Hepatitis C Virus NS5A Protein Modulates Transcription through a Novel Cellular Transcription Factor SRCAP

Abstract

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...

Hepatitis C Virus Inhibits Intracellular Interferon Alpha Expression in Human Hepatic Cell Lines *

“…Our finding that NS5A suppressed IRF-7-induced IFN-␣ promoter activation is in disagreement with the observations of Ghosh et al, who showed that NS5A activates IFN-␣ promoter in HepG2 cells, with an associated increase of IRF-3. 46 The discrepancy between our findings and the report by Ghosh et al may be due to the differences in the reagents used for IFN-␣ promoter activation. In our study, we used IRF-7 as an inducer, whereas Ghosh et al activated IFN-␣ promoter with Newcastle disease virus (NDV).…”

“…4,30,33,34,43,44 Among these HCV immunosuppressive proteins, NS5A has the ability to modulate a number of cell-cycle regulatory genes, 45,46 and has been implicated in the interference of IFN-mediated antiviral functions. 6 NS5A inhibits IFN-induced, double-stranded RNA-activated protein kinase, 31,32 which mediates the antiviral effects of IFN.…”

Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon

“…A second class is in NS5B (so far only one mutant has been described) which, in contrast, is resistant to the antiviral response possibly elicited by wt NS5A or, following the other hypothesis, which forms an active replication complex with wt NS5A. A number of reports have suggested various types of interaction between NS5A and cellular proteins, as well as specific interactions between NS5B and other NS proteins, including NS5A (Chung et al, 2000;Ghosh et al, 2000;He et al, 2002;Lan et al, 2002;Lin et al, 1997;Majumder et al, 2001;Tan et al, 1999;Tu et al, 1999). The two-hybrid system or immunoprecipitations are the most widely used experimental systems, with the majority of cases using the isolated NS5A polypeptide, tagged or not.…”

High Amino Acid Variability Within the Ns5a of Hepatitis C Virus (Hcv) Is Associated With Hepatocellular Carcinoma in Patients With Hcv–1B-Related Cirrhosis

“…71 In fact, HCV-NS5A seems to act over different cellular factors for the regulation of cellular metabolism, promoting cell growth and perturbing mitogenic signaling pathways. [72][73][74] In addition, HCV proteins also inhibit the JAK-STAT pathway that may be important in cellular proliferation. 76 Although the high frequency of NS5A-HCV mutated phenotypes among patients with HCC suggest that this region may be important in the development of the tumor; a significant proportion of HCV strains isolated from patients with HCC did not have a mutated PKR-bd.…”