Characterisation of RSV-F expression plasmids. A) Map of RSV-F expression plasmids. Wild type (wt) or codon optimised (syn) open reading frames of RSV-F are flanked by the human cytomegalovirus immediate early promoter/enhancer region (CMV) and the bovine growth hormone poly(A) signal (pA). Angled black arrows mark the transcriptional start point. The pIFwt and pIFsyn plasmids contain intron A and flanking untranslated exonic regions E1 and E2 of the cytomegalovirus immediate early gene. In pIΔIFwt the exon boundaries were precisely fused by deleting the intron. B) Northern blot analysis. 293T cells were transfected with the indicated plasmids containing the RSV-F ORF. As a negative control the empty vector (pcDNA3.1) was also transfected and processed in parallel. A total RNA extract from RSV-infected HEp2-cells served as a positive control. Cytoplasmic (C) or total (T) RNA was isolated from transfected cells, separated by agarose gel electrophoresis and used for subsequent Northern blot analysis. Size separated RNA was stained with ethidiumbromide (EtBr) revealing non-degraded 18S and 28S ribosomal RNA bands (18S shown as representative). A DIG-labelled probe spanning 780 bp of the RSV-F ORF was used for hybridisation. C) Western blot analysis. 293T cells were transfected with the indicated plasmids. Equal amounts of protein were separated on an acrylamide gel for subsequent detection of RSV-F expression in Western blot analysis using a monoclonal antibody against the F protein. As a positive control, RSV-infected HEp2 cells were processed in parallel.