mRNA Translation without eIFs Figure 2. Effect of arsenite on translation of SV sg-mRNA. Panel A. Effect of arsenite on SV infection. Uninfected or SV-infected BHK cells (10 pfu/cell) were treated with different concentrations of sodium arsenite for 30 min at 4 hpi. Proteins were then labelled with [35S]Met-Cys in presence of the same concentrations of sodium arsenite for 30 min. Samples were collected in the appropriate sample buffer and processed by SDS- PAGE, fluorography and autoradiography. Relative densitometry values obtained from capsid or actin bands are indicated below each lane. Panel B. Translation in SV-replicating BHK cells treated with arsenite. BHK cells were electroplated with 20 mg of rep C+Luc mRNA or transcription buffer as control. At 4 hpe cultures were treated with sodium arsenite for 30 min and then half of the cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min and the other half treated with arsenite only. Radioactive samples were examined by SDS-PAGE, fluorography and autoradiography (upper panel) and non-radioactive samples were used to analyze phosphorylation of eIF2a by isoelectric focusing (lower panel). Panel C. Translation of different mRNAs transfected in BHK cells treated with arsenite. BHK cells were electroporated with 20 mg of cap-Luc-poly(A) or C+Luc mRNA and at 30 mpe treated with different concentrations of arsenite for 1 hour. Half of the cultures were processed to measure luc activity (upper panel) and the other half to detect phosphorylation of eIF2 a by isoelectric focusing (lower panel). Panel D. Translation of sg-mRNA synthesized in the nucleus of BHK cells treated with arsenite. BHK cells were transfected with pcDNA-Luc or pcDNA C+Luc plasmids and, at 18 hpt, treated or not with arsenite for 30 min. Next, cultures were labelled with [35S]Met-Cys in presence or absence of arsenite for 30 min. One quarter of the samples were directly processed by SDS-PAGE, fluorography and autoradiography (upper panel). The remaining samples were first immunoprecipitated with anti-luc antibodies and then processed by SDS-PAGE, fluorography and autoradiography (lower panel). doi:10.1371/journal.pone.0004772.g002 I:C to induce phosphorylation of eIF2a. As observed in uninfected cells, phosphorylation of eIF2a abrogated translation of both cap- Luc-poly(A) and C+Luc mRNAs (data not shown). In conclusion, eIF2a is not necessary for the translation of mRNA C+Luc in SV- replicating cells, but this initiation factor is required for the translation of sg-mRNA in contexts other than infection. Translation of sg-mRNA variants lacking the AUG initiation codon Since translation of sg-mRNA does not require eIF2 in SV- infected cells, we speculated that a different initiation codon to the canonical model may be used for this mRNA. Perhaps, during infection, when eIF2a is phosphorylated the initiation of translation of sg-mRNA could operate at non-AUG codons. To investigate this possibility, a number of constructs were made containing AUGi and the next AUG present in the region coding for C protein changed to other codons. To maintain the predicted base pairing in this region, several modifications were introduced as depicted in figure 3A. In addition, we tested a variant that contains an altered hairpin structure (SV DDLP) [7]. The different viral genomic RNAs bearing altered initiation codons and the variant that contains a modified hairpin structure were electropo- rated into BHK cells and protein synthesis was estimated at PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4772