Production of Two Phosphoproteins from the NS5A Region of the Hepatitis C Viral Genome

Kaneko, Toshinobu; Tanji, Yasunori; Satoh, Shinya; Hijikata, Makoto; Asabe, Shinichi; Kimura, Kôichi; Shimotohno, Kunitada

doi:10.1006/bbrc.1994.2667

Cited by 204 publications

(183 citation statements)

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“…GST fusion proteins were purified and coprecipitation assay was performed as described (24). Whole-cell lysates (0.5 mg) or purified recombinant NS5A (5 ng) were Amino acid (a.a.) sequence alignment of NS5A from various HCV species revealed conservation (residues enclosed in boxes) of both class I and II proline-rich motifs (26). P denotes the highly conserved proline residue.…”

Section: Methodsmentioning

confidence: 99%

“…Several other nonsignaling SH2͞SH3-related proteins were also examined, but none demonstrated an interaction with NS5A (data not shown). The observed doublet band might represent basally and hyperphosphorylated forms of NS5A (26)(27)(28) or might be caused by other posttranslational modifications. Western blot analysis by using an anti-GST antibody indicated that all GST fusions were expressed efficiently (Fig.2 A Lower).…”

Section: Ns5amentioning

confidence: 99%

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NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling

Tan

Nakao

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

215

200

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Although hepatitis C virus (HCV) infection is an emerging global epidemic causing severe liver disorders, the molecular mechanisms of HCV pathogenesis remain elusive. The NS5A nonstructural protein of HCV contains several proline-rich sequences consistent with Src homology (SH) 3-binding sites found in cellular signaling molecules. Here, we demonstrate that NS5A specifically bound to growth factor receptor-bound protein 2 (Grb2) adaptor protein. Immunoblot analysis of anti-Grb2 immune complexes derived from HeLa S3 cells infected with a recombinant vaccinia virus (VV) expressing NS5A revealed an interaction between NS5A and Grb2 in vivo. An inactivating point mutation in the N-terminal SH3 domain, but not in the C-terminal SH3 domain, of Grb2 displayed significant diminished binding to NS5A. However, the same mutation in both SH3 regions completely abrogated Grb2 binding to NS5A, implying that the two SH3 domains bind in cooperative fashion to NS5A. Further, mutational analysis of NS5A assigned the SH3-binding region to a proline-rich motif that is highly conserved among HCV genotypes. Importantly, phosphorylation of extracellular signalregulated kinases 1 and 2 (ERK1͞2) was inhibited in HeLa S3 cells infected with NS5A-expressing recombinant VV but not recombinant VV control. Additionally, HeLa cells stably expressing NS5A were refractory to ERK1͞2 phosphorylation induced by exogenous epidermal growth factor. Moreover, the coupling of NS5A to Grb2 in these cells was induced by epidermal growth factor stimulation. Therefore, NS5A may function to perturb Grb2-mediated signaling pathways by selectively targeting the adaptor. These findings highlight a viral interceptor of cellular signaling with potential implications for HCV pathogenesis.

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Section: Methodsmentioning

confidence: 99%

Section: Ns5amentioning

confidence: 99%

NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling

Tan

Nakao

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

215

200

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“…It has two major forms, p56 and p58, which differ by the degree of phosphorylation of the serine residues. 135 However, other forms (p50, p53) have also been described in the Huh-7 hepatoma cell line. 136 Though expression of this protein in transgenic mice has not demonstrated direct oncongenicity, 137,138 NS5a has been associated with cell transformation in vitro.…”

Section: Hepatitis C Nonstructural Protein 5amentioning

confidence: 99%

Signal transduction cascades and hepatitis B and C related hepatocellular carcinoma

2006

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“…The NS5A gene of HCV encodes a 447 aa phosphoprotein (aa 1973 to 2419 of the prototype HCV type 1b, HCV-J (Kato et al, 1990)), which has differently phosphorylated forms of 56 and 58 kDa with modified serine residues (Kaneko et al, 1994). NS5A appears to play an important role in regulating HCV replication, although its precise function remains largely unclear.…”

Section: Introductionmentioning

confidence: 99%

HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis

Sheu²,

et al. 2002

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Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis and hepatocellular carcinoma. HCV NS5A, one of non-structural proteins of HCV, was suggested to play a role in oncogenic transformation. Since the tumor suppressor p53 is important for preventing neoplastic transformation, we investigated the functional effects of HCV NS5A on p53. In vitro and in vivo coimmunoprecipitation and confocal microscopy were used to determine the interaction of NS5A and p53. HCV NS5A binds directly to p53 and colocalizes p53 in the perinuclear region. NS5A inhibits transcriptional transactivation by p53 in a dose-dependent manner by use of a reporter assay. Down regulation of endogenous p21/waf1 expression, which is activated by p53 in Hep3B cells, by NS5A was demonstrated by using FLAG-and FLAG-NS5A Hep3B stable cell lines. The effect of NS5A on p53-mediated apoptosis was determined by flow cytometry in both NS5A permanently and transiently transfected Hep3B cells with exogenous p53. The p53-induced apoptosis was abrogated by NS5A and the inhibition effect correlates well with the binding ability of NS5A to p53. In addition, HCV NS5A protein interacts with and colocalizes hTAF II 32, a component of TFIID and an essential coactivator of p53, in vivo. These results suggest that HCV NS5A interacts with and partially sequestrates p53 and hTAF II 32 in the cytoplasm and suppresses p53-mediated transcriptional transactivation and apoptosis during HCV infection, which may contribute to the hepatocarcinogenesis of HCV infection.

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Production of Two Phosphoproteins from the NS5A Region of the Hepatitis C Viral Genome

Cited by 204 publications

References 0 publications

NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling

NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling

Signal transduction cascades and hepatitis B and C related hepatocellular carcinoma

HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis

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