mRNA Translation without eIFs Figure 5. Co-localization analyses of SV capsid protein and ribosomes. BHK cells were infected with SV (100 pfu/cell) and processed for immunofluorescence at 8 hpi. doi:10.1371/journal.pone.0004772.g005 requirements for eIFs observed in cell-free systems may differ from those found in the infected cells. If so, a reappraisal of previous experiments studying the mechanisms of viral mRNA translation in transfected cells or in cell-free systems should be undertaken. More surprises about the mechanism of initiation on the SV sg- mRNA appeared when AUGi was modified. Changes of AUG to other codons still allowed recognition of the original start site on the sg-mRNA, although the efficacy of translation diminished. The Met to Ala modification (AUG/GCG) was more efficiently recognized than the other modifications tested, but the important observation is that SV sg-mRNA still serves to initiate protein synthesis, even without an AUGi. These findings are in good agreement with the observation that hepatitis C virus mRNA is translated even when the initiator AUG codon is replaced by other codons [27]. Notably, Met/Ala SV sg-mRNA is unable to start translation at that site in uninfected cells or in cell-free systems and translation start at the first AUG codon. These results suggest that a different start codon is selected according to whether the mRNA has been transcribed in SV-replicating cells or in uninfected cells or in cell-free systems. Since the structure of the SV sg-mRNA is similar in both cases, i.e. infected or uninfected cells, we can conclude that the exact mechanism of translation of this mRNA depends on the context in which protein synthesis is examined. Therefore, the plasticity of the translation machinery is adapted to a given mRNA according to the environment. The hairpin loop present in the coding region of the SV sg- mRNA sequence is a translational enhancer [5,6], although its exact function during translation remains puzzling. In addition, this enhancing structure is crucial to signal the translation start codon, since its base pairing disruption gives rise to low accuracy for AUGi selection [5,7]. Similar elements have also been described for dengue and West Nile viruses [32,33]. In fact, it has been proposed that the hairpin element present in the capsid coding region of the dengue virus directs AUG selection [32]. Consistent with these findings, disruption of the hairpin structure in the Met-Ala variant abolishes the recognition of GCG and protein synthesis in this variant starts at the first AUG present in the sg-mRNA. Although inefficient, this mechanism used by SV sg-mRNA for initiation at a non-AUG codon is intriguing. We are tempted to speculate that perhaps this hairpin structure acts in a way akin to other structures found in IRES sequences; particularly the one studied with the dicistrovirus IRES [34,35]. In this case, a hairpin mimics a deacylated tRNA able to interact with the empty P site of the ribosome. The potential advantage for efficient translation conferred by the hairpin structure on sg-mRNA compared to mRNAs may provide new clues that help understand the molecular mechanism of the shut-off of host translation. Thus, certain eIFs might not be needed for sg-mRNA translation because of a viral protein. Both the sg-mRNA structure and viral proteins would determine the mechanism by which this viral mRNA is translated. PLoS ONE | www.plosone.org 8 March 2009 | Volume 4 | Issue 3 | e4772