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Effect of alpha interferon on the hepatitis C virus replicon.

ABSTRACT: Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.

SUBMITTER: Guo JT

PROVIDER: S-EPMC115097 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

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Effect of alpha interferon on the hepatitis C virus replicon.

Guo J T JT Bichko V V VV Seeger C C

Journal of virology 20010901 18

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from ...[more]

PMID: 11507197

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Project description:A lack of patient response to alpha interferon (?-IFN) plus ribavirin (RBV) treatment is a major problem in eliminating hepatitis C virus (HCV). We screened chemical libraries for compounds that enhanced cellular responses to ?-IFN and identified a triterpenoid, toosendanin (TSN). Here, we studied the effects and mechanisms of action of TSN on HCV replication and its effect on ?-IFN signaling. We treated HCV genotype 1b replicon-expressing cells and HCV-J6/JFH-infected cells with TSN, with or without ?-IFN, and the level of HCV replication was quantified. To study the effects of TSN on ?-IFN signaling, we detected components of the interferon-stimulated gene factor 3 (ISGF3), phosphorylated signal transducer and activator of transcription 1 (STAT1), and STAT2 by Western blotting analysis; expression levels of mRNA of interferon regulatory factor 9 using real-time reverse transcription-PCR (RT-PCR); and interferon-stimulated response element reporter activity and measured the expression levels of interferon-inducible genes for 2',5'-oligoadenylate synthetase, MxA, protein kinase R, and p56 using real-time RT-PCR. TSN alone specifically inhibited expression of the HCV replicon (50% effective concentration = 20.6 nM, 50% cytotoxic concentration > 3 ?M, selectivity index > 146). Pretreatment with TSN prior to ?-IFN treatment was more effective in suppressing HCV replication than treatment with either drug alone. Although TSN alone did not activate the ?-IFN pathway, it significantly enhanced the ?-IFN-induced increase of phosphorylated STATs, interferon-stimulated response element activation, and interferon-stimulated gene expression. TSN significantly increased baseline expression of interferon regulatory factor 9, a component of interferon-stimulated gene factor 3. Antiviral effects of treatment with ?-IFN can be enhanced by pretreatment with TSN. Its mechanisms of action could potentially be important to identify novel molecular targets to treat HCV infection.

Project description:To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.The alternative 22-nucleotide IRES, RNA-binding motif protein 3 IRES (Rbm3 IRES), was used to form a tricistronic HCV replicon, to facilitate constructing HCV-harboring stable cell lines and successive antiviral screening using a luciferase marker. Briefly, two sequential Rbm3 IRESes were inserted into bicistronic pUC19-HCV plasmid, consequently forming a tricistronic HCV replicon (pHCV-rep-NeoR-hRluc), initiating the translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating the translation of neomycin resistance gene. The sH7 cell lines, in which the novel replicon RNA stably replicated, were constructed by neomycin and luciferase activity screening. The intracellular HCV replicon RNA, expression of inserted foreign genes and HCV non-structural gene, as well as response to anti-HCV agents, were measured in sH7 cells and cells transiently transfected with tricistronic replicon RNA.The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in sH7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons. The average relative light unit in pHCV-rep-NeoR-hRluc group was approximately 2-fold of those in the pUC19-HCV-hRLuc and Tri-JFH1 groups (1.049 × 10(8) ± 2.747 × 10(7) vs 5.368 × 10(7) ± 1.016 × 10(7), P < 0.05; 1.049 × 10(8) ± 2.747 × 10(7) vs 5.243 × 10(7) ± 1.194 × 10(7), P < 0.05), suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES. The fold changes of 72 h/4 h relative light units in the pHCV-rep-NeoR-hRluc and pUC19-HCV-hRLuc groups were similar (159.619 ± 9.083 vs 163.536 ± 24.031, P = 0.7707), and were both higher than the fold change in the Tri-JFH1 group 159.619± 9.083 vs 140.811 ± 9.882, P < 0.05; 163.536 ± 24.031 vs 140.811 ± 9.882, P < 0.05), suggesting that the replication potency of the Rbm3 IRES tricistronic replicon matched the replication of bicistronic replicon and exceeded the potency of EMCV IRES replicon. Replication of tricistronic replicons was suppressed by ribavirin, simvastatin, atorvastatin, telaprevir and boceprevir. Interferon-alpha 2b could not block replication of the novel replicon RNA in sH7 cells. After interferon stimulation, MxA mRNA and protein levels were lower in sH7 than in parental cells.Tricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.

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Effect of alpha interferon on the hepatitis C virus replicon.

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Effect of alpha interferon on the hepatitis C virus replicon.

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