Abstract
Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain–derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl−) cells through genome editing and compared HCV production between Abl− cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl–NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.
Highlights
Hepatitis C virus (HCV), an enveloped positive-strand RNA virus and a member of the Flaviviridae family [1–3], is a frequent cause of acute/chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans
We have previously reported that knocking down Abl expression using shRNA or treatment with an Abl inhibitor suppresses HCV replication at the stage of viral particle assembly and that Abl is involved in the tyrosine phosphorylation of nonstructural protein 5A (NS5A) [25]
We found that administration of imatinib reduced viral particle production during the HCV life cycle in cultured hepatocytes infected with HCV (J6/JFH1)
Summary
Hepatitis C virus (HCV), an enveloped positive-strand RNA virus and a member of the Flaviviridae family [1–3], is a frequent cause of acute/chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. HCV binds to the target cell membrane and enters the cell via clathrin-mediated endocytosis [4, 5], where the viral particle releases genomic RNA, which is translated to a polyprotein of 3010 to 3014. The RNA replication complex synthesizes positive-strand RNA, which is assembled with structural proteins into a virus particle through budding into the ER lumen [1, 3]. NS5A is believed to be involved in HCV RNA replication and infectious virus particle formation and assembly [3]. Phosphorylation of Tyr in domain I is important for formation of the viral genome replication complex, and Src, a nonreceptor tyrosine kinase, is involved in this phosphorylation [18]. The N terminus of Abl contains a cap region (in the case of variant b of Abl, a myristoyl chain), which maintains the kinase in a locked and inactive conformation; the Src homology 3 (SH3) domain, SH2 domain, and catalytic tyrosine kinase domain (SH1). The presence of the myristate-binding protein, SH3-binding protein, SH2-binding protein, and Tyr412 phosphorylation in the activation loop of the kinase domain induces a change into the active “unclamped” conformation of Abl [21, 23, 24]
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