期刊
JOURNAL OF VIROLOGY
卷 76, 期 24, 页码 12513-12525出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.76.24.12513-12525.2002
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We functionally analyzed the role of metal ions in RNA-dependent RNA synthesis by three recombinant RNA-dependent RNA polymerases (RdRps) from GB virus-B (GBV), bovine viral diarrhea virus (BVDV), and hepatitis C virus (HCV), with emphasis on the HCV RdRp. Using templates capable of both de novo initiation and primer extension and RdRps purified in the absence of metal, we found that only reactions with exogenously provided Mg2+ and Mn2+ gave rise to significant amounts of synthesis. Mg2+ and Mn2+ affected the mode of RNA synthesis by the three RdRps. Both metals supported primer-dependent and de novo-initiated RNA by the GBV RdRp, while Mn2+ significantly increased the amount of de novo-initiated products by the HCV and BVDV RdRps. For the HCV RdRp, Mn2+ reduced the K-m for the initiation nucleotide, a GTP, from 103 to 3 muM. However, it increased de novo initiation even at GTP concentrations that are comparable to physiological levels. We hypothesize that a change in RdRp structure occurs upon GTP binding to prevent primer extension. Analysis of deleted proteins revealed that the C terminus of the HCV RdRp plays a role in Mn2+-induced de novo initiation and can contribute to the suppression of primer extension. Spectroscopy examining the intrinsic fluorescence of tyrosine and tryptophan residues in the HCV RdRp produced results consistent with the protein undergoing a conformational change in the presence of metal. These results document the fact that metal can affect de novo initiation or primer extension by flaviviral RdRps.
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