Figure 2

3'ss A7 is nonessential for RNA stability and Rev responsiveness. (A) Schematic drawing of the HIV-1 genome and of the subgenomic env expression plasmid SV E/X tat^- rev^-. LTR: long terminal repeat, SV40: SV40early promoter, pA: SV40 polyadenylation sequence. Nucleotide sequences of the 5'ss D4 and its mutations D4^- and -1G3U as well as the 3'ss A7 and its mutations A7^- and A7⁺ are shown beneath. The splice sites (grey squares, including the minor 3'ss 7a and 7b), the reported or supposed branch point sequence (bold, asterix indicates the branch point nucleotide) and the mutated nucleotides (underlined) are marked. In the 3'ss mutants the reading frame was kept unchanged except for position 703 (Val→Ala) in A7⁺. (B) HeLa-T4⁺ cells were transiently transfected with the subgenomic env expression plasmids (SV E/X tat ^- rev ^-) containing either the wild type 5'ss D4 or the non functional D4^- mutation combined with either the wild type 3'ss A7 or the A7^- mutation in presence or absence of a Rev expression plasmid (SVcrev) as indicated above the lanes. The poly(A)⁺ RNA was analyzed by Northern blotting. s: spliced, us: unspliced transcript. Transfection efficiency was monitored by co-transfection of a human growth hormone (hGH) expressing plasmid (pXGH5).