The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called "␤-flap" and a C-terminal segment (designated "linker") that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the ␤-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition irrespective of its incorporation in neosynthesized RNA. Hepatitis C virus (HCV)7 is a member of the Flaviviridae family that induces severe liver disease in humans (1). The viral genome is a single-stranded RNA of positive polarity containing a single open reading frame (ORF) flanked by two untranslated regions (UTRs), the 5Ј-UTR and 3Ј-UTR. The single ORF is translated into a large (ϳ3000 residues) polyprotein that is processed into some 10 mature proteins. Thus, the RNA-dependent RNA polymerase (RdRp) NS5b is cleaved from the C terminus of the polyprotein. In vivo the 591-residue NS5b is the central player in the synthesis of new genomic RNAs, in association with other viral and cellular proteins. This viral replication complex is associated with membranes (2) with the highly hydrophobic C-terminal 21 residues of NS5b forming a transmembrane helix (3). In vitro, NS5b has been shown to be capable of template-directed RNA synthesis on its own, requiring only divalent metals (magnesium or manganese) as cofactors. Indeed, NS5b can catalyze both de novo synthesis from a singlestranded template (4) and primer extension from the subsequent RNA duplex or from a preannealed template/primer duplex. The NS5b C-terminal transmembrane helix is dispensable for these activities, and C-terminal deletions of 21 residues (NS5b_⌬21) or more (NS5b_⌬47 to NS5b_⌬60) have been used in most activity and all crystallographic studies. The latter (5-7) has shown that the catalytic core of NS5b comprises residues 1-530 (Fig. 1E). They have also brought a puzzle to light; the 40-residue stretch (termed "linker" throughout this manuscript) between the catalytic core (fingers, palm, and thumb, Fig. 1) and the C-terminal membrane anchor occludes the catalytic cleft (5) in the crystal structures in which it is present (i.e. _⌬21 forms). The only reported exception is the case of the consensus subty...

The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.

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