Abstract
The hepatitis C virus (HCV) NS5A protein is phosphorylated by a cellular, serine/threonine kinase. To identify the major site(s) of NS5A phosphorylation, radiolabeled HCV-H NS5A phosphopeptides were purified and subjected to phosphoamino acid analysis and Edman degradation. These data identified the major intracellular phosphorylation site in the HCV-H NS5A protein as Ser(2321), a result verified by two additional, independent methods: (i) substitution of Ala for Ser(2321) and the concomitant disappearance of the major in vivo phosphorylated peptides and corresponding in vitro phosphorylated peptides; and (ii) comigration of the digestion products of a synthetic peptide phosphorylated on Ser(2321) with the major in vivo phosphorylated NS5A peptides. Site-directed mutagenesis of Ser(2321) suggested that phosphorylation of NS5A is dispensable for previously described interactions with NS4A and PKR, a cellular, antiviral kinase that does not appear to catalyze NS5A phosphorylation. The proline-rich nature of the amino acid sequence flanking Ser(2321) (PLPPPRS(2321) PPVPPPR) suggests that a proline-directed kinase is responsible for the majority of HCV NS5A phosphorylation, consistent with previous kinase inhibitor studies.
Highlights
The hepatitis C virus (HCV)1 nonstructural protein NS5A is phosphorylated primarily on serine residues [1, 2] by an unidentified cellular kinase
To identify the major NS5A phosphorylation sites unequivocally, total phosphorylated NS5A was digested with trypsin and chymotrypsin, and phosphopeptides that contained the majority of incorporated 32P when NS5A was phosphorylated in vivo were analyzed by Edman degradation
Identification of the Major HCV-H NS5A Phosphorylation Site as Ser2321 by Edman Degradation—To obtain direct evidence for the location of HCV NS5A phosphorylation sites, total phosphorylated NS5A was digested with trypsin and chymotrypsin, and the resulting peptides were resolved by a combination of reverse-phase high performance liquid chromatography (HPLC) and two-dimensional separation on thin layer cellulose plates
Summary
Plasmid Constructs—To introduce single S2118A, S2179A, S2210A, or S2321A mutations or a double S2201A/S2204A mutation into pTM3/ HCV 5A [2], polymerase chain reactions (PCRs) were first performed with one of the following mutant primers and an HCV NS5A N-terminal: (CMBL 476698; 5Ј-CGCCATGGGAGCCGGCTCCTGGCTA-3Ј) or C-terminal (CMBL 452810; 5Ј-GGCTCGAGCTAGCAGCACACGACATC-3Ј) primer: CMR 657 (5Ј-GAAAAATTCGGGCGccGGGATCTGGCAC-3Ј; S2118A), CMR 650 (GTTGACGTCCATGCTCACTGATCCCgCCCATATAACA-3Ј; S2179A), CMR 649 (5Ј-CTATGGCCAGCgCCTCGGCCgcCCAGCTGTCCGC-3Ј; S2201A/S2204A), CMR 651 (5Ј-AGCCAGCTGTCCGCTCCAgCTCTCAAGGCA-3Ј; S2210A), and CMR 648
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