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Mutational analysis of hepatitis E virus ORF1 "Y-domain": Effects on RNA replication and virion infectivity.

ABSTRACT: To investigate the role of non-structural open reading frame 1 "Y-domain" sequences in the hepatitis E virus (HEV) life cycle.Sequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions' infectivity was assayed on naïve HepG2/C3A cells.In silico analysis identified a potential palmitoylation-site (C336C337) and an ?-helix segment (L410Y411S412W413L414F415E416) in the HEV Y-domain. Molecular characterization of C336A, C337A and W413A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.This is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.

SUBMITTER: Parvez MK

PROVIDER: S-EPMC5292332 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Mutational analysis of hepatitis E virus ORF1 "Y-domain": Effects on RNA replication and virion infectivity.

Parvez Mohammad Khalid MK

World journal of gastroenterology 20170101 4

<h4>Aim</h4>To investigate the role of non-structural open reading frame 1 "Y-domain" sequences in the hepatitis E virus (HEV) life cycle.<h4>Methods</h4>Sequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed <i>in silico</i>. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. <i>In vitro</i> transcribed mRNA (<i>pSK-GFP</i>) constructs were transfected into S10-3 cel ...[more]

PMID: 28216965

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Project description:The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly.Hepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection.

Dataset Information

Mutational analysis of hepatitis E virus ORF1 "Y-domain": Effects on RNA replication and virion infectivity.

Publications

Mutational analysis of hepatitis E virus ORF1 "Y-domain": Effects on RNA replication and virion infectivity.

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