mRNA Translation without eIFs Figure 4. Subcellular localization of SV replicative complexes and different viral and cellular proteins. Panel A. Co-localization of viral transcription complexes and nucleocapsid aggregation sites in SV-infected cells. BHK cells were infected with SV (100 pfu/cell) and, at 7 hpi, the medium was supplemented with dactinomycin (2.5 mg/ml) for one hour. Cells were then transfected with a mixture of bromouridine (10 mM) and Lipofectamine 2000 reagent for 30 min. After this time, the transfection medium was replaced by 10% FCS supplemented with dactinomycin for 30 min. Immunofluorescence analysis was carried out as described in Materials and Methods. Panel B. Co-localization analyses of capsid protein and translation factors in SV-infected cells. BHK cells were infected with SV (100 pfu/cell) and, at 8 hpi, cells were processed for immunofluorescence. doi:10.1371/journal.pone.0004772.g004 infected cells during late viral protein synthesis. A similar picture also emerges for the participation of eIF2 during sg-mRNA translation. This factor is not required for the translation of SV sg- mRNA because it is highly phosphorylated in the infected cells. However in uninfected cells or in cell-free systems, phosphoryla- tion of eIF2 induced by arsenite or poly I:C treatment strongly blocks protein synthesis directed by SV sg-mRNA. The new concept that arises from these findings is that the structure of a given mRNA does not suffice to dictate its translation mechanism. Thus, a viral mRNA, such as SV sg-mRNA, follows the canonical mechanism for the initiation of translation in uninfected cells or in cell-free systems, whereas the same mRNA exhibits different eIF requirements in virus infected cells. Therefore, some viral mRNAs may exhibit a dual mechanism for their translation, i.e. the PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4772