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A view of the HCV polymerase at work
More than 3% of the world's population is infected with hepatitis C virus (HCV), a predisposing factor for life-threatening liver diseases such as cirrhosis and cancer. HCV encodes a polymerase called NS5B that catalyzes replication of the viral RNA genome. Drugs inhibiting NS5B have shown impressive antiviral activity in recent clinical trials. Appleby et al. (see the Perspective by Bressanelli) reveal the inner workings of HCV RNA replication by analyzing crystal structures of stalled NS5B polymerase ternary complexes during the initiation and elongation of RNA synthesis. They also define the way in which sofosbuvir, a drug with potent clinical efficacy, interacts with the NS5B active site.
Abstract
Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane–anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3′ terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site.
