Further insights into the roles of GTP and the C terminus of the hepatitis C virus polymerase in the initiation of RNA synthesis.
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.
Harrus
Déborah
D
From the Laboratoire de Virologie Moléculaire et Structurale, CNRS UPR3296, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
Ahmed-El-Sayed
Neveen
N
CNRS UMR 52342, IFR66, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.
Simister
Philip C
PC
From the Laboratoire de Virologie Moléculaire et Structurale, CNRS UPR3296, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
Miller
Steve
S
University of Kansas Medical Center, Kansas City, Kansas 66160.
Triconnet
Martine
M
From the Laboratoire de Virologie Moléculaire et Structurale, CNRS UPR3296, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
Hagedorn
Curt H
CH
University of Kansas Medical Center, Kansas City, Kansas 66160.
Mahias
Kathleen
K
CNRS UMR 52342, IFR66, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.
Rey
Félix A
FA
From the Laboratoire de Virologie Moléculaire et Structurale, CNRS UPR3296, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
Astier-Gin
Thérèse
T
CNRS UMR 52342, IFR66, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.
Bressanelli
Stéphane
S
From the Laboratoire de Virologie Moléculaire et Structurale, CNRS UPR3296, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. Electronic address: stephane.bressanelli@vms.cnrs-gif.fr.
D018360:Crystallography, X-Ray; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology; D012324:RNA-Dependent RNA Polymerase
D006160:Guanosine Triphosphate; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase