Fig. 1. MS identification of serine 249 phosphorylation. At the top, a schematic of the structure of NS5A is depicted, showing the location of the phosphopeptide. LCS, Low-complexity sequence. For MS analysis, HepG2 cells were seeded onto rat tail collagen- coated plates and transduced with BactTA and FBrepcon1neo at 800 p.f.u. per cell each, as described previously (McCormick et al., 2006a). Cells were lysed 24 h post-transduction and NS5A was purified by using protein G beads and a polyclonal sheep anti- NS5A serum (Macdonald et al., 2003). Following SDS-PAGE, the colloidal blue-stained band corresponding to p58 was excised and digested with trypsin. The tryptic digests were analysed by liquid chromatography–MS with precursor ion scanning on a 4000QTRAP system. Sums of the peptide masses detected by precursor ion scanning in the negative-ion mode across the 45 min HPLC separation are shown. The peptide was identified from the MS/MS fragmentation spectra by database searching and manual inspection of the spectra. amu, Atomic mass units. in Huh7 cells stably harbouring subgenomic replicons. HepG2 cells were therefore transduced with a BacMAM vector expressing the tetracycline transactivator (BactTA) together with FBrepcon1neo; NS5A was purified from cell lysates by using protein G–agarose and polyclonal sheep anti-NS5A serum, separated by SDS-PAGE and stained with colloidal blue stain. The p58 band was excised and submitted for MS analysis. Peptides from a trypsin digest were analysed on two systems independently: firstly, a 4000QTRAP system, where precursor ion scanning was used to identify phosphorylated peptides (Fingerprints Proteomics Facility, University of Dundee, UK) (Fig. 1), and secondly, a Waters Synapt HDMS system (Astbury Centre for Structural Molecular Biology, University of Leeds, UK) (data not shown). Potential phosphorylated peptides were then identified by MS/MS. In both cases, only one major phosphorylated peptide was identified. This was HDpSPDADLIEANLLWR [phospho- serine 249 (serine 2221 in polyprotein numbering)], which is situated at the junction of the first low-complexity sequence http://vir.sgmjournals.org NS5A phosphorylation site and domain II. This site has not been identified previously as a phosphorylation site, although it is situated close to a cluster of serine residues that have been postulated to be involved in hyperphosphorylation (serines 2197, 2201 and 2204) (Tanji et al., 1995). There are a number of reasons why other phosphorylated peptides were not identified, including inefficient ionization, low stoichiometry and failure of some peptides to be captured by the ion trap. Therefore, the failure to identify other phosphorylation sites does not mean that the BacMAM-expressed NS5A in HepG2 cells is not phosphory- lated on other residues. However, the identification of phospho-serine 249 by using two different systems in independent experiments suggests that this is a major site of phosphorylation within the hyperphosphorylated form of NS5A. In order to assess the potential significance of phosphor- ylation at this residue for viral genomic replication, we generated two mutant forms of the FK5.1 culture-adapted subgenomic replicon (Krieger et al., 2001) in which serine 249 was mutated to either an alanine (phosphoablative) or an aspartic acid (phosphomimetic) by PCR (oligonucleo- tide sequences are available upon request). We chose FK5.1 because the Con1 replicon is not culture-adapted and does not replicate at a sufficient level to allow discrimination between wild type and mutants with impaired replication efficiency. In vitro transcripts of the FK5.1 replicons were electroporated into Huh7 cells; cells were then selected for 3 weeks with 1 mg G418 ml21, prior to either staining for colony-forming assays or generation of stable polyclonal replicon-harbouring cell lines. Neither mutation had any effect on either the number of colonies (Fig. 2a) or the p56 : p58 ratio (Fig. 2b). In order to exclude the possibility that the replicons had reverted to the wild-type sequence, RNA was extracted from cells, reverse-transcribed, amp- lified using PCR, cloned and sequenced (data not shown). All clones examined contained the original mutation and had thus not reverted to wild type. These data were also confirmed in the context of a transient, luciferase-based FK5.1 subgenomic replicon. In vitro transcripts of the FK5.1 luciferase replicons were electroporated into Huh7 cells, and luciferase activity was measured at both 4 h post-transfection (to assess transfec- tion efficiency and translation of input RNA) and 72 h (to assess RNA replication) (Fig. 2c). Relative replication levels for both mutants were similar to those of the wild type. Serine 249 is almost completely conserved in all genotype 1 isolates of HCV; however, this residue is not conserved in other genotypes (Kuiken et al., 2005). Notably, the equivalent residue in the only isolate that has the capacity for replication in cell culture, JFH-1, is threonine. In addition to this, a serine is situated two residues N- terminally (serine 247) in JFH-1 NS5A (Fig. 3a). The data in Fig. 2 demonstrated that phosphorylation of serine 249 was dispensable for RNA replication; therefore, to test whether the presence of a phosphorylatable residue at this position was important for the role of NS5A in virus 2429