Abstract
The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and the regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A. We previously identified SRC proto-oncogene, nonreceptor tyrosine kinase (c-Src), as an essential host component of the HCV replication complex consisting of NS5A, the RNA-dependent RNA polymerase NS5B, and c-Src. Pulldown assays revealed an interaction between NS5A and the Src homology 2 (SH2) domain of c-Src; however, the precise binding mode remains undefined. In this study, using a variety of biochemical and biophysical techniques, along with molecular dynamics simulations, we demonstrate that the interaction between NS5A and the c-Src SH2 domain strictly depends on an intact phosphotyrosine-binding competent SH2 domain and on tyrosine phosphorylation within NS5A. Detailed analysis of c-Src SH2 domain binding to a panel of phosphorylation-deficient NS5A variants revealed that phosphorylation of Tyr-93 located within domain 1 of NS5A, but not of any other tyrosine residue, is crucial for complex formation. In line with these findings, effective replication of subgenomic HCV replicons as well as production of infectious virus particles in mammalian cell culture models were clearly dependent on the presence of tyrosine at position 93 of NS5A. These findings indicate that phosphorylated Tyr-93 in NS5A plays an important role during viral replication by facilitating NS5A's interaction with the SH2 domain of c-Src.
Highlights
The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and the regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A
The recombinantly produced family tyrosine kinase (Fyn) Src homology 2 (SH2) domain was included as a control because previous studies have shown that NS5A expressed in B-lymphocytes binds to recombinant Fyn–SH2 in a pulldown assay [33]
NS5A exists in a basally phosphorylated (p56) and a hyperphosphorylated (p58) form, and the phosphorylation state regulates the various functions of NS5A, which have to occur in a concerted manner during the life cycle of HCV
Summary
The hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and the regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A. Detailed analysis of c-Src SH2 domain binding to a panel of phosphorylation-deficient NS5A variants revealed that phosphorylation of Tyr-93 located within domain 1 of NS5A, but not of any other tyrosine residue, is crucial for complex formation In line with these findings, effective replication of subgenomic HCV replicons as well as production of infectious virus particles in mammalian cell culture models were clearly dependent on the presence of tyrosine at position 93 of NS5A. These findings indicate that phosphorylated Tyr-93 in NS5A plays an important role during viral replication by facilitating NS5A’s interaction with the SH2 domain of c-Src. The hepatitis C virus (HCV) is one of the leading causes for chronic liver diseases worldwide and accounts for about 30% of. NS5A is an oligomeric, multifunctional RNA-binding phosphoprotein, which is composed of an amphipathic N-terminal isopropyl -D-1-thiogalactopyranoside; LCS, low-complexity sequence; MD, molecular dynamics; NS, nonstructural; NVT, constant number of particles, volume, and temperature; OV, orthovanadate; rtPCR, real-time PCR; SDHA, succinate dehydrogenase complex, subunit A; SEC, size exclusion chromatography; SH, Src-homology; RMSD, root-mean-square deviation; PDB, Protein Data Bank; RMSF, root-mean-square fluctuation; EDC, 1-ethyl3-(dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide; Fyn, FYN proto-oncogene, Src family tyrosine kinase; c-Abl, ABL proto-oncogene 1, non-receptor tyrosine kinase; ELK, EPH receptor B1
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