enzyme digest of both amplicon and vector with BgIII and SacI restriction enzymes, and the ligated DNA used to transform E.coli Top10. The lacZ amplicon described previously was ligated to the intermediate pEGFP-C1/IRES vector following restriction en- zyme digest of both amplicon and vector with HindIII and ApaI restriction enzymes, and the ligated DNA used to transform E.coli Top10. Construction of JV 3Cmut and JV 3Cmut/polySTOP The JV 3C protease mutant was created by point mutation of the critical TGT encoded cysteine residue, within the GDCG active site motif, to a GGT encoded glycine residue by mutagenic overlap PCR using Bio-X-Act DNA polymerase. Three rounds of amplification using the JV full length cDNA clone as template were used to generate the final mutant protease cassette. Round 1 used the primers JV F1 (59-CGTCTCAGGGTTGATACT-39) and JV Mut 1 (59-GCAACCACCGTCACCAG-39), yielding a 222 bp amplicon (point mutation nucleotide shown in bold). Round 2 used the primers JV Mut 2 (59-CTGGTGACGGT- GGTTGC-39) and JV R2 (59-TTCCTGGGAGGAACAAGTT- 39), yielding a 651 bp amplicon. Amplicons generated in rounds 1 and 2 were pooled to serve as template for round 3 using the primers JV NF (59-ATGTCAACCACCACCAGC –39) and JV NR (59-AAGGGCTCCGGTGAAGG-39). This cassette contained two BclI restriction sites flanking the 3Cprotease active site, as also found in the wild-type full length clone. Restriction digest using BclI was used to remove the appropriate wild-type cassette from the JV full length clone. The mutant cassette was also digested with BclI prior to ligation to the BclI-digested JV full length clone. The ligated DNA was used to transform E.coli Top10, and was designated JV 3Cmut. Construction of JV 3Cmut/polySTOP was as follows: comple- mentary oligonucleotides with three translation termination codons (underlined) in each reading frame in sense and anti-sense orientations were desgined in such a way that upon annealing the duplex would contain blunt termini. The oligonucleotides were termed pSTOP Top (59-CTAGGTAAGTAAACGCGTCTACT- CACTCAC-39) and pSTOP Comp (59- GTGAGTGAGTA- GACGCGTTTACTTCAATAG-39). Each oligo (1 mg) was incubated with T4 polynucleotide kinase and ATP to phosphor- ylate the 59 termini, pooled and heated to 75uC for 15 min, and left to cool to room temperature to anneal the oligos. Following purification the polySTOP duplex was ligated to Eco47III digested JV 3Cmut, and ligated DNA was used to transform E.coli Top10. The duplex contained the unique restriction site MluI (shown in bold) to assist screening of recombinant clones. Construction of JV V5 The V5 epitope (N-Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly- Leu-Asp-Ser-Thr-C) is recognized by the anti-V5 monoclonal antibody (Invitrogen). Complementary oligonucleotides encoding the V5 epitope were designed in such a way as to generate SacII compatible termini following annealing (bold), and to preserve the reading frame when inserted into the SacII restriction site at nucleotide 123 within the 59 GS of the JV genome (underlined). The oligos were termed V5 Top (59-GGTAAGCCTATCCC- TAACCCTCTCCTCGGTCTCGATTCTACGAGC-39) and V5 Comp (59-TCGTAGAATCGAGACCGAGGAGAGGGT- TAGGGATAGGCTTACCGC-39). The oligos were phosphory- lated and annealed as described previously and the duplex ligated to the SacII digested JV full length clone. Ligated DNA was used to transform E.coli Top10. Analysis of the 59 GS of JV In vitro Transcription/Translation and RIPA analysis In vitro coupled transcription and translation was performed using the TNTH Coupled Reticulocyte Lysate System (Promega) as per the manufacturer’s instructions. Reactions were incubated at 30uC for 1–2 hr. For non-radiolabelled reactions the 35 S- Methionine was replaced with 1 mM unlabelled Methionine (2 ml). Reaction products (1–2 ml) were analysed by SDS-PAGE. Gels were stained and prepared for autoradiography by incubating for 30 min in a solution containing 32 g sodium salicylate, 100 ml methanol and 100 ml dH2O. Gels were dried under vacuum and the reaction products were detected by exposure to Kodak X- Omat scientific imaging film (Sigma) at 270uC for 16 hr followed by developing using a Kodak automated developer. Specific V5-tagged proteins synthesised by TNTH were precipitated from 5–10 ml of reaction product using the anti-V5 monoclonal antibody (Invitrogen) at the recommended dilution in 600 ml of 16 RIPA buffer (diluted from 10x stock: 10 mM Tris- HCl (pH 7.5), 1 mM EDTA, 0.15 mM NaCl, 0.1% SDS, 0.5% Empigen BB, 0.1 mM phenylmethylsulphonylfluoride) for 1 hr at 37uC. This was followed by a second incubation of tube for 2 hr rotating at room temperature with goat anti-mouse immuno- globulin G agarose beads (Sigma) to absorb the immune complexes. The beads were washed three times with 500 ml 16 RIPA buffer and once with 500 ml PBS. The beads were resuspended in sample buffer for analysis by SDS-PAGE and autoradiography as before. Transfection of CRFK cells with Bi-cistronic vectors Endotoxin-free preparations of plasmid DNA were prepared using the GenEluteTM Endotoxin free plasmid midi prep kit (Sigma). Crandall-Reese Feline Kidney cells (CRFKs) were seeded into a 12 well tray at approximately 40–50% confluence. CRFK cells were transfected with no DNA (negative control), pSV-b-Gal (control for b-galactosidase activity), pEGFP-C1/JV 59 GS/lacZ and pEGFP-C1/IRES/lacZ (control for IRES activity) using the SuperfectTM transfection reagent (Qiagen) as per the manufacturer’s recommendations. Following a 16 hour incubation the cells were observed for EGFP expression using a Leica Leitz DMRB fluorescence microscope. The cells were washed in PBS and fixed using a 0.5% solution of glutaraldehyde for 30 min at room temperature. The cells were incubated with an X-Gal stain solution: 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 1x X-Gal (Sigma) for 4 hours at 37uC and were observed for b-glactosidase activity by light microscopy. The experiment was performed more than once to confirm the results. RNA synthesis and transfection of CRFK cells JV V5 and JV FLC T7 cDNA plasmid constructs were linearised using NdeI (Invitrogen). Capped RNA was synthesised using the mMessage mMachineH Capped RNA Transcription kit (Ambion) according to the manufacturer’s instructions. CRFK cells were seeded into 6 well trays at approximately 50% confluence and were transfected with 2 mg purified RNA per well using Transmessenger transfection reagent (Qiagen) according to the manufacturer’s instructions. Immunological analysis For immunofluorescence CRFK cells were seeded onto 19 mm coverslips in 6 well trays and were transfected with RNA as described. Following a 24 hr incubation the coverslips were washed with PBS and fixed in 4% formaldehyde for 15 min at PLoS ONE | www.plosone.org 7 May 2008 | Volume 3 | Issue 5 | e2169