Identification of a C-Terminal Regulatory Motif in Hepatitis C Virus RNA-Dependent RNA Polymerase: Structural and Biochemical Analysis

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Abstract
The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-Å resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to ∼50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.