Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk.
Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-gamma1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities.
Inubushi
Sachiko
S
Division of Microbiology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Nagano-Fujii
Motoko
M
Division of Microbiology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Kitayama
Kikumi
K
Division of Microbiology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Tanaka
Motofumi
M
Division of Microbiology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
An
Chunying
C
Division of Microbiology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Yokozaki
Hiroshi
H
Division of Surgical Pathology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Yamamura
Hirohei
H
Hyogo Laboratory, Hyogo Prefectural Institute of Public Health and Environmental Sciences, Kobe 652-0032, Japan.
Nuriya
Hideko
H
Department of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan.
Kohara
Michinori
M
Department of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan.
Sada
Kiyonao
K
Division of Microbiology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Hotta
Hak
H
Division of Microbiology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D015536:Down-Regulation; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000737:chemistry / Q000821:virology; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D017384:Sequence Deletion; D000072377:Syk Kinase; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D047908:Intracellular Signaling Peptides and Proteins; D051966:Phospholipase C gamma; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; D017361:Viral Nonstructural Proteins; C000606896:SYK protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase