title: Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template.
abstract: 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 (1)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 (1)H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. (1)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.
authors.affiliation: Institute of Biochemistry and Biotechnology, Section of Microbial Biotechnology, ‡Institute of Physics, Section of Biophysics, §Institute of Biochemistry and Biotechnology, Section of Technical Biochemistry, Martin Luther University Halle-Wittenberg , D-06120 Halle/Saale, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Reich
authors.forename: Michael
authors.initials: M
authors.lastname: Kovermann
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
authors.forename: Paul
authors.initials: P
authors.lastname: Knick
authors.forename: René
authors.initials: R
authors.lastname: Geissler
authors.forename: Ralph Peter
authors.initials: RP
authors.lastname: Golbik
authors.forename: Jochen
authors.initials: J
authors.lastname: Balbach
authors.forename: Sven-Erik
authors.initials: SE
authors.lastname: Behrens
mesh_terms: D016174:Hepacivirus / Q000201:enzymology*; D055029:Inverted Repeat Sequences; D011485:Protein Binding; D059370:RNA Folding; D012330:RNA, Double-Stranded / Q000737:chemistry; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry*; D013816:Thermodynamics; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D014779:Virus Replication
chemical_list: D012330:RNA, Double-Stranded; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase