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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.

ABSTRACT: 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 HCV?E1, can propagate in packaging cells expressing E1 but results in only single-cycle infection in naive cells. By using the HCV?E1 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.

SUBMITTER: Tong Y

PROVIDER: S-EPMC5355622 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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

Tong Yimin Y Chi Xiaojing X Yang Wei W Zhong Jin J

Journal of virology 20170313 7

Hepatitis C virus (HCV) is an enveloped RNA virus belonging to the <i>Flaviviridae</i> 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 ...[more]

PMID: 28100619

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Project description:Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.IMPORTANCE Hepatitis C virus (HCV) infection is a major public health problem worldwide. It encodes two envelope proteins, E1 and E2, which play a major role in the life cycle of this virus. E2 has been extensively characterized, whereas E1 remains poorly understood. Here, we investigated E1 functions by using site-directed mutagenesis in the context of the viral life cycle. Our results identify unique phenotypes. Unexpectedly, two mutants clearly showed a shift in receptor dependence for cell entry, highlighting a role for E1 in modulating HCV particle interaction with a cellular receptor(s). More importantly, another mutant led to the assembly and release of viral particles devoid of genomic RNA. This unique phenotype was further characterized, and we observed a change in subcellular colocalization between HCV RNA and E1. This unique observation highlights for the first time cross talk between a viral envelope protein and genomic RNA during morphogenesis.

Dataset Information

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.

Publications

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

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