Journal of General Virology (1999), 80, 1179–1183. Printed in Great Britain .......................................................................................................................................................................................................... SHORT COMMUNICATION Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth Asish K. Ghosh, 1 Robert Steele, 1 Keith Meyer, 2 Ranjit Ray 2 and Ratna B. Ray 1, 2 Department of Pathology 1 and Division of Infectious Diseases and Immunology 2 , Saint Louis University, St Louis, MO 63104, USA The phosphoprotein NS5A of hepatitis C virus has recently been suggested to control PKR protein kinase for resistance to interferon. To investigate other functions of NS5A, studies were initiated on the regulation of transcription of important cellular genes and of cell growth by this protein. The results suggested that NS5A protein represses transcrip- tion of the cell cycle regulatory gene p21 WAF1 , while it activates the human proliferating cell nuclear antigen gene in murine fibroblasts and human hepatoma cells. Furthermore, introduction of NS5A into murine fibroblasts (NIH3T3) promoted anchorage-independent growth and tumour for- mation in nude mice. Thus, NS5A appears to exhibit a role in cell growth regulation. Hepatitis C virus (HCV) 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. The HCV genome contains a linear, positive-strand RNA molecule of  9500 nucleotides, with variability ap- parent in the viral genome and several genotypes described (Choo et al., 1989 ; Chamberlain et al., 1997 ; Simmonds, 1998). Information on the mechanism of HCV replication at the molecular level has so far depended upon the expression of its partial or entire genome and comparative analysis with genomes of distantly related flaviviruses or pestiviruses (Zanotto et al., 1996 ; Clarke, 1997). The HCV genome encodes a single polyprotein precursor that is cleaved by both host and viral proteases to generate at least ten individual proteins. The putative structural proteins (Core, E1 and E2p7) are located in the amino-terminal one-third of the polyprotein and the non- structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) are located in the carboxy-terminal two-thirds of the poly- protein. NS5A protein is generated as a mature product by the action of NS3 protease in conjunction with NS4A (Tanji et al., 1995). NS5A exists as phosphoproteins (p56 and p58) and the Author for correspondence : Ratna Ray (at the Department of Pathology). Fax 1 314 771 3816. e-mail rayrbslu.edu degree of phosphorylation accounts for the differences in size (Kaneko et al., 1994). Although the phosphorylation of NS5A occurs predominantly on serine residues, a low level of phosphorylation on threonine residues has also been observed (Reed et al., 1997). Apart from the probable role of NS5A in the replication cycle, it may be a critical factor in determining the susceptibility of the virus to interferon (Enomoto et al., 1995). NS5A protein localizes in the nuclear periplasmic mem- brane fraction (Tanji et al., 1995). Recent studies have suggested that two-thirds of the carboxy terminus of HCV NS5A protein functions as a transcriptional activator in yeast and mammalian cells in the context of the GAL4 system (Kato et al., 1997; Tanimoto et al., 1997). However, the full-length NS5A protein does not exert an effect in a similar experimental system. This interesting information led to our investigation of the effect of NS5A on the critical components [p21 WAF, p53 and pro- liferating cell nuclear antigen (PCNA)] involved in cell cycle regulation and its role in murine fibroblast growth regulation. Cell cycle progression is driven by the sequential activation of cyclin-dependent kinases (CDKs), which are subject to regulation by positive (cyclins) and negative (CDK-inhibitory proteins) effectors (Morgan, 1995). One such effector is the universal CDK inhibitor p21 WAF (Harper et al., 1993 ; Yang et al., 1995). p21 may participate in apoptosis and increased p21 expression correlates with enhanced cell death under certain conditions (el-Deiry et al., 1993; Sheikh et al., 1995). The p21 protein binds to and inhibits the activity of CDKs by preventing the phosphorylation of critical CDK substrates and blocking cell cycle progression (Harper et al., 1993 ; Xiong et al., 1993). To determine the role of HCV NS5A protein in p21 regulation, a transient transfection assay was performed as described previously (Ray et al., 1998). NIH Swiss mouse embryo fibroblasts (NIH3T3) and human hepatoma (HepG2) cells were transfected with 4 μg reporter construct (WWP–luc ; p21 promoter linked to luciferase gene) and different amounts of the effector construct (NS5A from HCV genotype 1a, under the control of a cytomegalovirus early promoter) or 4 μg pcDNA3 vector control by using a standard lipofectamine protocol (Life Technologies). Transfection efficiencies were normalized to an internal β-galactosidase control and luciferase activity was determined. Results from the luciferase assay with HepG2 or NIH3T3 cells suggested that the NS5A protein 0001-6095  1999 SGM BBHJ