Sustained activation of the Raf/MEK/Erk pathway in response to EGF in stable cell lines expressing the Hepatitis C Virus (HCV) core protein S Giambartolomei 1,2 , F Covone 1 , M Levrero 1,3 and C Balsano* ,1,2 1 Laboratory of Gene Expression, Fondazione Andrea Cesalpino, University of Rome `La Sapienza', Rome, Italy; 2 Department of Internal Medicine, University of L'Aquila, L'Aquila, Italy; 3 Department of Internal Medicine, University of Cagliari, Cagliari, Italy Chronic hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and hepatocellular carcinoma (HCC) worldwide. The HCV capside core is a multi- functional protein with regulatory functions that aects transcription and cell growth in vitro and in vivo. Here, we show that both HCV genotype 1a and 3 core proteins activate MEK1 and Erk1/2 MAP kinases and that the costitutive expression of the HCV core results in a high basal activity of Raf1 and MAP/kinase/ kinase, as determined by endogenous Raf1 in vitro kinase assay and immunodetection of hyperphosphory- lated Erk1 and Erk2 even after a serum starvation. Moreover, the activation of both Erk1/2 and the downstream transcription factor Elk-1 in response to the mitogenic stimulus EGF is signi®cantly prolonged. The sustained response to EGF in cells expressing the HCV core occurs despite a normal induction of the MAP phosphatases MKP regulatory feedback and is likely due to the costitutive activation of Raf-1 activity. The ability of HCV core proteins to directly activate the MAP kinase cascade and to prolong its activity in response to mitogenic stimuli may contribute to the neoplastic transformation of HCV infected liver cells. Oncogene (2001) 20, 2606 ± 2610. Keywords: HCV; core; Raf; Erk1/2; EGF, HCC Hepatitis C virus (HCV) is, worldwide, the main cause of chronic hepatitis, which often leads to liver cirrhosis and hepatocellular carcinoma (HCC). The HCV cap- side core protein, besides its essential role in the virus morphogenesis, also modulates many cellular functions (De Francesco, 1999). HCV core forms homo-multi- mers, binds RNA, associates with the endoplasmic reticulum and has been shown to interact with several cytoplasmic and nuclear proteins, incuding apolipo- protein II, lymphotoxin-b and TNF-a type 1 receptors, putative RNA helicases and nuclear ribonucleoprotein K (Barba et al., 1997, Mamiya and Worman, 1999; You et al., 1999; De Francesco, 1999). The functional consequences of these interactions are not well de®ned. HCV core has been shown to regulate transcription, to modulate cell susceptibility to many apoptotic stimuli, to transform rodent ®broblasts and to immortalize primary hepatocytes (Colombo, 1999; Hayashi et al., 1999; Jin et al., 2000; Lu et al., 1999; Ray et al., 2000). More interestingly, liver expression of HCV core in transgenic mice results in early steatosis (Moriya et al., 1997) and late HCC development (Moriya et al., 1998). A potential link between modulation of transcription and cell proliferation is provided by the reported ability of HCV core to activate the ERK/SRE signaling pathway in rodent ®broblasts (Tsuchihara et al., 1999), in human epithelial transformed cell lines (Shrivastava et al., 1998) and in a hepatoblastoma cell line (Aoki et al., 2000; Hayashi et al., 2000). Indeed, HCV core has been shown to bind 14-3-3 proteins and to activate Raf-1 (Aoki et al., 2000). The extracellular signal-regulated kinases (ERKs) signaling cascade is one of the evolutionarily conserved family of mitogen- activated protein (MAP) kinases that play an im- portant role in the regulation of growth, apoptosis and dierentiation (Garrington and Johnson, 1999). The p44 Erk1 and p42 Erk2 MAP kinases are activated by threonine 202 and tyrosine 204 residues phosphorila- tion by the upstream MAP kinase/kinases MEK1/2 which in turn are activated by the MAP/kinase/kinase/ kinase cRaf1 (Garrington and Johnson, 1999). The activation of Erk1/2 MAPKs in response to mitogenic stimuli is generally short-lived due to the induction of a number of feedback inhibitory pathways, including the upregulation of members of the dual speci®city MAP kinase phosphatases (MKP) family that inactivate the MAPK cascade at the level of the MAP kinases (Schaeer and Weber, 1999). MKP-1 phosphatase levels are rapidly induced after ERK1/2 activation both by transcriptional activation and by ERK1/2- phosphorylation dependent protein stabilization (Bron- dello et al., 1999). These mechanisms ensure a proper termination of the response to mitogens and the maintenance of cellular homeostasis. In this study we further investigate the role of HCV core in the regulation of the MAP kinases cascade and, in particular, whether and how HCV core may aect the kinetics of the Raf/MEK/Erk pathway activation in response to mitogens. Oncogene (2001) 20, 2606 ± 2610 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc *Correspondence: C Balsano, Fondazione A. Cesalpino, c/o I Clinica Medica, Policlinico Umberto I, Viale del Policlinico n. 155, 00161 Roma, Italy Received 11 January 2001; revised 6 February 2001; accepted 12 February 2001