In vitro translation in HeLa S3 cell extracts Translations were carried out in HeLa S3 cell extracts treated with micrococcal nuclease (a gift from E. Wimmer, Department of Molecular Genetics and Microbiology, Stony Brook University, NY, USA) and programmed with mRNAs synthesized by in vitro transcription. The preparation of extracts was essentially as described by Molla et al. [48]. Reactions were incubated at 30uC for 30 min in presence of 1 mg of MBP-2A [49] for each 10 ml HeLa S3 cell extracts to induce cleavage of eIF4G or in presence of 1 mg of MBP as control. To induce phosphorylation of eIF2a, extracts were treated with 0.5 mg/ml poly I:C also for 30 min. Subsequently, 100 ng of different transcript RNAs were added and reactions left to run for 1 hour at 30uC. Protein production was estimated by measuring luc activity. In the case of radioactive labelling, 0.7 mCi/ml of [35S]Met-Cys was added to the reaction mix and the synthesized proteins were analyzed by autoradiography of SDS-polyacrylamide gels. Analysis of protein synthesis by radioactive labelling BHK cells were seeded into 24-well plates at a concentration of about 105 cells/well. At the times indicated for each experiment, the media were removed and proteins were labelled for 30 min with 0.2 ml DMEM without methionine-cysteine supplemented with 2 ml EasyTagTM EXPRESS 35S Protein Labeling mix, [35S]Met-Cys (14 mCi/ml; Perkin Elmer) per well or with 0.2 ml DMEM without cysteine supplemented with 4 ml [35S]Cys (1 mCi/ml; Perkin Elmer) for 60 min. The cells were then collected in the appropriate gel loading buffer and analyzed by autoradiography of SDS-polyacrylamide gels. Measurement of luciferase activity Cells were lysed in a buffer containing 0.5% Triton X-100, 25 mM glycylglycine (pH 7.8), and 1 mM dithiothreitol. Lucifer- ase activity was determined using a Monolith 2010 luminometer (Analytical Luminescence Laboratory), using the Luciferase Assay System (Promega). Luc activity results are means6s.d. of three representative experiments performed in triplicate. IEF and western blot analysis Isoelectric focusing was carried out as described [7,50]. Rabbit anti-eIF2a antibody (Santa Cruz) was used to detect eIF2a. The other antibodies used in western blot experiments were rabbit antisera raised against firefly luciferase (Promega), rabbit anti- eIF4GI [51], rabbit antisera raised against the N-terminal and C- terminal region of eIF4GII (a gift from N. Sonenberg, McGill University, Montreal, Canada), and monoclonal anti-a-tubulin (Sigma). Moreover, anti-C antibody was obtained by immuniza- tion of a rabbit with purified nucleocapsids from SV infected BHK cells. Cell processing for electron microscopy (EM) At 8 hpi, cells were fixed with 2% glutaraldehyde in 0.2 M HEPES buffer (pH 7.4) for 1 h at room temperature and immediately scraped off the plate. For conventional EM, cells References mRNA Translation without eIFs were washed twice and resuspended in 0.2 M HEPES buffer (pH 7.4). After fixing, dehydrating, and infiltrating the cells with Epon, thin sections were obtained and stained with uranyl acetate and lead citrate. For immunoelectron microscopy, cells were processed by freeze substitution. Immunogold localization of SV capsid protein was done by placing the ultrathin sections on drops of different solutions. After a 30-min incubation with TBG (TBS (Tris-ClH 30 mM, ClNa 150 mM, pH 8.2) plus 0.1% BSA and 1% cold water fish skin gelatine), sections were floated for 1 h on a drop of anti-C antibodies diluted in TBG. Next grids were washed in TBS plus 0.1% BSA (365 min) and then exposed to 10 nm colloidal gold conjugated goat anti-rabbit IgG diluted in TBG for 1 hour. Grids were then washed consecutively with TBG, TBS, and distilled water (5 min each) before staining with a saturated solution of uranyl acetate followed by lead citrate. Immunofluorescence microscopy BHK cells were seeded on coverslips and infected with SV (100 pfu/cell). At 8 hpi cells were fixed in 4% PFA for 15 min, washed twice with PBS, and then permeabilized for 10 min with 0.2% Triton X-100 in PBS. All antibody incubations were carried out for 1 h in PBS containing 0.1% FCS and 0.1% Triton X-100. Coverslips were washed three times with PBS between primary and secondary antibody incubations, mounted in ProLong Gold anti-fade reagent (Invitrogen) and finally examined with a Radiance 2000 (Bio-Rad/Zeiss) confocal laser scanning micro- scope. Primary antibodies used were mouse monoclonal anti- bromodeoxyuridine BrdU (Affinity BioReagents), rabbit polyclon- al anti-C, mouse monoclonal against the carboxy terminal end of P ribosomal protein [52] (a gift from J.P. Garc´ıa Ballesta, Centro de Biolog´ıa Molecular Severo Ochoa, Madrid, Espa n˜a), goat polyclonal anti-eIF3 p110 (C-20) (Santa Cruz Biotechnology, Inc.), goat polyclonal anti-EF-2 (P-19) (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-eIF4E (P-2): sc-9976 (Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-eIF2a (FL-315), sc- 11386 (Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti- eIF4GI [51] and goat anti-TIA-1 (Acris Antibodies GmbH). Specific antibodies conjugated to Alexa 555 or Alexa 488 were used as secondary antibodies. Supporting Information Figure S1 Found at: doi:10.1371/journal.pone.0004772.s001 (4.07 MB EPS) Figure S2 Found at: doi:10.1371/journal.pone.0004772.s002 (8.12 MB EPS) Figure S3 Found at: doi:10.1371/journal.pone.0004772.s003 (6.01 MB EPS) Author Contributions Conceived and designed the experiments: LC. Performed the experiments: MAS AC JJB. Contributed reagents/materials/analysis tools: IV. Wrote the paper: MAS LC. 1. Strauss JH, Strauss EG (1994) The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58: 491–562. 2. Ou JH, Rice CM, Dalgarno L, Strauss EG, Strauss JH (1982) Sequence studies of several alphavirus genomic RNAs in the region containing the start of the subgenomic RNA. Proc Natl Acad Sci U S A 79: 5235–5239. 3. Li ML, Stollar V (2007) Distinct sites on the Sindbis virus RNA-dependent RNA polymerase for binding to the promoters for the synthesis of genomic and subgenomic RNA. J Virol 81: 4371–4373. 4. Frolov I, Schlesinger S (1994) Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells. J Virol 68: 1721–1727. 5. Frolov I, Schlesinger S (1994) Translation of Sindbis virus mRNA: effects of sequences downstream of the initiating codon. J Virol 68: 8111–8117. 6. Frolov I, Schlesinger S (1996) Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation. J Virol 70: 1182–1190. PLoS ONE | www.plosone.org 11 March 2009 | Volume 4 | Issue 3 | e4772