Figure 1

RTEm26-CTE is a potent combination of RNA transport elements. A) Structure of the gag reporter plasmid. The HIV-1 gag gene is flanked by the 5' and 3'LTRs providing promoter and polyadenylation signals, respectively. NLgag contains the major splice donor of HIV-1 located 5' to gag and a cryptic splice acceptor between RNA export elements and the 3'LTR and expresses HIV-1 gag [23, 24, 39]. The RTE structure [7] shows the nucleotide changes in mutant RTEm26 (nt 190–193 CACA changed to GCGG). The 226-nt RTE and the 173-nt CTE were inserted between the gag gene and the 3'LTR, generating the NLgagRTEm26-CTE. B) Expression of the gag reporter pNLgag plasmids, containing either no insert, RTEm26 or CTE alone, or the RTEm26-CTE combination. Cell extracts from transfected HeLa cells were analyzed for Gag production using an HIV-1 gag antigen capture assay. Gag expression is presented as fold induction as compared to the gag levels produced by pNLgag. Standard deviations are shown. C) Northern blots of total polyA-containing (top panel) and cytoplasmic (bottom panel) mRNAs from cells transfected with pNLgag or pNLgag containing RTEm26, CTE, or RTEm26-CTE were hybridized with a probe spanning the 3'end of the gag mRNAs [12]. Hybridization of the blot with a GFP probe serves as internal control of transfection efficiency and RNA preparation. The blots shown in the top and bottom panels are from two independent experiments. Note that the cytoplasmic poly-A mRNA samples are unequally loaded, and the CTE lane has 2.5-fold more GFP mRNA while the RTEm26-CTE lane has 60% of the GFP mRNA compared to the other lanes (no insert, RTEm26). The blots were quantitated using the STORM860 phosphoimager.