Another point of interest that should be taken into account to interpret our present results is the coupling between transcription and translation of viral mRNAs [22,36,37,38]. Thus, in SV- infected cells the only mRNAs that are translated in the late phase are those synthesized by the viral transcription machinery, while electroporated viral mRNAs with the same structure are ignored by the translational apparatus [22]. Several years ago it was proposed that cytopathic vacuoles constitute the sites where not only viral RNA synthesis but also translation takes place in SV- infected cells [39]. Certainly, we have now observed by electron microscopy that viral nucleocapsids are assembled at membranous structures in close connection with replication factories. Some of these membranous structures have invaginations that form spherules previously described as cytopathic vacuoles I. These vacuoles have numerous capsids attached and are located at the same sites as replicative complexes (Fig S3). Apart from these places very low C signal was evidenced in the cytoplasm by immuno-gold analyses (Fig S2). The co-localization of replication complexes and C protein was also determined by confocal microscopy (Fig 4A). In addition, our present findings reveal that some translation factors as eIF3 or eEF2 and ribosomes co-localize with the nucleocapsid aggregation sites (Fig 4B). This phenomenon may be due to the fact that protein synthesis takes place at discrete foci in SV-infected cells, where ribosomes are recruited and coincide with the sites in which viral genome replication is taking place. Consistent with our results, proteomic studies of replicative complexes evidence the presence of ribosomal proteins [40]. By contrast, eIF4G and eIF2 are excluded from SV replication sites, such that eIF4G localizes to stress granules as occurred in SFV- infected cells [8], while eIF2 presumably appears in the centrosomal region. The findings that viral transcription and translation are coupled and take place at discrete cytoplasmic regions, agrees well with the recent findings on vaccinia virus infected cells [41,42]. However, it should be stressed that the mechanism of viral translation may differ according to the animal virus considered. For instance eIF2 and eIF4G are excluded in SV-infected cells but could be necessary for translation of vaccinia virus mRNAs. In summary, it seems that translation of SV genomic mRNA gives rise to the formation of replication factories in modified membranous vesicles early during infection. These vesicles are concentrated in a perinuclear region as infection progresses. Translation components such as ribosomes and some factors accumulate close to these vesicles where they can participate in viral protein synthesis. Thus, viral transcription produces genomic RNA prone to encapsidation and sg-mRNA translation in a limited cellular space, favouring the interactions between viral RNAs and different cellular and viral proteins. Materials and Methods Cell line and viruses Baby hamster kidney (BHK-21) cells and SV were used to perform the experiments. SV virus stock was prepared from a pT7 SVwt infective cDNA clone (where wt is wild type) [43]. Viral infection of BHK cells was carried out in Dulbecco’s modified Eagle medium (DMEM) without serum for 40 min to permit virus attachment. This medium was then removed and infection continued in DMEM with 10% fetal calf serum. Plasmids and recombinant DNA procedures pT7 SVwt was used as the parental plasmid for all of the constructs. pT7 rep C+Luc and pT7 C+luc [22], pKs-Luc and pTM1-2A [23], pcDNA-Luc [44] and pTM1-Luc [45] have been described previously. pcDNA C+Luc was generated by cloning the mRNA Translation without eIFs sg-C+Luc sequence from pT7 rep C+Luc in pcDNA. To this end a double PCR product digested with Mlu I and Xho I was inserted into the same sites of pcDNA. The double PCR product was prepared as follows: for the first PCR primers 59Mlu pcDNA and 39joint pcDNA-L26S (the sequences are shown below) were used with pcDNA as DNA template, and for the other PCR, primers 59joint pcDNA-L26S and 39Xho SV were used with pT7 rep C+Luc as DNA template; a mixture of these products and 59Mlu I pcDNA and 39Xho SV as primers were then used for the second PCR. Mutants in the AUGi pT7 SV-C-Met, Arg/Lys,Lys; pT7 SV-C-Met/Ala and pT7 SV-C-Met/Cys were prepared by inserting the corresponding double PCR product digested with Hpa I and Aat II enzymes into the same sites of pT7 SVwt. pT7 SVwt was always used as DNA template and the oligonucleotides employed are shown below. The first PCR products were obtained using 59Hpa I SV as 59 oligonucleotide and one of the different 39mut oligonucleotides as 39 oligonucleotide; the other PCR product was prepared using one of the different 59mut oligonucleotides as 59 oligonucleotide and 39Aat II SV as 39 oligonucleotide. The double mutant pT7 SV DDLP/Met/Ala was prepared in the same way, but using 59Hpa I SV and 39mut Met/ Ala oligonucleotides with pT7 SV DDLP as DNA template and 59mut Met/Ala and 39Aat II SV with pT7 SV-C-Met/Ala as DNA templates for the first PCRs. The constructs pT7 C-wt, pT7 C-DDLP, pT7 C-Met/Ala and pT7 C-DDLP/Met/Ala were designed to produce the corresponding sg-mRNAs by in vitro transcription. To this end we used the oligonucleotides 59SacI- T7prom and 39Aat II SV and the different pT7 SV constructs as DNA template. These PCR products were subsequently digested with Sac I and Aat II enzymes and inserted into the same sites of pT7 SV rep C [46]. Oligonucleotides 59Mlu I pcDNA: ccgatatacgcgttac; 39joint pcDNA-L26S: gaaagt- tactatgctgactagttagccag agagctctg; 59joint pcDNA-L26S: ca- gagctctctggctaactagtcagcatagtacatttc; 39Xho SV: atta attcccctcgag- gaattccc; 59Hpa I SV: ggccgggcccgttaaccggtctgatgatc; 39Aat II SV: gttcttgac gtcgaacaatct;59mut Met/Ala: caccaccgcgaatagaggattctt- taacgcgctcggcc; 39mut Met/Ala: g gccgagcgcgttaaagaatcctc- tattcgcggtggtg; 59mut Met,Arg/Lys,Lys: caccaagaaaggattcttttact tgctcggccgc; 39mut Met,Arg/Lys,Lys: ggcggagcaagtaaaa- gaatcctcttttcttggtggtg; 59mut Met/Cys: caccaccacctgtaatagaggattctt- taaacagctcggccgcc; 39mut Met/Cys: ggcggccgagct gtttaaagaatcctc- tattacaggtggtggtg; 59SacI-T7prom: gcgcgcgagctctaatacgactcactat- agatagtc agcatagt. In vitro transcription and transfection Plasmids were used as templates for in vitro RNA transcription with T7 RNA polymerase (Promega). The transcription mixture always contained an m7G(59)ppp(59)G cap analog except when mRNAs containing EMCV IRES were prepared. When pKS-Luc, pTM1-Luc or pTM1-2A plasmids were used as templates, in vitro polyadenylation was performed with poly(A) polymerase (Invitro- gen). For transfection, subconfluent BHK cells were harvested, washed with ice-cold phosphate-buffered saline, and resuspended at a density of approximately 2.56106 cells/ml in the same buffer. Subsequently, 20 mg of in vitro-transcribed RNA were added to 0.4 ml cell suspension and the mixture was transferred to a 2-mm cuvette. Electroporation was carried out at room temperature by generating two consecutive 1.5-kV, 25-mF pulses with a Genepulser apparatus (Bio-Rad), as previously described [47]. Plasmids were transfected using Lipofectamine 2000 reagent (Promega) as recommended by the supplier. PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4772