HCV NS5A Protein Containing Potential Ligands for Both Src Homology 2 and 3 Domains Enhances Autophosphorylation of Src Family Kinase Fyn in B Cells

Abstract

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg176 to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr334 was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase

“…A recent study has shown that casein kinase I is also involved in HCV particle assembly through NS5A hyperphosphorylation (25). In addi-tion to serine and threonine phosphorylation, we have reported that overexpressed NS5A is tyrosine-phosphorylated in response to treatment with pervanadate, an inhibitor of protein tyrosine phosphatases (26). Moreover, we and others have reported that NS5A binds to non-receptor protein-tyrosine kinases such as c-Src, Fyn, Syk, and c-Abl in vitro (26 -28).…”

“…-We have previously reported that the SH3 domain of c-Abl binds to NS5A in vitro and that overexpressed NS5A is tyrosine-phosphorylated in response to pervanadate treatment (26). We therefore investigated the possibility that c-Abl promotes HCV particle assembly by phosphorylating NS5A.…”

An integrative approach for a network based meta-analysis of viral RNAi screens

“…c-Yes, a member of this family, is required for transportation of virions through the secretory pathway [ 76 ]. Several of the Src kinase family members are activated by HIV Nef [ 77 ], and also HCV NS5A induces phosphorylation events in the Src family [ 78 - 80 ].…”

Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression in U937 cells

“…In some experiments, prepared cell lysates were subjected to pull-down assays for analysing tyrosine phosphorylation of 3BP2 induced by the cross-linking of FcγRI. Pull-down assays using GST-Abl-SH3 (residues between 84 to 138 of mouse c-Abl) were performed as described previously 14 , 56 .…”