Hepatitis C Virus NS5A Protein Modulates Transcription through a Novel Cellular Transcription Factor SRCAP
Asish K. Ghosh, Mainak Majumder, Robert Steele, Peter Yaciuk, John Chrivia, Ranjit Ray, Ratna B. Ray
2000
Journal of Biological Chemistry
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
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... atoma (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 protein(s) by yeast two-hybrid screening. Results from this study provided important information regarding the association of NS5A protein with SRCAP, a most recently identified cellular transcriptional coactivator (14). EXPERIMENTAL PROCEDURES Yeast Two-hybrid Screening-The entire cDNA coding region of HCV NS5A (genotype 1a, H strain) was fused in frame with the Gal4 DNAbinding domain into the pGBT9 plasmid vector (CLONTECH) at the EcoRI/SalI restriction sites (pGBT9 -5A) and transformed into Saccharomyces cerevisiae yeast strain HF7c. The pGBT9 -5A positive yeast colonies were grown in appropriate liquid medium lacking tryptophan and were subsequently transformed with library plasmids fused to the Gal4 activation domain, constructed in pGAD plasmid vector (CLON-TECH) for screening of cellular partners. Colonies were selected on agar plates lacking histidine, tryptophan, and leucine over a 7-day period. Positive yeast transformants were picked up and replated for -galactosidase assay by colony-lift filter procedure. A positive interaction was determined by the appearance of blue colonies. The -galactosidase positive colonies were grown on a selective medium for plasmid isolation. Isolated plasmids were transformed into Escherichia coli KC8 strain and selected for the activation domain plasmids on M9-leu Ϫ agar plates. The potential NS5A interacting cDNA inserts were retransformed into HF7c yeast strain bearing pGBT9 -5A fusion gene and were grown on an appropriate selective medium for -galactosidase assay. Positive interacting cDNA clones were analyzed by nucleotide sequencing using an automated sequencer (Applied Biosystems). Nucleotide and predicted amino acid sequences were compared with known protein sequences deposited in the GenBank TM by BLAST analysis. Mammalian Two-hybrid Analysis-A mammalian expression plasmid encoding VP16, a hybrid polypeptide containing the transactivation domain of herpesvirus VP16 (15), was fused to NS5A (VP16 -5A). Gal-SRCAP expression plasmid DNA (14) was used in this study. HepG2 and NIH3T3 cells were cotransfected with 1 g of Gal4 responsive reporter gene (G5E1b-CAT), 2 g of VP16 -5A, and Gal-SRCAP or Gal-⌬SRCAP effector plasmids. CAT assay was performed as described
doi:10.1074/jbc.275.10.7184
pmid:10702287
fatcat:hadnkftuvbgkrmdt2bc27ki274