We have previously demonstrated that hepatitis C virus (HCV) NS5A protein promotes cell growth and transcriptionally regulates the p21/waf1 promoter, a downstream effector gene of p53. In this study, we investigated the molecular mechanism of NS5A-mediated transcriptional repression of p21/waf1. We observed that transcriptional repression of the p21/waf1 gene by NS5A is p53 dependent by using p53 wild-type (؉/؉) and null (؊/؊) cells. Interestingly, p53-mediated transcriptional activation from a synthetic promoter containing multiple p53 binding sites (PG13-LUC) was abrogated following expression of HCV NS5A. Additional studies using pull-down experiments, in vivo coimmunoprecipitation, and mammalian two-hybrid assays demonstrated that NS5A physically associates with p53. Confocal microscopy revealed sequestration of p53 in the perinuclear membrane and colocalization with NS5A in transfected HepG2 and Saos-2 cells. Together these results suggest that an association of NS5A and p53 allows transcriptional modulation of the p21/waf1 gene and may contribute to HCV-mediated pathogenesis.

Hepatitis C virus (HCV) is a major etiologic agent of chronic hepatitis worldwide and may lead to the development of hepatocellular carcinoma. However, the mechanism of development of chronic hepatitis or hepatocarcinogenesis by HCV remains unclear. In the present study, we have investigated the effect of nonstructural protein 5A (NS5A) on TNF- and Fas-mediated apoptosis in the liver of transgenic mice. For this purpose, transgenic mice were generated by targeting the HCV NS5A genomic region cloned under the control of a liver-specific apoE promoter. The transgenic animals were phenotypically similar to their normal littermates and did not exhibit a detectable histological change in the liver at 8-12 weeks of age. Intraperitoneal injection of recombinant TNF induced hepatic injury and apoptosis in normal mice. In contrast, transgenic mice expressing NS5A protein were protected against hepatic apoptosis after injection of TNF. However, injection of anti-Fas antibody into transgenic mice did not significantly influence hepatic apoptosis compared to the normal littermates. These results suggested distinct effects of TNF and anti-Fas antibody in transgenic mice expressing NS5A. We subsequently investigated the effect of NS5A in signaling pathways involved in these two cytokine-mediated apoptosis. A physical association between NS5A and TRADD was observed by pull-down assay, coimmunoprecipitation, and colocalization experiments. Furthermore, NS5A prevented the association between TRADD and FADD and blocked TRADD-mediated NF-kappaB activation. Together, our results suggest that NS5A impairs TNF-mediated apoptosis by interfering upstream of the signal transduction pathway and may play a role in HCV-mediated pathogenesis.

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Hepatitis C virus NS5A protein transcriptionally modulates cellular genes and promotes cell growth. NS5A is likely to exert its activity in concert with cellular factor(s). Using a yeast two-hybrid screen, we have demonstrated that NS5A interacts with the C-terminal end of a newly identified cellular transcription factor, SRCAP. The authenticity of this interaction was verified by a mammalian two-hybrid assay, in vitro pull-down experiment, and an in vivo coimmunoprecipitation assay in human hepatoma (HepG2) cells. An in vitro transient transfection assay demonstrated that SRCAP can efficiently activate transcription when recruited by the Gal4 DNA-binding domain to the promoter. However, down-regulation of p21 promoter activity by NS5A was enhanced following ectopic expression of SRCAP. Together these results suggest that the interaction of NS5A and SRCAP may be one of the mechanisms by which NS5A exerts its effect on cell growth regulation contributing to hepatitis C virus-mediated pathogenesis. Hepatitis C virus (HCV)1 is an important cause of morbidity and mortality worldwide, causing a spectrum of liver disease ranging from an asymptomatic carrier state to end-stage liver disease (1, 2). The HCV genome encodes a single polyprotein precursor that is cleaved by both host and viral proteases to generate structural and nonstructural proteins. The nonstructural protein 5A (NS5A) is generated as a mature protein by the action of NS3 protease in conjunction with NS4A (3,4). NS5A is localized in the nuclear periplasmic membrane (4) and exists as phosphoproteins (p56 and p58), with the degree of phosphorylation accounting for the difference in size (5-7). Phosphorylation status of NS5A differs among HCV genotypes (8). NS5A is phosphorylated by a cellular serine/threonine kinase, and Ser 2321 represents a major phosphorylation site (9). However, this phosphorylation site is dispensable for interactions with NS4A and PKR. Sequence comparison of the regions surrounding the phosphorylation sites indicates an extremely high level of conservation between different strains of the HCV, but the biological significance of phosphorylation is still undefined.Recent studies suggest that HCV NS5A protein transcriptionally modulates cellular genes, promotes cell growth (10, 11), and inhibits tumor necrosis factor-␣ mediated apoptotic cell death.2 There is also evidence that two-thirds of the NS5A protein from the C-terminal fused with Gal4 DNA-binding domain functions as a potent transcriptional activator (12, 13). Viral proteins may influence cellular genes, which in turn may be involved in the regulation of oncogenes or tumor suppressor genes. Inactivation of these genes may be a mechanism for the disruption of normal cell growth. Host factors are important components for the modulation of virus replication. Viruses also produce proteins that may interact with host factors for viral persistence by disrupting normal cell cycle. To further understand the functional role of HCV NS5A, we examined the interaction of NS5A with cellular...

We initially identified c-myc promoter-binding protein 1 (MBP-1) from a human cervical carcinoma (HeLa) cell expression library (25). MBP-1 is ubiquitously expressed in different human tissues (26) and is located at human chromosome 1p35-pter (31). This protein binds to the TATA box sequences of the c-myc P2 promoter. In vitro transient-transfection assay suggested that MBP-1 negatively regulates both human and mouse c-myc promoter activity (24, 25) through the N-terminal half (27,30). Further studies have shown that MBP-1 and TATA-binding protein (TBP) bind simultaneously in the minor groove of the c-myc P2 promoter (4). Ectopic expression of MBP-1 induces cell death and the reduction of c-myc expression (24) and regresses tumor growth (28). However, Bcl2, a cell survival gene (1, 29), protects against MBP-1-mediated apoptotic cell death, suggesting that, besides c-myc regulation, MBP-1 exerts a regulatory role on cell growth through other unknown mechanisms (24).A recent study indicated that MBP-1, when brought to the promoter by a Gal4 DNA-binding domain, can significantly repress transcriptional activity (7). Structure-function analysis of MBP-1 mutants in the context of the Gal4 DNA-binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338). This further suggests that MBP-1 can modulate cellular gene transcription through an alternative mechanism, besides blocking the c-myc transcription. To understand the regulatory role of MBP-1 in cell growth, it is important to characterize the interacting cellular protein(s) and to evaluate molecular mechanisms involved in MBP-1-mediated transcriptional repression. We undertook a search for cellular proteins that interact with MBP-1 using a two-hybrid interaction cloning strategy in yeast. An MBP-1 interacting protein (MIP-2A) was identified which relieves the transcriptional repression of MBP-1 and antagonizes MBP-1-mediated cell death. Thus, MBP-1 may regulate transcriptional modulation by forming a complex with MIP-2A and also exerts its effect for cell growth regulation. MATERIALS AND METHODSYeast two-hybrid screening. The coding region of MBP-1 was cloned in frame with the LexA DNA-binding domain into the pLexA plasmid vector (Clontech) at BamHI and XhoI restriction sites. The yeast strain EGY48 carrying both the Leu and LacZ reporter genes was transformed with Lex-MBP-1 plasmid DNA and selected positive clones on a synthetic dropout (sd) His Ϫ Ura Ϫ agar plate. The positive yeast colonies were tested for MBP-1 expression by Western blot analysis using anti-LexA monoclonal antibody (Clontech). Lex-MBP-1 positive yeast cells were grown in appropriate liquid medium lacking histidine and uracil and transformed with human cervical carcinoma (HeLa) cell cDNA library (kindly provided by Alain Nepveu, McGill University, Montreal, Quebec, Canada) constructed in pB42AD plasmid vector under the control of an inducible

Hepatitis C virus (HCV) is a major etiologic agent for chronic hepatitis worldwide often leading to the development of cirrhosis and hepatocellular carcinoma. However, the mechanism for development of chronic hepatitis or hepatocarcinogenesis by HCV remains unclear. HCV NS5A protein possesses many intriguing properties, including sequestration of p53 in the cytoplasm, downregulation of p21 protein, activation of STAT3, and inhibition of tumor necrosis factor-alpha-mediated apoptosis. Thus, we investigated whether this viral protein has oncogenic property in vivo. In the absence of an efficient cell culture system for virus growth and a suitable small animal model for HCV infection, transgenic FVB mice were generated by targeting the HCV NS5A genomic region cloned under the control of a liver-specific apoE promoter or mouse major urinary promoter (MUP). The apoE promoter is constitutively expressed in liver, on the other hand, the MUP is developmentally regulated and expressed in the liver after birth. Reverse transcription polymerase chain reaction and Western blot analysis indicated establishment of HCV NS5A transgene expression in several lines from both groups of mice. Immunohistochemical studies suggested the presence of NS5A in the cytoplasm of hepatocytes. The transgenic animals were phenotypically similar to their normal littermates and did not exhibit a major histological change within the liver up to 24 months of age. Our results suggested HCV NS5A protein is not directly cytopathic or oncogenic in this FVB transgenic mouse model, although this viral protein promotes cell growth in vitro. These animals will be a valuable model of HCV immunopathology as well as for evaluation of siRNA, interferon and other cytokine therapies.

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