eIF4G. Thus, influenza virus PB2 displaces eIF4E after its interaction with eIF4G [18], while adenovirus 100 K protein renders eIF4G unable to interact with Mnk1, leading to eIF4E dephosphorylation [19]. In addition, rotavirus NSP3 binds to both the 39 end of viral mRNAs and eIF4GI, displacing its interaction with PABP and promoting circularization of rotavirus mRNAs [20,21]. Although synthesis of SV late proteins does not need active eIF2, nor intact eIF4G or PABP, sg-mRNA in the cytoplasm of SV-infected cells will not necessarily be translated. This mRNA is not recognized by the protein-synthesizing machinery after its in vitro synthesis and electroporation in SV- infected cells. Moreover, when genuine sg-mRNAs synthesized in the infected cells are extracted and then electroporated back into the cells, they are excluded from translation in SV-infected cells but can direct protein synthesis in uninfected cells [22]. In the present work we show that SV sg-mRNA is translated according to the canonical model in uninfected cells or in cell-free systems, while this mRNA does not need certain initiation factors for translation when synthesized by the viral transcription machinery. In addition, a different initiation site is selected when mRNA is synthesized in the infected cells and when it is directly electropo- rated into uninfected cells or translated in cell-free systems. In both cases, the primary structure of SV sg-mRNA is the same, but the cellular context dictates the exact mode of initiation. We also show that, in infected cells, some translation factors and ribosomes migrate to transcription sites whereas others are excluded. These findings are consistent with the concept that translation is tightly coupled to transcription in virus infected cells [22]. Results Intact eIF4G is necessary for translation of SV sg-mRNA in uninfected BHK cells, but is dispensable in SV-infected cells We previously reported that SV sg-mRNA can be translated when eIF4G is cleaved by viral proteases [14]. Initially two types of mRNAs were generated by in vitro transcription, that is, sg- C+Luc mRNA from pT7 C+Luc plasmid [22] and the replicon rep C+Luc [22]. After transfection, rep C+Luc gives rise to the sg- C+Luc mRNA using the SV transcription machinery. In the case of sg-C+Luc mRNA from pT7 C+Luc plasmid, after electropo- ration, sg-mRNA will be translated in uninfected cells and whereas in the other it will be translated in an environment that resembles the infected cells because there is viral replication and transcrip- tion. Translation of C+Luc mRNA renders a fusion protein that releases C protein and luciferase (luc) through proteolytic activity of C protein. To induce cleavage of translation initiation factor eIF4G, PV 2Apro was expressed on electroporation of synthesized IRES-2A mRNA. This mRNA contains the EMCV IRES followed by the PV 2Apro gene (IRES-2A) [23,24]. BHK cells were then electroporated with IRES-2A mRNA or transcription buffer as control. At 2 hours post-electroporation (hpe), cells were once again electroporated with in vitro transcribed C+Luc mRNA using cap-Luc-poly(A) or IRES-Luc-poly(A) mRNAs as controls (Fig. 1A). eIF4GI and eIF4GII are both already proteolyzed by 2Apro at 2 hpe (Fig 1A). Under these conditions cap-Luc-poly(A) mRNA translation was strongly inhibited at 4 hpe, suggesting that cap- and poly(A)-dependent translation was hampered in IRES-2A electroporated cells (Fig 1A). A similar situation occurs with C+Luc mRNA translation, which is also deeply inhibited after eIF4G cleavage (Fig 1A). These results support the idea that SV sg-mRNA is translated by a cap- and poly(A)-dependent mechanism in uninfected BHK cells. mRNAs that contain EMCV IRES are able to drive translation even when eIF4G is cleaved by mRNA Translation without eIFs 2Apro [24]. Luc activity measured in cells electroporated with IRES-Luc increased consistently throughout the experimental period, irrespective of 2Apro-expression and eIF4G cleavage (Fig 1A). Similar findings were obtained in HeLa cells (data not shown). In addition, the effect of eIF4G cleavage on the translation of C+Luc mRNA was assayed in HeLa S3 cell extracts. To achieve eIF4G cleavage before mRNA addition, 1 mg of purified MBP- 2Apro was added to extracts for 30 min at 30uC, followed by translation of the different mRNAs. Luc activity was then estimated (Fig 1B, upper panel), along with hydrolysis of eIF4G (Fig 1B, lower panel). As with uninfected cells, cleavage of eIF4G strongly inhibits translation both of cap-Luc-poly(A) and C+Luc mRNAs whereas protein synthesis directed by IRES-Luc mRNA was stimulated. We then determined the translational behaviour of C+Luc mRNA produced from the replicon rep C+Luc. In this case cells were first electroporated with rep C+Luc mRNA prepared in vitro and electroporated once again with IRES-2A mRNA two hours later. At 1, 3 or 5 hours after the second electroporation cultures were collected and luc production was determined by measuring luc activity (Fig 1C) and also by western blot analysis employing anti-luc antibodies (Fig 1C). The integrity of eIF4GI and eIF4GII was analyzed in parallel (Fig 1C). Translation of sg-C+Luc mRNA synthesized from the replicon rep C+Luc was not impaired when eIF4G was cleaved by 2Apro expression (Fig 1C). These findings are in good agreement with the results obtained in SV-infected cells [14]. Taken together these data support the notion that SV sg-mRNA is translated differentially in infected and uninfected cells, as regards requirement for eIF4G integrity. Differential inhibition of SV sg-mRNA translation by arsenite in uninfected or SV-infected cells Arsenite is widely used to induce phosphorylation of eIF2a, leading to the inhibition of translation [13,25,26]. Furthermore, culture cells infected by SV exhibit high levels of phosphorylated eIF2a at times when viral structural proteins are being synthesized [7,9]. We therefore compared the eIF2a requirement for the translation of C+Luc mRNA both in uninfected and SV-replicating cells. First, we assayed the effect of arsenite on SV infection by analyzing protein synthesis in cultures treated with different concentrations of this compound (Fig 2A). Arsenite treatment blocked protein synthesis in control BHK cells in a dose-dependent manner. Thus, actin synthesis decreases by 48 and 83% on treatment with 50 and 200 mM arsenite respectively. However, in infected cells, synthesis of C protein is only reduced by 8 and 29%, respectively, after the same treatment. Moreover, as a consequence of arsenite activity at the endoplasmic reticulum, viral glycoprotein processing was affected, such that the precursor PE26KE1 was not cleaved to produce mature products PE2, 6K and E1. The activity of arsenite on the translation of C+Luc mRNA was subsequently tested. To this end in vitro synthesized rep C+Luc or C+Luc mRNAs were electroporated into BHK cells. As with SV- infected cells, addition of arsenite had no effect on the translation of sg-mRNA derived from rep C+Luc (Fig 2B, upper panel). The phosphorylation state of eIF2a was analyzed in both control and rep C+Luc electroporated cells in the absence or presence of arsenite (Fig 2B, lower panel). This compound induced phosphorylation of eIF2a in control BHK cells. Moreover, in agreement with previous findings [7] almost total phosphorylation of eIF2a appeared in rep C+Luc transfected cells, both in absence or presence of arsenite. The translation of C+Luc mRNA directly transfected into uninfected cells was quantified by measuring luc activity at 90 minutes post-electroporation (mpe). Interestingly, translation of C+Luc mRNA was strongly blocked by arsenite in uninfected cells PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4772