Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication T. Kohashi, 1 * S. Maekawa, 1,2 * N. Sakamoto, 1 M. Kurosaki, 1 H. Watanabe, 1 Y. Tanabe, 1 C.-H. Chen, 1 N. Kanazawa, 1 M. Nakagawa, 1 S. Kakinuma, 1 T. Yamashiro, 1 Y. Itsui, 1 T. Koyama, 1 N. Enomoto 2 and M. Watanabe 1 1 Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Yushima, Bunkyo, Tokyo, Japan; and 2 First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Shimokato, Chuo, Yamanashi, Japan Received July 2005; accepted for publication October 2005 SUMMARY. The number of amino acid substitutions in the interferon sensitivity-determining region (ISDR) in the non- structural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon (IFN) 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 as to how different 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 repli- cation. While no colonies were formed in the colony for- mation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (>200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong re- sponse to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection. Keywords: HCV replicon, NS5A, ISDR. INTRODUCTION Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide [1]. Interferon (IFN)-alpha remains the key anti-viral agent against HCV, even in the era of combination therapy with pegylated-IFN and ribavirin [2,3]. However, because the eradication rate does not exceed half of the patients treated overall, the molecular mechanism(s) enabling HCV to sur- vive during IFN treatment should be elucidated to enable the development of more effective therapeutic modalities. The interferon sensitivity-determining region (ISDR) at position 2209–2248 of NS5A in the HCV genome was ori- ginally identified as the viral genomic element in which codon changes were closely related to the clinical IFN response, i.e. the more substitutions in the ISDR, the more favourable the IFN response [4–6]. The close correlation of the ISDR mutations with IFN responses has been confirmed by other researchers and by a recent meta-analysis [7–10]. Amino acid substitutions in the ISDR were also associated with serum HCV-RNA levels, indicating that ISDR sequences have an important role in HCV replication. Because HCV clearance was strongly associated with the expression of numerous IFN-stimulated genes induced by endogenous IFN in the natural course of infection, the IFN-response and viral replication capacity cannot be considered separately [11]. It is thought that clearance and replication of HCV is con- trolled by a Ôtug of warÕ between IFN and HCV. Thus, the NS5A protein might perturb this process according to the ISDR sequence. A variety of putative NS5A functions have been postula- ted to date on the basis of studies in vitro. These include binding to TRADD [12], Grb2 [13], p21 [14,15], amphi- physin II [16] and other proteins that may influence the pathogenesis of HCV through their anti-viral effects, modu- lating apoptosis, signal transduction, or regulating the cell *Both these authors contributed equally to this study. Abbreviations: ISDR, interferon sensitivity-determining region; NS5A, nonstructural 5A; HCV, hepatitis C virus; IFN, interferon. Correspondence: Dr Shinya Maekawa, First Department of Medicine, Faculty of Medicine, University of Yamanashi, 1110, Shimokato, Chuo, Yamanashi 409-3898, Japan. E-mail: maekawa@yamanashi. ac.jp Journal of Viral Hepatitis, 2006, 13, 582–590 doi:10.1111/j.1365-2893.2006.00739.x Ó 2006 The Authors Journal compilation Ó 2006 Blackwell Publishing Ltd