Characterisation of VSV-G expression plasmids. A) Map of VSV-G expression plasmids. Wild type (wt) or codon optimised (syn) open reading frames of VSV-G 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 pIGwt vector contains intron A and flanking untranslated exonic regions E1 and E2 of the cytomegalovirus immediate early gene. In pIΔIGwt the exon boundaries were precisely fused by deleting the intron. B) Northern blot analysis. Cells were cotransfected with the indicated VSV-G expression plasmids, a codon optimised HIV-1 gag-pol expression plasmid (Hgpsyn) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Poly(A) RNA was isolated from transfected cells and analysed by Northern blot with a probe spanning the transcribed region of the BGH poly(A) signal present on all VSV-G transcripts and the positive 5 kb HIV-1 gag-pol transcript. C) Western blot analyses. Cells were cotransfected with the indicated VSV-G expression plasmids, an SIV gag-pol expression plasmid (SgpΔ2) and the lentiviral vector construct VICGΔBH containing a GFP expression cassette. Monoclonal antibodies to HIV-1 p24 capsid protein, which is cross reactive to SIV p27, or to VSV-G, respectively, were used for detection of the viral proteins in lysates of transfected cells.