Identification of nucleotide binding sites in the poliovirus RNA polymerase

Richards, Oliver C.; Hanson, Jeffrey L.; Schultz, Steve C.; Ehrenfeld, Ellie

doi:10.1021/bi00019a005

Cited by 16 publications

(24 citation statements)

References 32 publications

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“…5. Interestingly, Gly-64 is likely to be near Lys-61, a side chain previously shown to be susceptible to nucleotide cross-linking (28)(29)(30). We propose that Gly-64 is near a nucleotide-binding site in the fingers domain, and that mutation of this glycine to the bulkier serine may reduce the space for base mispair formation or reduce the flexibility of the fingers domain.…”

Section: Discussionmentioning

confidence: 83%

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

Pfeiffer

Kirkegaard

2003

Proc. Natl. Acad. Sci. U.S.A.

384

442

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Ribavirin is a nucleotide analog that can be incorporated by viral polymerases, causing mutations by allowing base mismatches. It is currently used therapeutically as an antiviral drug during hepatitis C virus infections. During the amplification of poliovirus genomic RNA or hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. This observation has led to the hypothesis that ribavirin's antiviral activity results from error catastrophe caused by increased mutagenesis of viral genomes. Here, we describe the generation of ribavirin-resistant poliovirus by serial viral passage in the presence of increasing concentrations of the drug. Ribavirin resistance can be caused by a single amino acid change, G64S, in the viral polymerase in an unresolved portion of the fingers domain. Compared with wild-type virus, ribavirinresistant poliovirus displays increased fidelity of RNA synthesis in the absence of ribavirin and increased survival both in the presence of ribavirin and another mutagen, 5-azacytidine. Ribavirin-resistant poliovirus represents an unusual class of viral drug resistance: resistance to a mutagen through increased fidelity.

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Section: Discussionmentioning

confidence: 83%

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

Pfeiffer

Kirkegaard

2003

Proc. Natl. Acad. Sci. U.S.A.

384

442

View full text Add to dashboard Cite

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“…7A), decreasing it by ϳ80% as compared with the wild type VPg. Because chemical cross-linking of the oxidized nucleotide to a protein may occur nonspecifically via exposed lysine, some residual binding was expected (24).…”

Section: Resultsmentioning

confidence: 99%

“…The surplus periodate was reduced with glycerol. Oxidized UTP was cross-linked to PVA VPg by modifying the procedures used previously (23,24). Crosslinking mixture contained 0.02 Ci of oxidized [␣-32 P]UTP, 10 mM HEPES, pH 7.4, 5 mM MgCl 2 or MnCl 2 , 3 mM sodium cyanoborohydride (NaCNBH 3 ), and 3 g of PVA VPg in a final volume of 30 l. Reactions were incubated at 0°C for 60 min.…”

Section: Identification Of the Amino Acid Involved In Formation Of Thmentioning

confidence: 99%

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Puustinen

Mäkinen

2004

Journal of Biological Chemistry

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“…Although K m for GTP of 3D pol is very low, high concentrations (ϳ2 mM) were required for stoichiometric crosslinking and maximum protection from heat denaturation (37). Two NTP binding sites were mapped, one near the amino terminus (residues 57-74) outside of the polymerase core region and the other in the central region overlapping with motif B (residues 266 -286) (38). Only the amino-terminal NTP binding site was shown to be crucial for RNA replication in that a leucine substitution for the highly conserved lysine residue at position 61 completely abolished RdRp activity (39,40).…”

Section: Discussionmentioning

confidence: 99%

Selective Stimulation of Hepatitis C Virus and Pestivirus NS5B RNA Polymerase Activity by GTP

Lohmann

Overton

Bartenschlager

1999

Journal of Biological Chemistry

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NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G) 12 but not with poly(A)-oligo(U) 12 . Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, in particular those affecting the GDD motif (motif C) strongly reduced or completely abolished activation by GTP, suggesting that the same NTP-binding site is used for stimulation and RNA synthesis. Since GTP did not affect the overall RNA binding properties or the elongation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis. Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pestivirus classical swine fever virus, but not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses. The hepatitis C virus (HCV)1 is an RNA virus that causes acute and chronic liver disease (for reviews see Refs. 1-3). A high proportion of infected patients fail to clear the virus and contract chronic infection, which may lead to liver cirrhosis and, eventually, to hepatocellular carcinoma. Currently it is estimated that 100 -200 million people worldwide suffer from a chronic HCV infection. Thus, HCV became a focus of intensive research worldwide.Based on similarities of genome organization and virus particle structure with the flaviviruses like yellow fever virus and the animal pathogenic pestiviruses like the classical swine fever virus (CSFV), HCV has been classified as the separate genus Hepacivirus in the family Flaviviridae (4).These viruses have in common an enveloped virus particle and a single-stranded RNA genome of positive polarity encoding a polyprotein that is cleaved by host cell signalases and viral proteinases. In the case of HCV, the genome has a length of ϳ9600 nucleotides. Its single long open reading frame is flanked at the 5Ј-and 3Ј-ends by nontranslated regions of about 340 and 230 nucleotides in length, respectively. The 5Ј nontranslated region is important for efficient translation of the viral polyprotein and functions as an internal ribosome entry site (5-7). The 3Ј nontranslated region most likely required for RNA replication carries a 3Ј-terminal highly conserved sequence forming stable secondary and probably also higher order structures (8 -11).HCV polyprotein processing is accomplished by a combination of host and viral proteinase...

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Identification of nucleotide binding sites in the poliovirus RNA polymerase

Cited by 16 publications

References 32 publications

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Selective Stimulation of Hepatitis C Virus and Pestivirus NS5B RNA Polymerase Activity by GTP

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