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Cytopathic and noncytopathic interferon responses in cells expressing hepatitis C virus subgenomic replicons.

ABSTRACT: Hepatitis C virus (HCV) is the only known positive-stranded RNA virus that causes persistent lifelong infections in humans. Accumulation of HCV RNA can be inhibited with alpha interferon (IFN-alpha) in vivo and in culture cells. We used cell-based assay systems to investigate the mechanisms responsible for the cytokine-induced inhibition of HCV replication. The results showed that IFN-alpha could suppress the accumulation of viral RNA by a noncytopathic pathway and could also induce apoptosis of virally infected cells in a concentration- and cell line-dependent fashion. Whereas the noncytopathic IFN-alpha response depended on a functional Jak-STAT signal transduction pathway, it did not appear to require double-stranded RNA-dependent pathways. Moreover, we found that functional proteasomes were required for establishment of the IFN-alpha response against HCV. Based on the results described in this study we propose a model for the mechanism by which IFN-alpha therapy suppresses HCV replication in chronic infections by both cytopathic and noncytopathic means.

SUBMITTER: Guo JT

PROVIDER: S-EPMC224980 | biostudies-literature | 2003 Oct

REPOSITORIES: biostudies-literature

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Cytopathic and noncytopathic interferon responses in cells expressing hepatitis C virus subgenomic replicons.

Guo Ju-Tao JT Zhu Qing Q Seeger Christoph C

Journal of virology 20031001 20

Hepatitis C virus (HCV) is the only known positive-stranded RNA virus that causes persistent lifelong infections in humans. Accumulation of HCV RNA can be inhibited with alpha interferon (IFN-alpha) in vivo and in culture cells. We used cell-based assay systems to investigate the mechanisms responsible for the cytokine-induced inhibition of HCV replication. The results showed that IFN-alpha could suppress the accumulation of viral RNA by a noncytopathic pathway and could also induce apoptosis of ...[more]

PMID: 14512527

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Project description:AimTo 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.MethodsThe 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.ResultsThe 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.ConclusionTricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.

Dataset Information

Cytopathic and noncytopathic interferon responses in cells expressing hepatitis C virus subgenomic replicons.

Publications

Cytopathic and noncytopathic interferon responses in cells expressing hepatitis C virus subgenomic replicons.

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