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Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template

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2014

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Reich, Stefan
Lilie, Hauke
Knick, Paul
Geissler, René
Golbik, Ralph Peter
Balbach, Jochen
Behrens, Sven-Erik

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Biochemistry. 2014, 53(44), pp. 7002-7012. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi5006656

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The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D 1H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3'(-)SL by 1H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. 1H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3'(-)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.

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ISO 690REICH, Stefan, Michael KOVERMANN, Hauke LILIE, Paul KNICK, René GEISSLER, Ralph Peter GOLBIK, Jochen BALBACH, Sven-Erik BEHRENS, 2014. Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template. In: Biochemistry. 2014, 53(44), pp. 7002-7012. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi5006656
BibTex
@article{Reich2014-11-11Initi-44422,
  year={2014},
  doi={10.1021/bi5006656},
  title={Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template},
  number={44},
  volume={53},
  issn={0006-2960},
  journal={Biochemistry},
  pages={7002--7012},
  author={Reich, Stefan and Kovermann, Michael and Lilie, Hauke and Knick, Paul and Geissler, René and Golbik, Ralph Peter and Balbach, Jochen and Behrens, Sven-Erik}
}
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    <dcterms:abstract xml:lang="eng">The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D &lt;sup&gt;1&lt;/sup&gt;H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3'(-)SL by &lt;sup&gt;1&lt;/sup&gt;H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. &lt;sup&gt;1&lt;/sup&gt;H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3'(-)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.</dcterms:abstract>
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