Identification of a novel phosphorylation site in hepatitis C virus NS5A.
Hepatitis C virus (HCV) NS5A protein is phosphorylated on multiple residues; however, despite extensive study, the precise identity of these sites has not been determined unambiguously. In this study, we have used a combination of immunoprecipitation and mass spectrometry to identify these phosphorylation sites. This analysis revealed the presence of a major phosphorylated residue within NS5A from the genotype 1b Con1 isolate - serine 249 (serine 2221 in polyprotein numbering). However, mutation of this residue (or the corresponding threonine in the JFH-1 isolate) to either a phosphomimetic (aspartate) or a phosphoablative (alanine) residue resulted in no phenotype. We conclude that phosphorylation of this residue, in the context of a highly culture-adapted HCV genome, does not play a role in either viral RNA replication or virus assembly. It is possible that it might be important in an aspect of virus biology that is not recapitulated faithfully in the Huh-7 cell-culture system.
Nordle Gilliver
Anna
A
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Griffin
Stephen
S
Harris
Mark
M
D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D012694:Serine; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase