OHGIMOTO Shinji Kobe Univ, Grad Sch Med, Assistant, 大学院・医学系研究科, 助手 (80292853)
NAGANO Motoko Kobe Univ, Grad Sch Med, Assistant, 大学院・医学系研究科, 助手 (90304089)
ISHIDO Satoshi Kobe Univ, Grad Sch Med, Associate Professor, 大学院・医学系研究科, 助教授 (10273781)
|Budget Amount *help
¥9,700,000 (Direct Cost: ¥9,700,000)
Fiscal Year 2002: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥6,000,000 (Direct Cost: ¥6,000,000)
1. Correlation between ISDR mutations and HCV RNA titers:
HCV strains obtained from patients in Surabaya, Indonesia, and their serum HCV RNA titers were examined. The number of amino acid mutations in IFN sensitivity-determining region (ISDR) and PKR-binding domain of NS5A of HCV-1b, -1c and -2a were inversely correlated with serum HCV RNA titers. HCV strains with 4 more mutations in ISDR had significantly lower HCV RNA titers than those with fewer mutations.
2. Interaction between HCV NS5A and 2',5'-oligoadenylate synthetase.
Possible interaction between HCV NS5A and 2',5'-oligoadenylate synthetase (2-5AS), a key molecule for IFN-mediated antiviral action, was studied. NS5A and 2-5AS were colocalized in cultured cells, especially in the perinuclear region of the cytoplasm. NS5A physically interacted with 2-5AS in cultured cells, with an N-terminal region of 148 residues (aa1-148) of NS5A, which lacks ISDR and PKR-binding domain, being responsible for the interaction. On the other hand, two separate regions of 2-5AS were independently involved in the interaction with NS5A. Single point mutations were introduced to the 37th residue of NS5A(1-148), such as Phe-to-Leu (F37L), F37N, F37S and F37Y, and effects of those mutations on the degree of interaction with 2-5AS were studied. The results showed that the F37L mutation increased binding to 2-5AS while the F37N mutation decrease it. Virus rescue assay revealed that NS5A(1-148) counteracted antiviral activity of IFN to practically the same extent as that achieved by the full-length NS5A. Interestingly, NS5A(1-148)F37N, which bound to 2-5AS less efficiently, had weaker activity to counteract IFN antiviral activity compared with the wild-type NS5A(1-148).
3. Summary of the present study.
Our present results collectively suggest that NS5A counteracts IFN antiviral activity not only through an ISDR-dependent mechanism but also through an ISDR-independent mechanism, which possibly involves interaction with 2-5AS.