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Identification of alpha interferon-induced envelope mutations of hepatitis C virus in vitro associated with increased viral fitness and interferon resistance.

ABSTRACT: Alpha interferon (IFN-?) is an essential component of innate antiviral immunity and of treatment regimens for chronic hepatitis C virus (HCV) infection. Resistance to IFN might be important for HCV persistence and failure of IFN-based therapies. Evidence for HCV genetic correlates of IFN resistance is limited. Experimental studies were hampered by lack of HCV culture systems. Using genotype (strain) 1a(H77) and 3a(S52) Core-NS2 JFH1-based recombinants, we aimed at identifying viral correlates of IFN-? resistance in vitro. Long-term culture with IFN-?2b in Huh7.5 cells resulted in viral spread with acquisition of putative escape mutations in HCV structural and nonstructural proteins. Reverse genetic studies showed that primarily amino acid changes I348T in 1a(H77) E1 and F345V/V414A in 3a(S52) E1/E2 increased viral fitness. Single-cycle assays revealed that I348T and F345V/V414A enhanced viral entry and release, respectively. In assays allowing viral spread, these mutations conferred a level of IFN-? resistance exceeding the observed fitness effect. The identified mutations acted in a subtype-specific manner but were not found in genotype 1a and 3a patients, who failed IFN-? therapy. Studies with HCV recombinants with different degrees of culture adaptation confirmed the correlation between viral fitness and IFN-? resistance. In conclusion, in vitro escape experiments led to identification of HCV envelope mutations resulting in increased viral fitness and conferring IFN-? resistance. While we established a close link between viral fitness and IFN-? resistance, identified mutations acted via different mechanisms and appeared to be relatively specific to the infecting virus, possibly explaining difficulties in identifying signature mutations for IFN resistance.

SUBMITTER: Serre SB

PROVIDER: S-EPMC3838148 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Identification of alpha interferon-induced envelope mutations of hepatitis C virus in vitro associated with increased viral fitness and interferon resistance.

Serre Stéphanie B N SB Krarup Henrik B HB Bukh Jens J Gottwein Judith M JM

Journal of virology 20130918 23

Alpha interferon (IFN-α) is an essential component of innate antiviral immunity and of treatment regimens for chronic hepatitis C virus (HCV) infection. Resistance to IFN might be important for HCV persistence and failure of IFN-based therapies. Evidence for HCV genetic correlates of IFN resistance is limited. Experimental studies were hampered by lack of HCV culture systems. Using genotype (strain) 1a(H77) and 3a(S52) Core-NS2 JFH1-based recombinants, we aimed at identifying viral correlates of ...[more]

PMID: 24049176

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Project description:AIM:Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN-alpha), which results in an efficient reduction of the viral load only in 20-40% of treated patients. Mutations at HBV precore prevail in different clinical status of HBV infection. The roles of precore mutation in the progression of chronic hepatitis and interferon sensitivity are still unknown. The aim of this study was to explore if there was any relationship between HBV precore mutation and sensitivity to interferon in vitro. METHODS:HBV replication-competent recombinant constructs with different patterns of precore mutations were developed. Then the recombinants were transiently transfected into hepatoma cell line (Huh7) by calcium phosphate transfection method. With or without IFN, viral products in culture medium were collected and quantified 3 d after transfection. RESULTS:We obtained 4 recombinant constructs by orientation-cloning 1.2-fold-overlength HBV genome into pUC18 vector via the EcoRI and Hind III and PCR mediated site-directed mutagenesis method. All the recombinants contained mutations within precore region. Huh7 cells transfected with recombinants secreted HBsAg and HBV particles into the cell culture medium, indicating that all the recombinants were replication-competent. By comparing the amount of HBV DNA in the medium, we found that HBV DNA in medium reflecting HBV replication efficiency was different in different recombinants. Recombinants containing precore mutation had fewer HBV DNAs in culture medium than wild type. This result showed that recombinants containing precore mutation had lower replication efficiency than wild type. HBV DNA was decreased in pUC18-HBV1.2-WT recombinants after IFN was added while others with precore mutations were not, indicating that HBV harboring precore mutation was less sensitive to IFN in cell culture system. CONCLUSION:These data indicate that HBV harboring precore mutation may be resistant to IFN in vitro.

Dataset Information

Identification of alpha interferon-induced envelope mutations of hepatitis C virus in vitro associated with increased viral fitness and interferon resistance.

Publications

Identification of alpha interferon-induced envelope mutations of hepatitis C virus in vitro associated with increased viral fitness and interferon resistance.

Similar Datasets

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