Open AccessRegulation of De Novo -Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions
Author(s) -
Sreedhar Chinnaswamy,
Ayaluru Murali,
P. Li,
Koki Fujisaki,
C. Cheng Kao
Publication year - 2010
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.02446-09
Subject(s) - ns5b , biology , rna dependent rna polymerase , rna polymerase , polymerase , rna , primer extension , hepatitis c virus , de novo synthesis , rna polymerase i , microbiology and biotechnology , virology , biochemistry , enzyme , virus , gene , hepacivirus
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins.In vitro , the RdRp can initiatede novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Δ1 loop and thumb domain in NS5B are required forde novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affectsde novo initiation. We have shown that an increasing enzyme concentration increasesde novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increasede novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulatede novo initiation. Our results support a model in which thede novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of ade novo initiation-competent HCV RdRp.