Analyzing the role of NS5A protein in regulation of interferon sensitivity using HCV replicon system.

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The number of amino acid substitutions in the interferon sensitivity-determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon response and viral load. Several HCV replicon-based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear how the variety of ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons, and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in colony formation assay using HCV replicons with few mutations (0, 1, and 3) in the ISDR, numerous colonies (>200) appeared using constructs with six mutations. Introduction of various distinct ISDR sequences with multipl … More e mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically altered replication, including codon 2209 which was closely associated in patients with a strong response to IFN. ISDR sequences associated with a clinical interferon response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.Next, we studied expression profiles of ISGs in cells supporting subgenomic HCV replication (Huh7/Rep), and screened their activities to suppress HCV replication. Real-time PCR analyses showed that the expression levels of 23 ISGs were significantly lower in Huh7/Rep than naive Huh7 cells due to transcriptional suppression of the interferon-stimulated response element (ISRE). Furthermore, the expression level of ISGs was also decreased in the cured Huh7 cells in which replicon had been eliminated (cHuh7), indicating adaptation of the cells to support HCV replication by downregulating ISGs. On the other hand, expression of HCV replicon was significantly suppressed by overexpression of several ISGs including PKR, MxA, IRF-9, GBP-1, IFI-6-16, IFI-27, 25OAS and IRF-1. Knock down of GBP-1, IFI-6-16 and IFI-27 by short hairpin RNA resulted in increase of HCV replication. Less