A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria Stephen D. C. Griffin, 1 Ruth Harvey, 3 3 Dean S. Clarke, 1 Wendy S. Barclay, 2 Mark Harris 1 and David J. Rowlands 1 Correspondence David J. Rowlands D.J.Rowlands@bmb.leeds.ac.uk 1 Astbury Centre of Molecular Biology, School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK 2 School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK 3 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK Received 8 September 2003 Accepted 6 November 2003 We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy. INTRODUCTION Hepatitis C virus (HCV) is now the leading indicator for liver transplant surgery in the West and, according to World Health Organization figures, currently infects over 3 % of the world population. Mild or often asymptomatic acute infection leads to persistence in the majority of cases with most patients being unaware that they carry the virus. In a high proportion of patients, chronic infection eventually leads to the future onset of liver disorders such as cirrhosis or, in the worst cases, hepatocellular carcinoma: a highly aggressive tumour with a poor prognosis (Choo et al., 1992). Treatment in the clinic is currently limited to anti-viral chemotherapy involving a combination of type 1 interferon and the guanosine analogue ribavirin, though this regime is expensive, poorly tolerated and ineffective against some of the more common viral genotypes. Vaccine development is hindered by the lack of good in vitro and in vivo models of infection, the antigenic heterogeneity of the virus and its ability to avoid immune defences. Consequently, the identification of new viral drug targets is especially impor- tant in the search for control strategies for the disease. HCV is the prototype member of the Hepacivirus genus of the family Flaviviridae (Robertson et al., 1998). The virus is enveloped and has a single-stranded positive-sense RNA genome of around 9?6 kb, which is replicated in the cytosol via a negative-strand intermediate. An internal ribosome entry site (IRES) drives translation of a single polyprotein of approximately 3000 amino acids which is then proteo- lytically cleaved by cellular and viral proteases into the mature virus gene products: Core-E1-E2-p7-NS2-NS3- NS4A-NS4B-NS5A-NS5B (Clarke, 1997). Core, with the two viral glycoproteins E1 and E2, comprise the structural proteins of the virion. The non-structural (NS) proteins are thought to form a ribonucleoprotein complex with the virus genome that associates with intracellular membranes and is the site of RNA replication effected by the NS5B protein (Schmidt-Mende et al., 2001; Egger et al., 2002). The p7 protein of HCV lies at the junction between the structural and non-structural regions of the virus poly- protein (Lin et al., 1994; Mizushima et al., 1994), though it is not known whether p7 is a virion component. The protein is small (63 amino acids) and highly hydrophobic Published ahead of print on 14 November 2003 as DOI 10.1099/ vir.0.19634-0. 3Present address: PHLS Central Public Health Laboratory, Colindale, London NW9 5HT, UK. 0001-9634 G 2004 SGM Printed in Great Britain 451 Journal of General Virology (2004), 85, 451–461 DOI 10.1099/vir.0.19634-0