Further Insights into the Roles of GTP and the C Terminus of the
Hepatitis C Virus Polymerase in the Initiation of RNA Synthesis |
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Authors: | Déborah Harrus Neveen Ahmed-El-Sayed Philip C Simister Steve Miller Martine Triconnet Curt H Hagedorn Kathleen Mahias Félix A Rey Thérèse Astier-Gin Stéphane Bressanelli |
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Institution: | From the ‡Laboratoire de Virologie Moléculaire et Structurale, CNRS UPR3296, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.;§CNRS UMR 52342, IFR66, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France, and ;the ¶University of Kansas Medical Center, Kansas City, Kansas 66160 |
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Abstract: | The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase
essential for replication of the viral RNA genome. In vitro and
presumably in vivo, NS5b initiates RNA synthesis by a
de novo mechanism. Different structural elements of NS5b
have been reported to participate in RNA synthesis, especially a so-called
“β-flap” and a C-terminal segment (designated
“linker”) that connects the catalytic core of NS5b to a
transmembrane anchor. High concentrations of GTP have also been shown to
stimulate de novo RNA synthesis by HCV NS5b. Here we describe a
combined structural and functional analysis of genotype 1 HCV-NS5b of strains
H77 (subtype 1a), for which no structure has been previously reported, and J4
(subtype 1b). Our results highlight the linker as directly involved in lifting
the first boundary to processive RNA synthesis, the formation of the first
dinucleotide primer. The transition from this first dinucleotide primer state to
processive RNA synthesis requires removal of the linker and of the
β-flap with which it is shown to strongly interact in crystal
structures of HCV NS5b. We find that GTP specifically stimulates this transition
irrespective of its incorporation in neosynthesized RNA. |
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Keywords: | Crystal Structure Hepatitis Virus Positive-strand RNA Viruses RNA Polymerase RNA Synthesis |
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