by cellular proteases and secreted to cell culture media (E2 661 p), while the E1 protein is retained into the cell due to its transmembrane sequence. The purification of E2 661 p from culture media was facilitated by a His tag introduced in its amino terminus. Both E2 661 and E2 661 p glycoproteins shared very similar structural features, monitored by spectroscopic and antigenic studies. Moreover, their functional properties, tested by means of CD81 binding, were almost indistinguishable, indicating that the E2 ectodomain constitutes an independent folding unit.

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Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein

Cited by 51 publications

References 65 publications

Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans -Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis

Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans -Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

Production and characterization of the ectodomain of E2 envelope glycoprotein of hepatitis C virus folded in the presence of full-length E1 glycoprotein

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