Downloaded from www.microbiologyresearch.org by IP: 54.82.48.176 On: Mon, 01 Aug 2016 11:12:35 Review Hepatitis C virus NS5A: tales of a promiscuous protein Andrew Macdonald3 and Mark Harris Correspondence Mark Harris mharris@bmb.leeds.ac.uk School of Biochemistry & Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK The non-structural 5A (NS5A) protein of hepatitis C virus (HCV) has been the subject of intensive research over the last decade. It is generally accepted that NS5A is a pleiotropic protein with key roles in both viral RNA replication and modulation of the physiology of the host cell. Our understanding of the role of NS5A in the virus life cycle has been hampered by the lack of a robust in vitro system for the study of HCV replication, although the recent development of the subgenomic replicon has at least allowed us to begin to dissect the involvement of NS5A in the process of viral RNA replication. Early studies into the effects of NS5A on cell physiology relied on expression of NS5A either alone or in the context of other non-structural proteins; the advent of the replicon system has allowed the extrapolation of these studies to a more physiologically relevant cellular context. Despite recent progress, this field is controversial, and there is much work to be accomplished before we fully understand the many functions of this protein. In this article, the current state of our knowledge of NS5A, discussing in detail its direct involvement in virus replication, together with its role in modulating the cellular environment to favour virus replication and persistence, are reviewed. The effects of NS5A on interferon signalling, and the regulation of cell growth and apoptosis are highlighted, demonstrating that this protein is indeed of critical importance for HCV and is worthy of further investigation. Hepatitis C virus Hepatitis C virus (HCV) is the only member of the genus Hepacivirus, within the family Flaviviridae. Viral isolates are further classified into six genotypes (1–6) and 30 subtypes, the most intensively studied of which are genotypes 1a and 1b. It is estimated that 170 million individuals are infected with this virus (Anonymous, 1999), and in 80 % of cases the virus establishes a chronic infection, resulting in fibrosis, cirrhosis and, increasingly, hepatocellular carcinoma (HCC). Current therapy, which consists of a combination of pegy- lated alpha interferon (IFN-a) and ribavirin, gives a res- ponse rate of between 48 % (genotypes 1, 4, 5 and 6) and 88 % (genotypes 2 and 3) (Poynard et al., 2003). The genome is a positive-sense 9?6 kb RNA molecule, compris- ing 59 and 39 untranslated regions (UTRs) flanking a single open reading frame encoding a polyprotein of ~3000 aa (Fig. 1). A second translation product, the F protein, is produced by ribosomal frameshift (Xu et al., 2003). Little is known about the function of this protein, although it has been shown to localize to the endoplasmic reticulum (ER). The 59 UTR contains an internal ribosome entry site (IRES), allowing cap-independent initiation of translation. The polyprotein is cleaved into 10 polypeptides by cellular and viral proteinases. At the N terminus are three struc- tural proteins – Core and the glycoproteins E1 and E2. Following E2 is p7, a protein that oligomerizes to form cation channels (Griffin et al., 2003) and has been speculated to be incorporated into virus particles. The C-terminal two- thirds of the polyprotein comprise the six non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. Two of these are functionally well characterized: NS3 complexed with NS4A is a proteinase that cleaves between the non- structural proteins (it also has helicase activity); NS5B is the viral RNA-dependent RNA polymerase. The others presumably play as yet undefined roles in virus replication; however, an increasing body of literature points to a role for these proteins in perturbing cell signalling and mediating immune evasion. In this regard, NS5A is of great interest, and here we will review our current knowledge of this protein. Structure of the NS5A protein A major limitation in our understanding of NS5A is the paucity of structural information. The protein is predicted to be predominantly hydrophilic and to contain no trans- membrane helices. A number of structural features of the protein have, however, been derived experimentally. 3Present address: MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK. Published online ahead of print on 8 June 2004 as DOI 10.1099/ vir.0.80204-0. 0008-0204 G 2004 SGM Printed in Great Britain 2485 Journal of General Virology (2004), 85, 2485–2502 DOI 10.1099/vir.0.80204-0