HCV NS5A has three domains, which have multiple roles in the viral life cycle. We previously found that NS5A is able to down-regulate HCV RNA translation through a mechanism requiring the polyU/UC region within the viral 3'UTR to which NS5A binds. In this study, we further investigated the role of domain I in modulating viral translation. Using a series of deletion and substitution mutants, we identified a number of positively charged residues that played a role in this modulatory effect, most prominently R112. The R112A mutation negated the ability of domain I and full-length NS5A to modulate viral translation. Additionally, the R112A mutation impeded domain I binding to the polyU/UC RNA, suggesting a mechanism for this down-regulatory effect. Finally, the R112A mutation rendered HCV replication deficient. These results collectively point to a crucial role for the R112 residue of NS5A in the modulation of HCV life cycle by NS5A. (C) 2015 Elsevier Inc. All rights reserved.