Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) has acquired a unique β-hairpin in the thumb subdomain which protrudes toward the active site. We report here that this β-hairpin plays an important role in positioning the 3' terminus of the viral RNA genome for correct initiation of replication. The presence of this β-hairpin interferes with polymerase binding to preannealed double-stranded RNA (dsRNA) molecules and allows only the single-stranded 3' terminus of an RNA template to bind productively to the active site. We propose that this β-hairpin may serve as a "gate" which prevents the 3' terminus of the template RNA from slipping through the active site and ensures initiation of replication from the terminus of the genome. This hypothesis is supported by the ability of a β-hairpin deletion mutant that utilizes dsRNA substrates and initiates RNA synthesis internally. The proposed terminal initiation mechanism may represent a novel replication strategy adopted by HCV and related viruses.
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