glycoproteins but not into C protein. Notably, although ineffi- ciently incorporated radioactive cysteine into SV-C-Met/Cys, it could be detected both in glycoproteins and in C protein. The mobility of the labelled C protein suggests that translation initiates at the UGU codon. We subsequently examined C synthesis using sg-mRNAs prepared in vitro in both uninfected cells and Hela S3 extracts. In the first case the different sg-mRNAs were electroporated into BHK cells and, at three hpe, C production was determined by western blot analysis (Fig 3C, upper panel). In the second case translation was examined by radioactive labelling of Hela S3 extracts programmed with different mRNAs (Fig 3C, lower panel). The results from both experiments coincide. Only one product is synthesized from each mRNA analyzed and these products are always generated from translation initiation at the first AUG present at their respective sequences. Once again, as occurred after eIF4G cleavage or arsenite treatment, a different transla- tional behaviour of sg-mRNA is observed according to whether it is transcribed by the viral machinery or directly electroporated into uninfected cells or translated in HeLa cell extracts. In conclusion, canonical initiation takes place when the sg-mRNA is translated out of the infection context, while perhaps initiation may occur even at non-AUG codons during infection. Modifica- tions of AUGi to other codons in hepatitis C virus mRNA has little effect on its translability [27]. In this regard, SV sg-mRNA shares some similarities with the behaviour observed in hepatitis C virus mRNA. Although Met/Ala mutant is a weak initiator at GCG, it is notable that this variant is still capable of significantly recognizing the GCG codon. We reasoned that the hairpin present in the C sequence probably pointed to the site where initiation starts, even if the AUG codon has been replaced by GCG. To analyze this possibility a new construct was generated that combines DDLP and Met/Ala mutations. BHK cells were electroporated with the different replicons prepared in vitro and C production was analyzed by western bloting (Fig 3D, left panel). Significantly, synthesis of C protein with the new variant SV-C-DDLP/Met/Ala differs from SV-C-Met/Ala because only the fastest migrating C product is synthesized. Therefore, disruption of DLP structure abrogates translation initiation at the non-canonical GCG codon. Thus, the DLP structure could signal the translation start codon in the infection context. In agreement with the above experiments one product could be detected (Fig 3D, right panel) when translation of this new sg-mRNA was tested in uninfected cells only. Notably, truncated C production was higher from C –DDLP/Met/Ala mRNA than from C-Met/Ala mRNA. Perhaps, because the first AUG from C-Met/Ala mRNA is in the hairpin sequence that contains a very stable secondary structure (see Fig 3A) in a canonical initiation, this hairpin may hamper the scanning process to select the AUGi, whereas the non-structured DDLP sequence would make the AUGi more accessible. Location of several eIFs and ribosomes in SV-infected cells We recently provided evidence that transcription and transla- tion are coupled in SV-infected cells [22]. In such a case, we would predict that viral translation takes place in cytoplasmic regions close to transcription and replication factories. Indeed, electron microscopy of SV-infected cells shows that nucleocapsids are assembled around membranous structures localized at discrete sites in the cytoplasm (Fig S2). Some of these membranous structures resemble replication factories (Fig S3). Our first goal was therefore to determine whether viral nucleocapsids co-localize with active transcription sites. To this end, SV-infected cells were mRNA Translation without eIFs labeled with bromouridine (BrU) in presence of actinomycin D at 6 hpi. Fixed cells were then incubated with specific antibodies against C protein and BrU and analyzed by immunofluorescence (Fig 4, upper panel). Transcription sites detected by anti-BrU antibodies did indeed co-localize with C protein. The next step was to examine co-localization of different translation factors with C protein (Fig 4, lower panel). The subcellular distribution of p- 110 subunit of eIF3 in SV-infected cells differs from that observed in uninfected BHK cells. In uninfected cells eIF3 is uniformly distributed throughout the cytoplasm, whereas this factor is concentrated in the region of the nucleus in SV-infected cells. Moreover, eIF3 co-localizes with SV C protein. Translation elongation factor eEF2 has a localization and behaviour similar to eIF3, whereas eIF4E does not modify its distribution. To determine whether ribosomes are redistributed during infection, a monoclonal antibody against the carboxy terminal end of P ribosomal protein was employed. Figure 5 shows that ribosomes appear concentrated near the nucleus in SV-infected cells but are spread throughout the entire cytoplasm in control BHK cells. In SV-infected cells, most of the cytoplasm is devoid of ribosomes, which are concentrated close to the nucleus, near and overlapping the C protein signal (see in more detail lower panels in Fig 5). These findings suggest that components of the protein synthesizing machinery are redistributed after SV infection, localizing to a perinuclear region enriched in C protein, where viral transcription is taking place. As shown in this work and in previous articles, synthesis of SV late proteins in infected cells can take place without operative eIF4G and eIF2a [7,14]. For this reason, it was of interest to analyze the distribution of these initiation factors after SV infection. Both eIF2a and eIF4GI modified their location in SV- infected cells, as compared to the uninfected counterparts. Thus, eIF2a concentrates in a region near the nucleus devoid of ribosomes, presumably the centrosomal region (Fig 6, upper panel). On the other hand, most of eIF4GI is found in cytoplasmic granules (Fig 6, upper panel), which are most probably stress granules since both eIF4GI and TIA markers co-localize in SV- infected cells (Fig 6, lower panel). The presence of eIF2a and eIF4GI in places other than those enriched in ribosomes and other translation factors is consistent with the idea that these two factors do not participate in the initiation of translation of SV sg-mRNA. Discussion Viruses have evolved special mRNA structures that confer high translatability under conditions where cellular protein synthesis has been abated. The most studied of these structures are IRESs, which direct the internal initiation of translation instead of the typical cap recognition step [16,28,29]. Viral mRNAs that contain an IRES element exhibit special requirements as regards the integrity of initiation factors. In particular, eIF4G cleaved by some picornavirus or retrovirus proteases can still participate in the initiation of translation on mRNAs bearing an IRES element [30,31]. Interestingly, SV sg-mRNA, which is devoid of an IRES and contains a cap structure at its 59 end, is still translated when eIF4G is cleaved by poliovirus 2Apro or HIV PR [14]. Most notably, we now present evidence that the SV sg-mRNA requires eIF4G to be intact when it is translated in uninfected cells or in cell-free systems. These findings support the idea that this sg- mRNA is translated following the canonical mechanism in uninfected cells, such that there is a cap recognition step. Cleavage of eIF4G abrogates the cap recognition necessary for translation of SV sg-mRNA in uninfected cells. Surprisingly, the eIF4F complex and therefore cap recognition do not seem to be operative in SV- PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4772