Research Article
Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A

https://doi.org/10.1016/j.jbc.2022.101804Get rights and content
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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.

Keywords

protein tyrosine kinase
phosphotyrosine
host factors against virus infection
assembly of infectious virus particle
Abl tyrosine kinase
hepatitis C virus
viral protein
nonstructural protein 5A

Abbreviations

Abl
Abelson
Abl
Abl-deficient cell
AFK
Abl family kinase
cDNA
complementary DNA
DAA
direct-acting antiviral
DOC
sodium deoxycholate
DSL
detergent-soluble lysate
edR
editing-resistant
ER
endoplasmic reticulum
HCV
hepatitis C virus
HCVcc
cell culture–adapted HCV
HEK 293T
human embryonic kidney 293T cell line
IgG1
immunoglobulin G1
KD
kinase-dead
mAb
monoclonal antibody
NS5A
nonstructural protein 5A
pTyr
phosphotyrosine
SH3
Src homology 3
TCID50
median tissue culture infectious dose

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