Document Detail


Two crucial early steps in RNA synthesis by the hepatitis C virus polymerase involve a dual role of residue 405.
MedLine Citation:
PMID:  22532694     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 and then processively copies the whole RNA template. Dissections of de novo RNA synthesis by genotype 1 NS5B proteins previously established that there are two successive crucial steps in de novo initiation. The first is dinucleotide formation, which requires a closed conformation, and the second is the transition to elongation, which requires an opening of NS5B. We also recently published a combined structural and functional analysis of genotype 2 HCV-NS5B proteins (of strains JFH1 and J6) that established residue 405 as a key element in de novo RNA synthesis (P. Simister et al., J. Virol. 83:11926-11939, 2009; M. Schmitt et al., J. Virol 85:2565-2581, 2011). We hypothesized that this residue stabilizes a particularly closed conformation conducive to dinucleotide formation. Here we report similar in vitro dissections of de novo synthesis for J6 and JFH1 NS5B proteins, as well as for mutants at position 405 of several genotype 1 and 2 strains. Our results show that an isoleucine at position 405 can promote both dinucleotide formation and the transition to elongation. New structural results highlight a molecular switch of position 405 with long-range effects, resolving the implied paradox of how the same residue can successively favor both the closed conformation of the dinucleotide formation step and the opening necessary to the transition step.
Authors:
Nathalie Scrima; Célia Caillet-Saguy; Michel Ventura; Déborah Harrus; Thérèse Astier-Gin; Stéphane Bressanelli
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-04-24
Journal Detail:
Title:  Journal of virology     Volume:  86     ISSN:  1098-5514     ISO Abbreviation:  J. Virol.     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-06-08     Completed Date:  2012-08-15     Revised Date:  2013-06-25    
Medline Journal Info:
Nlm Unique ID:  0113724     Medline TA:  J Virol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  7107-17     Citation Subset:  IM    
Affiliation:
Laboratoire de Virologie Moléculaire et Structurale, CNRS UPR 3296, Gif-sur-Yvette, France.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Substitution
Crystallography, X-Ray
Hepacivirus / enzymology*,  physiology*
Humans
Models, Molecular
Mutagenesis, Site-Directed
Mutant Proteins / chemistry,  genetics,  metabolism
Mutation, Missense
Protein Conformation
RNA Replicase / chemistry,  genetics,  metabolism*
RNA, Viral / biosynthesis*
Viral Nonstructural Proteins / chemistry,  genetics,  metabolism*
Virus Replication*
Chemical
Reg. No./Substance:
0/Mutant Proteins; 0/NS-5 protein, hepatitis C virus; 0/RNA, Viral; 0/Viral Nonstructural Proteins; EC 2.7.7.48/RNA Replicase
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