mRNA Translation without eIFs different times by radioactive labelling (Fig 3B, upper panel) and also by western blot analysis using anti-C antibodies (Fig 3B, lower panel). As expected, wt SV RNA was the most efficient and synthesized C as a unique product. SV DDLP rendered three products that in total represent 28% of C product from wt SV, as estimated by densitometric analysis of the labelled sample at 10 hpe. The two products with higher molecular weight migrate very closely to each other, but they can be separated by using longer electrophoresis times (data not shown). Most probably, these C-related products are the result of a loss of fidelity in AUGi selection during the initiation of translation. Their sizes are consistent with alternative initiation at the first three AUGs of C sequence. The SV-C-Met,Arg/Lys,Lys variant rendered two products, the one with the lower mobility corresponds in size to wt C and represents 7% of protein C synthesized at 10 hpe. Gel mobility of this product suggests that it is synthesized by initiation at the non-canonical AAG codon (see scheme in Fig 3A). The most abundant C product synthesized by SV-C-Met,Arg/Lys,Lys (19% of wt C) is consistent with a translation initiation from the first AUG of its sequence, that is the third AUG in the wt C (see scheme in Fig 3A). SV-C-Met/Ala and SV-C-Met/Cys synthe- sized the lower amounts of C products at 10 hpe, 9 and 5% of wt C respectively. However, they synthesized almost exclusively products with similar mobility to wt C protein. These C products are presumably derived from initiation at non-canonical GCG or UGU codons, respectively. Analyses and quantification by western blot analysis yielded similar results to those obtained by radioactive labelling (Fig 3B, lower panel). However, one significant difference was that the products synthesized by translation initiation at the non-canonical codons AAG from SV-C-Met,Arg/Lys,Lys or UGU from SV-C-Met/Cys do not accumulate in cells. As wt C protein does not contain any cysteine, this variant will produce a radioactive C product when labelled with radioactive cysteine if the UGU codon is used. BHK cells electroporated with wt SV and SV-C-Met/Cys RNAs were labelled at 5 hpe with [35S]-Cys (Fig S1). wt SV efficiently incorporated radioactive cysteine into its Figure 3. Translation of SV sg-mRNA containing AUGi replaced by other codons. Panel A. Schematic representation of the first 228 nt from the 59end of SV sg-mRNA that include the leader sequence and the translation enhancing motif. The mutations introduced in the different constructs are indicated. Panel B. Synthesis of C protein from different SV variants with modified start codons of sg-mRNA. BHK cells were electroporated with the different in vitro transcribed mRNAs and, at the times indicated, cultures were labelled with [35S]Met-Cys for 30 min. Samples were processed by SDS-PAGE, fluorography and autoradiography (upper panel) and also by western blot analysis to detect C products with anti-C antibodies (lower panel). Panel C. Synthesis of C protein from sg-mRNAs with altered AUGi electroporated into BHK cells or translated in HeLa cell extracts. BHK cells were electroporated with the different in vitro prepared sg-mRNAs and, at 3 hpe, C production was analyzed by western blotting (upper panel). Translation was carried out in HeLa S3 extracts programmed with the different mRNAs for 1 h at 30uC in presence of 0.7 mC/ml [35S]Met-Cys. The synthesized proteins were analyzed by autoradiography of SDS-polyacrylamide gels (lower panel). Panel D. Synthesis of C protein from sg-mRNAs that have disrupted the DLP structure. BHK cells were electroporated with different SV genomes or with sg-mRNAs synthesized by in vitro transcription. Cultures were collected at 4 hpe for replicons and at 3 hpe for in vitro transcribed sg-mRNAs. C products were analyzed by western blotting with anti-C antibodies. Samples electroporated with sg-mRNAs (Right panel) were exposed ten times more than samples electroporated with replicons (Left panel). doi:10.1371/journal.pone.0004772.g003 PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4772