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Response of hepatitis C virus to long-term passage in the presence of alpha interferon: multiple mutations and a common phenotype.

ABSTRACT: Cell culture-produced hepatitis C virus (HCV) has been subjected to up to 100 serial passages in human hepatoma cells in the absence or presence of different doses of alpha interferon (IFN-?). Virus survival, genetic changes, fitness levels, and phenotypic traits have been examined. While high initial IFN-? doses (increasing from 1 to 4 IU/ml) did not allow HCV survival beyond passage 40, a gradual exposure (from 0.25 to 10 IU/ml) allowed the virus to survive for at least 100 passages. The virus passaged in the presence of IFN-? acquired IFN-? resistance as evidenced by enhanced progeny production and viral protein expression in an IFN-? environment. A partial IFN-? resistance was also noted in populations passaged in the absence of IFN-?. All lineages acquired adaptative mutations, and multiple, nonsynonymous mutations scattered throughout the genome were present in IFN-?-selected populations. Comparison of consensus sequences indicates a dominance of synonymous versus nonsynonymous substitutions. IFN-?-resistant populations displayed decreased sensitivity to a combination of IFN-? and ribavirin. A phenotypic trait common to all assayed viral populations is the ability to increase shutoff host cell protein synthesis, accentuated in infections with IFN-?-selected populations carried out in the presence of IFN-?. The trait was associated with enhanced phosphorylation of protein kinase R (PKR) and eIF2?, although other contributing factors are likely. The results suggest that multiple, independent mutational pathways can confer IFN-? resistance to HCV and might explain why no unified picture has been obtained regarding IFN-? resistance in vivo.

SUBMITTER: Perales C

PROVIDER: S-EPMC3700284 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Response of hepatitis C virus to long-term passage in the presence of alpha interferon: multiple mutations and a common phenotype.

Perales Celia C Beach Nathan M NM Gallego Isabel I Soria Maria Eugenia ME Quer Josep J Esteban Juan Ignacio JI Rice Charles C Domingo Esteban E Sheldon Julie J

Journal of virology 20130501 13

Cell culture-produced hepatitis C virus (HCV) has been subjected to up to 100 serial passages in human hepatoma cells in the absence or presence of different doses of alpha interferon (IFN-α). Virus survival, genetic changes, fitness levels, and phenotypic traits have been examined. While high initial IFN-α doses (increasing from 1 to 4 IU/ml) did not allow HCV survival beyond passage 40, a gradual exposure (from 0.25 to 10 IU/ml) allowed the virus to survive for at least 100 passages. The virus ...[more]

PMID: 23637397

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Project description:Background & aimsPegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3).MethodsPegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining.ResultsPegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates.ConclusionsOur comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness.Impact and implicationsUnderstanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

Project description:UNLABELLED:Combination treatment with pegylated-interferon-alpha (PEG IFN-alpha) and ribavirin, the current recommended therapy for chronic hepatitis C virus (HCV) infection, results in a sustained virological response (SVR) in only about half of patients. Because genes involved in the interferon-alpha pathway may affect antiviral responses, we analyzed the relationship between variants in these genes and SVR among participants in the Hepatitis C Antiviral Long-Term treatment Against Cirrhosis (HALT-C) trial. Patients had advanced chronic hepatitis C that had previously failed to respond to interferon-based treatment. Participants were treated with peginterferon-alpha2a and ribavirin during the trial. Subjects with undetectable HCV RNA at week 72 were considered to have had an SVR. Subjects with detectable HCV RNA at week 20 were considered nonresponders. We used TaqMan assays to genotype 56 polymorphisms found in 13 genes in the interferon-alpha pathway. This analysis compares genotypes for participants with an SVR to nonresponders. The primary analysis was restricted to European American participants because a priori statistical power was low among the small number (n = 131) of African American patients. We used logistic regression to control the effect of other variables that are associated with treatment response. Among 581 European American patients, SVR was associated with IFNAR1 IVS1-22G (adjusted odds ratio, 0.57; P = 0.02); IFNAR2 Ex2-33C (adjusted odds ratio, 2.09; P = 0.02); JAK1 IVS22+112T (adjusted odds ratio, 1.66; P = 0.04); and ADAR Ex9+14A (adjusted odds ratio, 1.67; P = 0.03). For the TYK2-2256A promoter region variant, a borderline association was present among European American participants (OR, 1.51; P = 0.05) and a strong relationship among African American patients; all 10 with SVR who were genotyped for TYK2 -2256 carried the A variant compared with 68 of 120 (57%) nonresponders (P = 0.006). CONCLUSION:Genetic polymorphisms in the interferon-alpha pathway may affect responses to antiviral therapy of chronic hepatitis C.

Project description:The negative regulators in the interferon (IFN) signaling pathway inhibit intrahepatic immune response, resulting in suboptimal therapeutic response to IFNα treatment in chronic hepatitis B (CHB) patients. Identifying the key negative factors and elucidating the regulating mechanism are essential for improving anti-HBV (hepatitis B virus) efficacy of IFNα. From the Gene Expression Omnibus (GEO) database, we downloaded and analyzed gene expression profiles of CHB patients with different responses to IFNα (GSE54747), and found that innate immune status was associated with the IFNα-based therapeutic response in CHB patients. Through PCR array, we found higher baseline level of IFN-induced transmembrane protein 2 (IFITM2) mRNA and lower baseline level of IFNα mRNA in peripheral blood mononuclear cells (PBMCs) of CHB patients with suboptimal response to IFNα treatment. Increased IFITM2 protein was also found in the serum of IFNα nonresponsive patients. With further experiments, we found that overexpressing IFITM2 in Huh7 cells suppressed endogenous IFNα synthesis by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK), TANK-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3); knocking out IFITM2 enhanced activation of the endogenous IFNα synthesis pathway, exhibiting better inhibition on HBV replication. We also found that IFITM2 protein was shuttled by exosomes to dendritic cells (DCs), the main source of endogenous IFNα. Exosome-mediated transport of IFITM2 inhibited synthesis of endogenous IFNα in DCs whereas the inhibitory effect was abolished when IFITM2 was knocked out. Furthermore, we demonstrated that both palmitoylation inhibitor and mutation on 70/71 sites of IFITM2 protein influenced its incorporation into exosomes. Mutated IFITM2 protein increased the effect of IFNα against HBV. Conclusion: Exosome-mediated transport of IFITM2 to DCs inhibits IFNα pathway activation and blocks anti-HBV efficacy of exogenous IFNα. The findings provide an explanation to the suboptimal response of CHB patients to IFNα treatment.

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Response of hepatitis C virus to long-term passage in the presence of alpha interferon: multiple mutations and a common phenotype.

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Response of hepatitis C virus to long-term passage in the presence of alpha interferon: multiple mutations and a common phenotype.

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