The bovine viral diarrhea virus (BVDV) RNA-dependent RNA polymerase can initiate RNA replication by a de novo mechanism without a primer. The structure of BVDV polymerase, determined to 2.9-Å resolution, contains a unique N-terminal domain, in addition to the fingers, palm, and thumb domains common to other polymerases. The structure of BVDV polymerase complexed with GTP, which is required for de novo (primer-independent) initiation, shows that GTP binds adjacent to the initiation NTP, suggesting that the GTP mimics a vestigial RNA product. Comparison of five monomers in two different crystal forms showed conformational changes in the fingertip region and in the thumb domain that may help to translocate the RNA template and product strands during elongation. The putative binding sites of previously reported BVDV inhibitors are also discussed.T he function of a polymerase is to synthesize the complementary RNA or DNA molecule from a template strand. Thus, the polymerase requires binding sites for the template, nucleoside triphosphates (NTPs or dNTPs), and the nascent polynucleotide product. Also required are mechanisms to catalyze the addition of NTP or dNTP to the nascent chain, to recognize the next complementary NTP molecule, and to move the template and product by one nucleotide in readiness for the next elongation event. Finally, the polymerase, in conjunction with other viral and cellular enzymes, such as a helicase in a replication complex, requires a mechanism for initiating and terminating synthesis of the new product polynucleotide.RNA replication in positive-sense single-stranded (ss)RNA viruses is initiated at or near the 3Ј end of the template using either primer-dependent or primer-independent (de novo) mechanisms. Poliovirus, for example, utilizes a genome-linked protein as a primer for initiation of RNA synthesis (1). In Flaviviridae, on the other hand, de novo initiation is the likely mechanism used during virus replication in infected cells (2). In de novo initiation, the second NTP is added (with the release of its pyrophosphate moiety) directly to the 3Ј-OH of the first initiation NTP without the need of a primer. This nucleotidyl transfer reaction is then repeated with subsequent NTPs to generate the complementary RNA product. De novo initiation by Flaviviridae RNA polymerases requires (i) a template RNA with a virus-specific initiation nucleotide at the 3Ј end, (ii) a complementary initiation NTP, and (iii) GTP (3-6). Because base pairing between the template RNA and individual NTPs may not be sufficient to allow the formation of a stable initiation complex, it is likely that de novo initiation requires specific molecular interactions among the template RNA, NTPs, the RNA polymerase, and possibly other viral and host proteins. Although not all RNA-dependent RNA polymerases (RdRps) require GTP for initiating RNA synthesis, other ligands or structural components can act similarly as GTP and help to position the 3Ј-OH group of the priming nucleotide ready for nucleophilic attack.The Flavivirida...
HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log 10 HCV copies/g total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 Å of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10-to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four-to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.Hepatitis C virus (HCV) is an enveloped, positive-sense, single-stranded RNA virus of approximately 9.6 kb that possesses an RNA-dependent RNA polymerase (RdRp), NS5B. Like that in many RNA viruses, this RNA replicase lacks a proofreading mechanism. The mutation rate of the HCV RdRp is estimated to be 10
The poliovirus RNA-dependent RNA polymerase required an oligouridylate primer or a HeLa cell protein (host factor) to initiate RNA synthesis on poliovirion RNA in vitro. The polymerase synthesized template-sized product RNA in the oligouridylate-primed reaction. In the host factor-dependent reaction, the largest product RNA synthesized by the polymerase was twice the size of the template RNA. About half of the product RNA recovered from this reaction was shown to exist in the form of a snapback sequence. Time-course reactions and pulse-chase experiments showed that the product RNA was only slightly larger than the template RNA at early reaction times and that with time it increased in size to form the dimer-sized product RNA. Inhibition of the elongation reaction by adding only [ft-32P]UTP and ATP resulted in the formation of template-sized product RNA. The dimer-sized product RNA was unaffected by phenol extraction or proteinase K treatment but was converted to template-sized molecules by S1 nuclease. Dimer-sized poliovirus RNA that was sensitive to S1 nuclease was also isolated from poliovirus-infected cells. The results from this study indicate that the labeled negative-strand product RNA synthesized in vitro was covalently linked to the positive-strand template RNA. Thus, in vitro, the primer-dependent poliovirus RNA polymerase may initiate RNA synthesis in the presence of the host factor by using the 3' end of the template RNA as a primer.
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