A single-amino-acid mutation in hepatitis C virus NS5A disrupts physical and functional interaction with the transcription factor HNF-1α

Matsui, Chieko; Sianipar, Imelda Rosalyn; Minami, Nanae; Deng, Lin; Hotta, Hak; Shoji, Ikuo

doi:10.1099/vir.0.000179

Cited by 8 publications

(17 citation statements)

References 20 publications

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“…Immunoprecipitation and immunoblot analyses were performed as described previously . The primary antibodies used were: mouse monoclonal antibodies against HCV NS3, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Millipore), Bim (H-5, Santa Cruz Biotechnology), Tim23 and Bax (BD Biosciences); rabbit polyclonal antibodies against PARP, btubulin (Cell Signaling Technology) and HCV NS5A (Matsui et al, 2015); and rabbit monoclonal antibody against Bim (Cell Signaling Technology). Normal rabbit IgG served as a control.…”

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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“…Immunoprecipitation analysis revealed that the mutant NS5AV121A(1-126) and NS5AV124A (1-126) failed to interact with FLAG-OTUD7B. We recently reported that the mutant NS5A V121A has lost the interaction with HNF-1a and that the NS5A-HNF-1a interaction is crucial for degradation of HNF-1a (22). Okamoto et al have reported that NS5A Val 121 is important for the interaction with FKBP8 and crucial for HCV replication (31).…”

Section: Discussionmentioning

confidence: 95%

“…We also determined the specific aa residues of NS5A required for the interaction with OTUD7B. Immunoprecipitation analysis revealed that the mutant NS5AV121A and NS5AV124A failed to interact with FLAG‐OTUD7B. We recently reported that the mutant NS5A V121A has lost the interaction with HNF‐1α and that the NS5A‐ HNF‐1α interaction is crucial for degradation of HNF‐1α .…”

Section: Discussionmentioning

confidence: 99%

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Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B

Sianipar

Matsui

Minami

et al. 2015

Microbiology and Immunology

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Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin-proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A-binding protein. Co-immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh-7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B-binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A-OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.

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“…HCV NS5A, a proline-rich, hydrophilic phosphoprotein, functions as a key regulator of HCV RNA replication and assembly (27). NS5A can interact with a wide variety of host cell proteins, including hepatocyte nuclear factor 1a, apolipoprotein A1, 2 0 -5 0 oligoadenylate synthase, La, heat shock protein 27, PKR, FK506-binding protein and ovarian tumor protein 7B (25,26,(28)(29)(30)(31)(32)(33)(34). Here, we demonstrated a specific interaction between unconjugated ISG15 and NS5A via domain I of NS5A (Fig.…”

Section: Hepatitis C Virus Infection Triggers Intrahepatic Synthesis mentioning

confidence: 99%

Unconjugated interferon‐stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I

Minami

Abe

Deng

et al. 2017

Microbiology and Immunology

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Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is induced by type I INF. Although several groups have reported ISGylation of the HCV NS5A protein, it is still unclear whether ISGylation of NS5A has anti- or pro-viral effects in hepatitis C virus (HCV) infection. In the present study, the role of ISGylation-independent, unconjugated ISG15 in HCV infection was examined. Immunoprecipitation analyses revealed that ISG15 interacts specifically with NS5A domain I. ISG15 mutants lacking the C-terminal glycine residue that is essential for ISGylation still interacted with NS5A protein. Taken together, these results suggest that unconjugated ISG15 affects the functions of HCV NS5A through protein-protein interaction.

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A single-amino-acid mutation in hepatitis C virus NS5A disrupts physical and functional interaction with the transcription factor HNF-1α

Cited by 8 publications

References 20 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

Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B

Unconjugated interferon‐stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I

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