Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk

Inubushi, Sachiko; Nagano-Fujii, Motoko; Kitayama, Kikumi; Tanaka, Motofumi; An, Chunying; Yokozaki, Hiroshi; Yamamura, Hirohei; Nuriya, Hideko; Kohara, Michinori; Sada, Kiyonao; Hotta, Hak

doi:10.1099/vir.0.83510-0

Cited by 24 publications

(32 citation statements)

References 63 publications

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“…Human adult liver autopsy materials and surgically resected liver tissue of patients with or without HCV infection were obtained with written informed consent. Immunohistochemical staining was performed as described previously (Inubushi et al, 2008;Kasai et al, 2009). The primary antibodies used were rabbit antibodies against Bim or cleaved caspase 3 (Cell Signaling Technology).…”

Section: Methodsmentioning

confidence: 99%

HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis

Deng

Chen

Tanaka

et al. 2015

Journal of General Virology

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We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the proapoptotic protein Bim and the protein expression of three major splice variants of Bim (Bim EL , Bim L and Bim S ). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.

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Section: Methodsmentioning

confidence: 99%

HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis

Deng

Chen

Tanaka

et al. 2015

Journal of General Virology

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“…The VAP-A expression vector, pEF-FLAG-VAP-A, was kindly provided by Y. Matsuura (17). Generation of NS5A deletion mutants has been described previously (20). The NS5A derived from HCV JFH1 (genotype 2a) was cloned in the pEF1/Myc-His vector (Invitrogen, Carlsbad, CA) at the KpnI and XbaI sites by using a PCR-amplified fragment using primers described in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

Role of Oxysterol Binding Protein in Hepatitis C Virus infection

Amako

Sarkeshik

Hotta

et al. 2009

J Virol

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Hepatitis C virus (HCV) RNA genome replicates within the ribonucleoprotein (RNP) complex in the modified membranous structures extended from endoplasmic reticulum. A proteomic analysis of HCV RNP complexes revealed the association of oxysterol binding protein (OSBP) as one of the components of these complexes. OSBP interacted with the N-terminal domain I of the HCV NS5A protein and colocalized to the Golgi compartment with NS5A. An OSBP-specific short hairpin RNA that partially downregulated OSBP expression resulted in a decrease of the HCV particle release in culture supernatant with little effect on viral RNA replication. The pleckstrin homology (PH) domain located in the N-terminal region of OSBP targeted this protein to the Golgi apparatus. OSBP deletion mutation in the PH (⌬PH) domain failed to localize to the Golgi apparatus and inhibited the HCV particle release. These studies suggest a possible functional role of OSBP in the HCV maturation process.Hepatitis C virus (HCV) infection is one of the leading causes of chronic hepatitis. HCV infection is associated with cirrhosis, steatosis, and hepatocellular carcinoma (33). The HCV RNA genome of ϳ9.6 kb is translated via an internal ribosome entry site element on the rough endoplasmic reticulum (ER) as a polyprotein precursor of about 3,010 amino acids that is co-and posttranslationally processed by cellular and viral proteases into mature structural and nonstructural (NS) proteins (33). HCV replicates within ribonucleoprotein (RNP) complexes associated with modified ER membranous structures (15). Recent work implicated lipid droplets that emanate from the ER as sites of RNA replication (28,44). Almost all of the HCV NS proteins along with a variety of cellular factors are associated with the RNP complexes engaged in viral RNA replication (37). It is likely that these NS proteins not only participate in replication process but also are involved in the various steps of virion morphogenesis and assembly. Membrane-associated RNP complexes are generally composed of viral proteins, replicating RNA, host proteins, and altered cellular membranes (1). In this respect, a growing body of evidence implicates the functional role of NS5A in early steps of virion assembly and morphogenesis (3,27,45). NS5A is a phosphoprotein that migrates in sodium dodecyl sulfate gels as 56-kDa (basally phosphorylated) and 58-kDa (hyperphosphorylated) forms of proteins. The C-terminal domain III region of NS5A and the phosphorylated residue (Ser 457 ) are important for virion maturation (3, 27, 45). NS5A domain III contains the binding site for viral core protein, indicating the possible involvement of NS5A protein in virus assembly (27). NS5A anchors to the ER membrane by an N-terminal hydrophobic ␣-helix, and this attachment is needed for its key role(s) in viral replication (10). Studies suggest that phosphorylation of NS5A plays a functional role in viral replication (12). The hyperphosphorylated NS5A reduces its interaction with the human vesicle-associated membrane protein-associate...

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“…To express a series of NS5A deletion mutants as hemagglutinin (HA)-tagged proteins, each fragment was amplified by PCR and cloned into the NotI site of pCAG-HA. pEF1A-NS5A (Con1)-myc-His was used as a template (18). The primer sequences used in this study are available from the authors upon request.…”

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confidence: 99%

“…Expression plasmids for core protein, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B were described previously (9,10,18). To express E1 and E2 (E1/E2), the cDNA fragment of nucleotides (nt) 825 to 2676 derived from the HCV Con1 strain was amplified by PCR using the plasmid pFKI389neo/core-3=/Con1 (a kind gift from R. Bartenschlager) as a template.…”

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confidence: 99%

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Hepatitis C Virus Infection Suppresses GLUT2 Gene Expression via Downregulation of Hepatocyte Nuclear Factor 1α

Matsui

Shoji

Kaneda

et al. 2012

J Virol

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Hepatitis C virus (HCV) is the main cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV is a single-stranded, positive-sense RNA virus that is classified into the Flaviviridae family, Hepacivirus genus (21). More than 170 million people worldwide are chronically infected with HCV. The 9.6-kb HCV genome encodes a polyprotein of approximately 3,010 amino acids (aa). The polyprotein is cleaved co-and posttranslationally into at least 10 proteins by viral proteases and cellular signalases: the structural proteins core, E1, E2, and p7 and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (21).Persistent HCV infection causes not only intrahepatic diseases but also extrahepatic manifestations, such as type 2 diabetes. Clinical and experimental data suggest that HCV infection is an additional risk factor for the development of diabetes (26,29,30). HCV-related glucose metabolic changes and insulin resistance have significant clinical consequences, such as accelerated fibrogenesis, reduced virological response to alpha interferon (IFN-␣)-based therapy, and increased incidence of hepatocellular carcinoma (29). Therefore, the molecular mechanism of HCV-related diabetes needs to be clarified.We have sought to identify a novel mechanism of HCV-induced diabetes. We previously demonstrated that HCV suppresses hepatocytic glucose uptake through downregulation of cell surface expression of glucose transporter 2 (GLUT2) in a human hepatoma cell line (19). The uptake of glucose into cells is conducted by facilitative glucose carriers, i.e., glucose transporters (GLUTs). GLUTs are integral membrane proteins that contain 12 membrane-spanning helices. To date, a total of 14 isoforms have been identified in the GLUT family (24). GLUT2 is expressed in the liver, pancreatic ␤-cells, hypothalamic glial cells, retina, and enterocytes. Glucose is transported into hepatocytes by GLUT2 (34). We previously reported that GLUT2 expression was reduced in hepatocytes obtained from HCV-infected patients (19). We also demonstrated that GLUT2 mRNA levels were lower in HCV replicon cells and in HCV J6/JFH1-infected cells than in the control cells. GLUT2 promoter activity was suppressed in HCV-replicating cells. However, the molecular mechanism of HCV-induced suppression of GLUT2 gene expression remains to be elucidated.In the present study, we aimed to clarify molecular mechanisms of HCV-induced suppression of GLUT2 gene expression. We analyzed transcriptional regulation of the GLUT2 promoter in HCV replicon cells. We demonstrate that HCV infection downregulates hepatocyte nuclear factor 1␣ (HNF-1␣) expression at both transcriptional and posttranslational levels, resulting in suppression of GLUT2 promoter. We propose that HCV-induced downregulation of HNF-1␣ may play a crucial role in glucose metabolic disorders caused by HCV. MATERIALS AND METHODSCell culture. The human hepatoma cell line Huh-7.5 (4) was kindly provided by Charles M. Rice (The Rockefeller University, New York, NY).

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Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk

Cited by 24 publications

References 63 publications

HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis

HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis

Role of Oxysterol Binding Protein in Hepatitis C Virus infection

Hepatitis C Virus Infection Suppresses GLUT2 Gene Expression via Downregulation of Hepatocyte Nuclear Factor 1α

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