Journal of General Virology (2010), 91, 2428–2432 DOI 10.1099/vir.0.023614-0 Short Communication Correspondence Mark Harris m.harris@leeds.ac.uk Received 13 May 2010 Accepted 26 June 2010 Identification of a novel phosphorylation site in hepatitis C virus NS5A Anna Nordle Gilliver, Stephen Griffin3 and Mark Harris Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK Hepatitis C virus (HCV) NS5A protein is phosphorylated on multiple residues; however, despite extensive study, the precise identity of these sites has not been determined unambiguously. In this study, we have used a combination of immunoprecipitation and mass spectrometry to identify these phosphorylation sites. This analysis revealed the presence of a major phosphorylated residue within NS5A from the genotype 1b Con1 isolate – serine 249 (serine 2221 in polyprotein numbering). However, mutation of this residue (or the corresponding threonine in the JFH-1 isolate) to either a phosphomimetic (aspartate) or a phosphoablative (alanine) residue resulted in no phenotype. We conclude that phosphorylation of this residue, in the context of a highly culture- adapted HCV genome, does not play a role in either viral RNA replication or virus assembly. It is possible that it might be important in an aspect of virus biology that is not recapitulated faithfully in the Huh-7 cell-culture system. Hepatitis C virus (HCV) is estimated to infect some 123 million individuals (Shepard et al., 2005), and establishes a chronic infection that can ultimately result in liver fibrosis, cirrhosis or hepatocellular carcinoma. Combination therapy comprising pegylated alpha inter- feron (IFN-a) and ribavirin is only successful in approxi- mately 50 % of patients. HCV, a member of the family Flaviviridae, is an enveloped virus with a positive-sense RNA genome 9.6 kb in length and containing a single ORF. An internal ribosome entry site mediates cap-independent translation of the 3000 aa polyprotein, which is cleaved co- and post-translationally by host-cell and viral proteases to release the structural proteins (core, E1, E2 and p7) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). NS5A has been shown to have many functions: foremost, as a component of the RNA replication complex, it is absolutely required for viral RNA replication. Structural analysis has revealed that NS5A comprises three domains separated by short low-complexity regions (Tellinghuisen et al., 2004) (Fig. 1, top). The structure of domain I has been determined; it coordinates a zinc ion and is postulated to dimerize (Love et al., 2009; Tellinghuisen et al., 2005). Domains II and III are less structured and more flexible; in particular, domain III can accommodate a GFP insert at the C terminus with no adverse effects (Appel et al., 2005; McCormick et al., 2006b; Moradpour et al., 2004). By SDS-PAGE and Western blotting, two forms of the protein with different apparent mobilities can be 3Present address: Leeds Institute of Molecular Medicine, Faculty of Medicine and Health, University of Leeds, Leeds LS9 7TF, UK. 2428 observed: these correspond to alternatively phosphorylated forms of NS5A – a basally phosphorylated form (apparent molecular mass 56 kDa) and a hyperphosphorylated form (58 kDa). Proline-directed kinases such as casein kinase II (CKII) have been implicated in basal phosphorylation (Reed et al., 1997), and CKIa has been implicated in hyperphosphorylation (Quintavalle et al., 2007); however, there is little consensus as to either the locations or the number of phosphorylation sites on both forms of NS5A. Basal phosphorylation sites have been shown to be present in both domains II and III (Tanji et al., 1995) and hyperphosphorylation sites have also been mapped to domain II (Katze et al., 2000; Tanji et al., 1995). Interestingly, inhibition of hyperphosphorylation either pharmacologically or by mutation enabled replication of a non-culture-adapted genotype 1b subgenomic replicon (Appel et al., 2005; Neddermann et al., 2004). In order to assign phosphorylation sites unambiguously within NS5A, we adopted a mass-spectrometric (MS) approach. We showed previously that a recombinant baculovirus containing a tetracycline-responsive mammalian promoter (a BacMAM vector termed FBrepcon1neo; McCormick et al., 2006a) could be used to drive high levels of expression of a Con1-derived genotype 1b NS3-5B subgenomic replicon in HepG2 cells. Importantly, HepG2 supported the replication of a subgenomic replicon delivered via the baculovirus route, as judged by induction of an IFN-b response that was not seen following delivery of a GND- mutant (replication-defective) replicon (McCormick et al., 2004). Furthermore, NS5A expressed from this vector exhibited both basal and hyperphosphorylated species (p56 and p58) (McCormick et al., 2006a), similar to those observed 023614 G 2010 SGM Printed in Great Britain