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

Shota Yamauchi,Kenji Takeuchi,Kazuyasu Chihara,Xuedong Sun,Chisato Honjoh,Hatsumi Yoshiki,Hak Hotta,Kiyonao Sada

doi:10.1074/jbc.m115.666859

Abstract

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr(330) (Tyr(2306) in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr(330).

Highlights

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) regulates viral RNA replication and virus particle assembly
Results c-Abl Is Involved in HCV Particle Assembly—Treatment with the Abl inhibitor imatinib prevents the spread of HCV infection in cell culture [36] but does not appear to affect virus entry [42]
To determine which step in the HCV life cycle is blocked by treatment with imatinib, Huh-7.5 cells were infected with cell culture-grown HCV (HCVcc) of the J6/JFH1 chimeric genome [37, 40] and incubated in the presence or absence of imatinib

Summary

Introduction

Background: HCV NS5A regulates viral RNA replication and virus particle assembly. Results: Phosphorylation of NS5A by c-Abl is required for efficient production of infectious HCV particles but not for viral RNA replication. Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. We show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase.

Results

Conclusion