De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

Abstract

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a K m for GTP of 3.5 M, all of the mutations except one had K m s that were three-to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.Hepatitis C Virus (HCV), a member of the flaviviridae family of positive-strand RNA viruses, infects up to 3% of the world's population (1). A prime target for antiviral treatment in HCV is the NS5B protein, the RNA-dependent RNA polymerase (RdRp) responsible for viral RNA synthesis. The RdRp is one of the subunits expected to participate in HCV replication and is responsible for the initiation and elongation of viral RNA synthesis. Initiation of RNA synthesis in infected cells likely starts de novo; that is, by use of a 1-nucleotide (nt) primer (3,14). This mode of RNA synthesis requires a specialized site in the RdRp, called the I site, that specifically recognizes the initiating nucleoside triphosphate (designated NTPi, with GTP being preferred by the recombinant HCV RdRp) (14,23,25). A second NTP-binding pocket, the I ϩ 1 site, binds a nucleotide for phosphoryl transfer as dictated by the template base (14). Cocrystal structures of the HCV RdRp and nucleotides have been reported previously (3, 21), identifying a number of residues at D225, R48, R158, R386, R394, and S367 that interact with specific moieties of the initiation GTP (Fig. 1A). In this structure, it is not known whether the GTP binds to the I site or the I ϩ 1 site. NTPi binding to the I site is base specific, while binding of the second NTP to the I ϩ 1 site should be directed by the template (25). Because the RdRp-GTP st...

Functional Analysis of RNA Binding by the Hepatitis C Virus RNA-dependent RNA Polymerase

“…Notably, the Arg-158 in peptide 155-168 (colored yellow in Fig. 5B) was previously determined to interact with the ␣-phosphate of GTP and shown to be important for RdRp activity (13,25). We did not observe residues within the novel ␤-loop to be among the regions cross-linked to RNA.…”

Screening of the Yeast yTHC Collection Identifies Essential Host Factors Affecting Tombusvirus RNA Recombination

“…Mutagenesis of the replicase proteins has led to altered recombination frequencies or altered the sites of recombination (15,30,47), suggesting that many recombination events are due to template switching (replicase jumping) by the viral replicase (22,27,39). In vitro template-switching experiments confirmed the abilities of purified viral RNA-dependent RNA polymerases or partially purified viral replicases to switch templates (8,25,52). Nonreplicative RNA recombination events have also been demonstrated for a small group of RNA viruses (11,18).…”

Hepatitis C Virus Replication in Transfected and Serum-Infected Cultured Human Fetal Hepatocytes

“…29,30 Core protein expression occurred in clusters of HFHs transfected with WT RNA surrounded by cells that exhibited little or no immunoreactivity and was localized to the cell cytoplasm in a punctated pattern ( Figure 2E). However, no staining of core protein was detected in cultures that were transfected with the 3Ј-UTR mutant RNA ( Figure 2F).…”