Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 291, Issue 8, Pages 3918-3931Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M115.675413
Keywords
antibody; Hepatitis C virus (HCV); phosphoproteomics; phosphorylation; proteomics; casein kinase; serine 235
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Funding
- National Health Research Institutes [NHRI-EX104-10213BI]
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The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase I (CKI) directly phosphorylated Ser-235 in vitro. Inhibition of CKI reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKI probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein.
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