A. Nordle Gilliver, S. Griffin and M. Harris Fig. 2. Role of serine 249 phosphorylation in genotype 1b RNA replication. (a) Huh7 cells (4 106) were electroporated with the indicated in vitro-transcribed FK5.1 RNAs (2 mg), selected in the presence of G418 (1 mg ml”1) for 3 weeks and colonies were stained with Coomassie brilliant blue. Number of colonies produced is presented as a percentage of the initial number of electroporated cells. (b) Western blot analysis of lysates from stable cell lines harbouring the indicated FK5.1 replicons with sheep polyclonal antiserum to either NS3 (Aoubala et al., 2001) or NS5A (Macdonald et al., 2003). (c) Huh7 cells were electro- porated with replicon RNA and harvested into passive lysis buffer (Promega) at the indicated time points (black bars, 4 h; grey bars, 72 h). Luciferase activity (relative luciferase units; RLU) was measured as described previously (Macdonald et al., 2003). In both graphs, error bars show SEM; data from three independent experiments are shown. assembly (Appel et al., 2008; Hughes et al., 2009; Masaki et al., 2008; Tellinghuisen et al., 2008a), we mutated either serine 247 or threonine 249 to either alanine or aspartic acid in the context of full-length JFH-1 virus (Wakita et al., 2005), previously modified to contain synonymous muta- tions generating unique restriction sites flanking NS5A (Hughes et al., 2009). Somewhat surprisingly, neither mutation had a substantial effect on either virus assembly 2430 Fig. 3. Role of serine 247/threonine 249 phosphorylation in assembly and release of infectious HCV. (a) Sequence of the region surrounding serine 249 in Con1, J4 and JFH-1 NS5A. Mutated residues are shown in bold. Huh7 cells (4 106) were electroporated with the indicated in vitro-transcribed virus RNAs (10 mg) and virus release into the culture supernatant (black bars) was measured by focus-forming assay (Hughes et al., 2009). Intracellular virus titres (grey bars) were measured by focus- forming assay following cell disruption by repetitive freeze–thaw at 72 h post-transfection (p.t.). (b) Huh7 cells were electro- porated with the indicated virus RNAs and harvested at 48 h p.t. by lysis in Glasgow lysis buffer (Harris & Coates, 1993). Protein (10 mg) was analysed by Western blotting with antiserum to NS5A or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as indicated. or release (Fig. 3a). HCV protein synthesis in infected cells (Fig. 3b) and the NS5A p56 : p58 ratio were also unaffected. We conclude that phosphorylation of these residues is not important for either virus genome replication or particle assembly; however, we cannot rule out the possibility that, unlike Con1, in the context of FK5.1 or JFH-1 these residues are not phosphorylated. To test this, it would be necessary to express either the FK5.1 subgenomic replicon or full-length JFH-1 in HepG2 cells using a BacMAM vector and analyse the phosphorylation status of NS5A. Such experiments are under way in our laboratory. What, then, might be the role of phosphorylation of serine 249? It is possible that, in the context of the culture- adapted FK5.1 subgenomic replicon or the highly effi- ciently replicating JFH-1 genome, there is no requirement Journal of General Virology 91