Analysis of the 59 GS of JV and, following incubation, were assayed for EGFP and lacZ expression. Both constructs were able to direct translation of the EGFP cistron effectively as expected (Figure 2a and Figure2d). The use of an authentic EMCV IRES to direct translation of the lacZ cistron was also effective (Figure 2e), with levels of b-galactosidase activity comparable to those of the b-galactosidase reporter (Figure 2f). However, no b-galactosidase activity was detected from cells transfected with the pEGFP-C1/JV59GS/lacZ construct (Figure 2b), demonstrating that the JV 59GS was unable to initiate translation, and therefore, in this context, did not possess any IRES-like functions. Translation initiation in JV ORF1 As it was clear that the JV 59GS did not posses any IRES-like functions it was necessary to determine the location of translation initiation within ORF1. This was predicted be the ATG encoding methionine at nucleotide position 22, as it is situated in a favourable context for translation initiation [7]. To investigate this multiple translation termination codons (polySTOP) were inserted into the JV genome within the 3B-encoding region, downstream of the 59GS, to halt translation at a defined point. In vitro transcription and translation of this construct would, in theory, yield a product whose size would relate to the initiation codon used within the 59GS (Figure 3). To address the unlikely event of translation read-through or re-initiation downstream of the polySTOP, which would result in subsequent translation of the 3C protease and cleavage of the truncated ORF1 polyprotein, a mutation was made within the active site encoding region of the 3C protease within JV ORF1, to prevent any viral mediated cleavage of ORF1 translation products (JV 3Cmut/polySTOP). A point mutation of the critical cysteine residue within the highly conserved GDCG motif to a glycine residue was performed, and this approach has been described for the successful inactivation of other norovirus’ 3C activity [28]. In vitro transcription and translation analysis was performed on JV wild type (Figure 4, lane 2), JV 3Cmut (Figure 4, lane 3) and JV 3Cmut/polySTOP (Figure 4, lane 4). The mutation of the critical cysteine residue Figure 2. Fluorescence microscopy images and light microscopy images following x-gal staining of CRFK cells transfected with: A and B–pEGFP-C1/JV 59 GS/lacZ, C–no DNA, D and E–pEGFP-C1/IRES/lacZ, F–pSV b-Gal reporter. doi:10.1371/journal.pone.0002169.g002 PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2169