Hepatitis C Virus NS5A Physically Associates with p53 and Regulates p21/waf1 Gene Expression in a p53-Dependent Manner

Majumder, Mainak; Ghosh, Asish K.; Steele, Robert; Ray, Ranjit; Ray, Ratna B.

doi:10.1128/jvi.75.3.1401-1407.2001

Cited by 236 publications

(181 citation statements)

References 41 publications

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“…Consistent with our results using NS5A from HCV genotype 1b, Majumder et al (2001) showed that NS5A from HCV genotype 1a also physically associates with p53 and down-regulates p21/waf1 gene expression in a p53 dependent manner just before our submission. By using the mammalian two-hybrid system, they showed that the p53-interacting domain of NS5A is located within the first 150 amino acid residues.…”

Section: Discussionsupporting

confidence: 79%

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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Section: Discussionsupporting

confidence: 79%

HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis

Sheu²,

et al. 2002

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“…Besides HCV core protein, other two HCV-encoded viral products, nonstructural protein 3 (NS3; Ishido and Hotta, 1998) and nonstructural protein 5A (NS5A; Majumder et al, 2001;Lan et al, 2002), have been recently demonstrated capable of interacting with the C-terminus of p53 and thereby deregulate p53 normal functions. Both NS3 and NS5 viral proteins, when being overexpressed in cells, cause the downregulation of p53-dependent transactivational activity.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, this may also be the case for HCV. Previous studies indicate that of 10 viral proteins encoded by the HCV genome, HCV core protein, and nonstructural protein NS3 and NS5A physically interact with p53, albeit their effects on p53 transcriptional activity vary and are not conclusive yet (Ishido and Hotta, 1998;Ray et al, 1998;Lu et al, 1999;Otsuka et al, 2000;Arima et al, 2001;Kwun et al, 2001;Majumder et al, 2001). In the case of HCV core protein, the evidence of in vivo association and cellular localization is not available.…”

Section: Introductionmentioning

confidence: 99%

Modulation of p53 transcription regulatory activity and post-translational modification by hepatitis C virus core protein

Kao

Chen

Chen³

et al. 2004

Oncogene

115

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Oncogenic virus proteins often target to tumor suppressor p53 during virus life cycle. In the case of hepatitis C virus (HCV) core protein, it has been shown to affect p53-dependent transcription. Here, we further characterized the in vitro and in vivo interactions between HCV core protein and p53 and showed that these two proteins colocalized in subnuclear granular structures and the perinuclear area. By use of a reporter assay, we observed that while low level of HCV core protein enhanced the transactivational activity of p53, high level of HCV core protein inhibited this activity. In both cases, however, HCV core protein increased the p53 DNA-binding affinity in gel retardation analyses, likely due to the hyperacetylation of p53 Lys 373 and Lys 382 residues. Additionally, HCV core protein, depending on its expression level, had differential effects on the Ser 15 phosphorylation of p53. Moreover, HCV core protein could rescue p53-mediated suppressive effects on both RNA polymerase I and III transcriptions. Collectively, our results indicate that HCV core protein targets to p53 pathway via at least three means: physical interaction, modulation of p53 gene regulatory activity and post-translational modification. This feature of HCV core protein, may potentially contribute to the HCV-associated pathogenesis.

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“…As a hot spot for highly adaptive mutations, NS5A is an attractive target for initial examination. Although the role of NS5A in the HCV life cycle is unknown, many NS5A interactors have been reported, including RNA-regulated protein kinase (16, 17), apolipoproteins (18), p53 (19,20), Grb2 (21,22), and amphiphysin II (23), but none have been shown to have a defined role in the viral life cycle.One previously reported NS5A-interacting protein is human vesicle-associated membrane protein-associated protein A (hVAP-A), a widely expressed, endoplasmic reticulum͞Golgi-localized protein involved in intracellular vesicle trafficking. This interaction was originally identified in a yeast two-hybrid screen with NS5A as bait and further validated in biochemical and immunofluorescence colocalization experiments (24).…”

mentioning

confidence: 99%

Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication

Evans

Rice

Goff

2004

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

294

287

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The study of the hepatitis C virus (HCV) has been hindered by the lack of in vitro model systems. The recent development of HCV subgenomic RNA replicons has permitted the study of viral RNA replication in cell culture; however, the requirements for efficient replication of replicons in this system are poorly understood. Many viral isolates do not function as replicons and most require conserved changes, termed adaptive mutations, to replicate efficiently. In this report, we focus on the HCV nonstructural protein 5A (NS5A), a frequent locus for adaptive mutation. We found the interaction between NS5A and human vesicle-associated membrane protein-associated protein A (hVAP-A), a cellular target N-ethylmaleimide-sensitive factor attachment protein receptor, to be required for efficient RNA replication: NS5A mutations that blocked interaction with hVAP-A strongly reduced HCV RNA replication. Further analyses revealed an inverse correlation between NS5A phosphorylation and hVAP-A interaction. A subset of the previously identified adaptive mutations suppressed NS5A hyperphosphorylation and promoted hVAP-A binding. Our results support a model in which NS5A hyperphosphorylation disrupts interaction with hVAP-A and negatively regulates viral RNA replication, suggesting that replicon-adaptive mutations act by preventing the phosphorylation-dependent dissociation of the RNA replication complex.A pproximately 3% of the worldwide population is infected by the hepatitis C virus (HCV) (1), a positive-strand RNA virus of the family Flaviviridae. Infections frequently become chronic, persisting for the life of the individual, often leading to liver cirrhosis or cancer. The HCV genome is a single-stranded, positivestrand (coding), uncapped RNA Ϸ9,600 nt in length, which encodes a single Ϸ3,000-aa polypeptide, cleaved by cellular and viral proteases into at least 10 mature viral peptides ( Fig. 1 A) (reviewed in refs. 2 and 3). This genome is replicated through a minus-strand RNA intermediate in the cytoplasm of an infected cell.Study of HCV has been hindered by the lack of convenient model systems capable of robust viral replication. The development of HCV subgenomic RNA replicons has enabled study of viral RNA replication in cell culture (4). In this system, hepatocarcinoma Huh7 cells are transfected with a modified HCV RNA encoding the neomycin phosphotransferase gene in place of the structural region of the viral polyprotein (Fig. 1B). Replication of this RNA through the action of the remaining nonstructural (NS) proteins confers G418-resistance to the host cell. The replication potential of particular replicons can be scored as the frequency of G418-resistant colonies derived from each RNA.The determinants for replicon efficiency are poorly understood. Not all HCV isolates make functional subgenomic replicons. Six closely related viral genotypes, classified by sequence homology, comprise the HCV genus (5). Genotype 1, including subtypes 1a and 1b, is the most abundant worldwide (6). Of the six known functional replicons, four...

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Hepatitis C Virus NS5A Physically Associates with p53 and Regulates p21/waf1 Gene Expression in a p53-Dependent Manner

Cited by 236 publications

References 41 publications

HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis

HCV NS5A interacts with p53 and inhibits p53-mediated apoptosis

Modulation of p53 transcription regulatory activity and post-translational modification by hepatitis C virus core protein

Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication

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