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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• 作者：Stefan Reich ; Michael Kovermann ; Hauke Lilie ; Paul Knick ; Ren茅 Geissler ; Ralph Peter Golbik ; Jochen Balbach ; Sven-Erik Behrens
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：November 11, 2014
• 年：2014
• 卷：53
• 期：44
• 页码：7002-7012
• 全文大小：440K
• ISSN：1520-4995

文摘

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鈥?鈭?SL by ¹H NMR, the base pair at the stem鈥檚 edge was identified as the most stable component of the structure. ¹H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3鈥?鈭?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.