Analysis of anti-interferon mechanism by HCV NS5A protein using HCV replicon system

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Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh-7 cells. We have extended these results to other isolated HCV clones, and we have constructed another HCV replicon from HC-J4, one of the chimpanzee-infectious clones. An HCV replieon (RpJ4) was constructed from a chimpanzee-infectious clone, HC-J4, which consists of HCV-5'-UTR, neomycin phosphotransferase gene, the encephalomyocarditis virus IRES, HCV-non-structural region, NS3 to NS5B, and HCV-3'-UTR. The adaptive mutations known to be required for HCV-Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC-J4) 2197 serine to praline, aa.2201 serine to deletion, and aa.2204 serine to isoleucine (RpJ4-S2197P, RpJ4-S22001del, and RpJ4-S2204I). RpJ4/ISDRmutant and RpJ4-S2201del/ISDRmutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). Replieon RNA was transfec … More ted into Huh-7 cells, and stable replicon-expressing cell lines were established by G418 selection. After transfection to naive Huh-7 cells, RpJ4 and RpJ4/ISDRmutants did not produce any G418-resistant colonies. In contrast, G418-resistant cells were transduced efficiently by the introduction of RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDRmutants, with the RpJ4-S2201del/ISDR mutant being most efficient. The HCV replioon derived from HC-J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR Moreover, additional introduction of mutations into ISDR further enhances its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV-1b replications, for both the HCV-Con1 and the HC-J4 replicons.Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the IFN-mediated antiviral system in cells expressing an HCV replieon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor-1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replieon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replieon. Moreover in cured Huh7 cells, from which the HCV replicon had been eliminated, expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of ISG-mediated antiviral responses. Less