Heterogeneity of CK2 phosphorylation sites in the NS5A protein of different hepatitis C virus genotypes q Francesca Dal Pero 1, * ,  , Giovanni Di Maira 1,2,  , Oriano Marin 1,2 , Gladis Bortoletto 1 , Lorenzo A. Pinna 1,2 , Alfredo Alberti 1,3 , Maria Ruzzene 1,2 , Martina Gerotto 1 1 Venetian Institute of Molecular Medicine (VIMM), University of Padova, Via Orus, 2, Padova 35129, Italy 2 Department of Biological Chemistry, University of Padova, Italy 3 Department of Clinical and Experimental Medicine, University of Padova, Italy Background/ Aims: The hepatitis C virus NS5A protein is phosphorylated by several cellular kinases, including casein kinase 2 (CK2). Little is known about CK2 phosphorylation of NS5A from different HCV genotypes and clinical isolates. Methods: NS5A from patients with HCV-1a (24 cases), HCV-1b (9) or HCV-3 (16) was analyzed by direct sequencing and CK2 phosphorylation sites were defined using a well-validated prediction rule. In vitro phosphorylation assays were performed using recombinant CK2 and synthetic peptides or full-length NS5A. In vivo phosphorylation by endogenous CK2 of NS5A expressed in hepatoma cells was also investigated. Results: The mean number of CK2 sites within full-length NS5A, was significantly higher in HCV-3 compared to HCV- 1a (P < 0.01) and HCV-1b (P < 0.01). The number of CK2 sites was more homogeneous in HCV-3 variants compared to HCV-1a and HCV-1b variants (P < 0.05). The number of predicted CK2 sites correlated with the degree of in vitro and in vivo phosphorylation of NS5A by CK2. Conclusions: CK2-dependent phosphorylation of NS5A is heterogeneous among different HCV genotypes and clinical isolates. This might have an influence on virus biology and pathogenicity. Ó 2007 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: HCV; Hepatitis C virus; NS5A; Non structural 5A; CK2; Casein kinase 2; Phosphorylation 1. Introduction The hepatitis C virus (HCV), one of the major causes of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide, expresses several structural and non-structural proteins. Among different HCV proteins, the non-structural 5A protein (NS5A) has a key role in the virus life cycle being involved in viral RNA replica- tion [1]. NS5A has perinuclear cytoplasmic location and associates with the endoplasmic reticulum where, with other non-structural viral proteins, it forms the replica- tive complex [1–3]. NS5A and its truncated forms [4–8], have been impli- cated in the modulation of several cellular mechanisms. Some of the truncated forms migrate to the nucleus [9] where they act as a transcriptional activator with conse- quences on cell growth, apoptosis, and lipid metabolism [10,11]. NS5A can also alter major cellular pathways by directly interacting with cellular proteins [12–14]. Finally, NS5A can hamper the antiviral treatment response by blocking interferon effectors including PKR [4] and 2 0 –5 0 OAS [15]. Many NS5A functions depend on protein phosphor- ylation [16], being NS5A the main phosphoprotein of HCV. Two phosphorylated forms of NS5A, p56 and 0168-8278/$32.00 Ó 2007 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2007.07.020 Received 25 January 2007; received in revised form 27 June 2007; accepted 16 July 2007; available online 24 September 2007 Associate Editor: M.U. Mondelli q The authors declare that they do not have anything to disclose regarding conflict of interest with respect to this manuscript. * Corresponding author. Tel.: +39 049 7923224; fax: +39 049 7923250. E-mail address: francesca.dalpero@unipd.it (F. Dal Pero).   These authors contributed equally to this work. www.elsevier.com/locate/jhep Journal of Hepatology 47 (2007) 768–776