Analysis of the 59 GS of JV Figure 3. A-JV ORF1 polyprotein showing mutation to the 3C protease at amino acid 1132 and insertion of the polySTOP cassette at amino acid 930, as found in the JV 3Cmut/polySTOP construct. B-The possible in vitro transcription and translation products from the 6 in- frame Met residues within the 59GS and their respective predicted molecular weights. M1 represents the initiation codon found at nucleotide 22 within the JV genome. doi:10.1371/journal.pone.0002169.g003 within the 3C region of JV successfully inactivated the 3C protease, thus a large, .200kDa uncleaved polyprotein is yielded following TNTH. The major product generated by JV 3Cmut/ polySTOP was calculated to be 103kDa in size. Based on computer predictions this is in agreement with the initiation of translation occurring at nucleotide 22, which demonstrates that the JV N-term protein is translated in full in vitro. At this time it is not possible to determine whether translation of intracellular VPg- bound viral RNA initiates at nucleotide 22, although it is likely given the favourable context in which the initiation codon is situated. Immune detection of JV N-term As the JV N-term was found to be translated in vitro attempts were made to express and purify the protein in bacteria for immunisation so that the protein could be identified by radio- immune precipitation assay (RIPA), as it was possible that the N- term protein was migrating on gels aberrantly and possibly co- migrating with 2C. Attempts to express the protein in bacteria were unsuccessful due to toxicity. Therefore, the 14aa V5 epitope encoding sequence was cloned in frame into the JV cDNA construct at nucleotide position 123 (JV V5). The V5 epitope originates from the P and V proteins of the SV5 paramyxovirus Figure 4. Analysis of TNTH products for JV (lane 2), JV 3Cmut (lane 3) and JV 3Cmut/polySTOP (lane 4), on a 7% SDS PAGE gel. Molecular weight marker representation is displayed in lane 1. The major band of approximately 103 kDa in lane 4 is indicative of translation initiation at nucleotide 22. doi:10.1371/journal.pone.0002169.g004 PLoS ONE | www.plosone.org 4 May 2008 | Volume 3 | Issue 5 | e2169