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Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus.

ABSTRACT: Recent genetic studies suggested that viral nonstructural (NS) proteins play important roles in morphogenesis of flaviviruses, particularly hepatitis C virus (HCV). Adaptive and compensatory mutations occurring in different NS proteins were demonstrated to promote HCV production in cell culture. However, the underlying molecular mechanism of NS proteins in HCV morphogenesis is poorly understood. We have isolated a cell culture-adapted HCV of genotype 2a (JFH1) which grew to an infectious titer 3 orders of magnitude higher than that of wild-type virus. Sequence analysis identified a total of 16 amino acid mutations in core (C), E1, NS2, NS3, NS5A, and NS5B, with the majority of mutations clustered in NS5A. Reverse genetic analysis of these mutations individually or in different combinations demonstrated that amino acid mutations in NS2 and NS5A markedly enhanced HCV production. Additionally, mutations in C, E1, NS3, and NS5B synergistically promoted HCV production in the background of NS2 and NS5A mutations. Adaptive mutations in NS5A domains I, II, and III independently enhanced HCV production, suggesting that all three domains of NS5A are important for HCV morphogenesis. More importantly, adaptive mutations greatly enhanced physical interactions among HCV structural and NS proteins, as determined by studies with coimmunoprecipitation and mammalian two-hybrid assays. Collectively, these findings demonstrate that adaptive mutations can enhance specific protein-protein interactions among viral structural and NS proteins and therefore promote the assembly of infectious HCV particles.

SUBMITTER: Jiang J

PROVIDER: S-EPMC3416171 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus.

Jiang Jieyun J Luo Guangxiang G

Journal of virology 20120606 17

Recent genetic studies suggested that viral nonstructural (NS) proteins play important roles in morphogenesis of flaviviruses, particularly hepatitis C virus (HCV). Adaptive and compensatory mutations occurring in different NS proteins were demonstrated to promote HCV production in cell culture. However, the underlying molecular mechanism of NS proteins in HCV morphogenesis is poorly understood. We have isolated a cell culture-adapted HCV of genotype 2a (JFH1) which grew to an infectious titer 3 ...[more]

PMID: 22674987

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Project description:While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties.Characterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability.

Project description:BackgroundDespite the high prevalence of genotype 1b hepatitis C virus (HCV) among patients, a cell culture system that permits entire viral life cycle of genotype 1b isolates is limited. To develop a cell-cultured hepatitis C virus (HCVcc) of genotype 1b, the proper combination of HCV genomic variants and host cells is essential. HCV genomes isolated from patients with distinctive symptoms may provide the variants required to establish an HCVcc of genotype 1b.ResultsWe first established subgenomic replicons in Huh7 cells using HCV cDNAs isolated from two patients: one with fulminant hepatitis after liver transplantation (TPF1) and another with acute hepatitis and moderate symptoms (sAH). Replicons established from TPF1 and sAH showed mutations in NS4B and in NS3 and NS5A, respectively. Using these replication machineries, we constructed HCV genomic RNAs for each isolate. Virus infectivity was evaluated by a focus-forming assay, which is dependent on the intracellular expression of core antigen, and production of virus particles was assessed by density-gradient centrifugation. Infectious virus was only observed in the culture medium of cells transfected with TFP1 HCV RNA. A chimeric genome with the structural segment (5'-untranslated region [UTR] through NS2) from sAH and the replication machinery (NS3 through 3'-UTR) from TPF1 exhibited greater infectivity than did TFP1, despite formation of deficient virus particles in sAH, suggesting that this genomic segment potentiates virus particle formation. To identify the responsible variants, infectious virus formation was assessed in a chimeric genome carrying parts of the sAH structural segment of the TPF1 genome. A variant in NS2 (M170T) was identified that enhanced infectious virus formation. HCVcc carrying an NS2 gene encoding the M170T substitution and adaptive mutations in NS4B (referred to as TPF1-M170T) infected naïve cured Huh7 cells in a CD81-dependent manner.ConclusionsWe established a novel HCVcc of genotype 1b in Huh7 cells by introducing an amino acid variant in NS2 and adaptive mutations in NS4B from HCV genomic RNA isolated from a patient with fulminant HCV after liver transplantation.

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Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus.

Publications

Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus.

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