Reduced packaging of unspliced RNAs into virions upon opening of the 5′ TAR structure. (A) C33A cells were transfected with 5′ TAR-mutated HIV-rtTA plasmids and cultured with dox for 48 h. The TAR mutations did not affect virus protein and particle production  and the culture supernatant contained similar amounts of virus particles (based on the CA-p24 level). Virus particles were purified from culture supernatant samples by ultracentrifugation. Viral RNA isolated from equal amounts of cells and purified virions (based on CA-p24) was analyzed by RT/PCR using primers (as indicated in Figure 2A) that specifically detect unspliced (primers 1 + 2), single spliced (3 + 4) and double spliced transcripts (3 + 5). (B) The cellular and virion RNA was subsequently analyzed by denaturing Northern blotting. The position of the 18 S and 28 S rRNA bands (determined by ethidium-bromide staining; not shown), and the unspliced (9 kb), single spliced (4 kb) and double spliced (2 kb) viral transcripts are indicated. M: mock transfected cells. The arrow at the right side indicates the position of the double spliced RNAs that are visible in the A5 and B5 virion samples. *, 12-kb unspliced RNAs. As previously observed [17,], part of the viral transcripts produced from the HIV-rtTA plasmids is not polyadenylated at the 3′ LTR, which results in RNAs that are extended with vector sequences. Most of these read-through RNAs will be polyadenylated at the SV40 polyadenylation signal that was inserted immediately downstream of the 3′ LTR [17.]. However, a fraction of the transcripts is also not polyadenylated at this position, but at the downstream 5′ LTR in the circular constructs, which results in the production of 12-kb RNA fragments. In agreement with this interpretation, we previously observed that removal of the SV40 polyadenylation signal increased the 12-kb RNA level [17.]. **, 20-kb unspliced RNAs. As previously shown, the A and B mutations reduce the activity of the adjacent polyadenylation signal by stabilizing the polyA hairpin [17.]. Accordingly, the A5 and B5 mutation reduce polyadenylation of the read-through transcripts at the 5′ LTR. Subsequent polyadenylation of these read-through transcripts at the 3′ LTR or adjacent SV40 polyadenylation site can explain the production of 20-kb RNAs. These 12-kb and 20-kb RNAs form a small part of all viral RNAs, and their presence in both cells and virions is proportional to the presence of unspliced RNAs, which indicates that they are packaged with similar efficiency as the unspliced RNAs. (C) The profiles of the lanes from the Northern blots shown in panel B were analyzed with 1-D geI analysis software (ImageQuantTL). The position of the unspliced (us), single spliced (ss), and double spliced (ds) viral transcripts, and the 12-kb (*) and 20-kb (**) RNAs are indicated. The inserts in the right panels zoom in on the position of the double spliced RNAs, to demonstrate their presence in the A5 and B5 virion samples (indicated with arrow).