Unspliced and spliced viral RNAs with a stable TAR hairpin are more efficiently packaged than RNAs with an opened TAR structure. (A) C33A cells were transfected with wild-type HIV-rtTA (TARm), the B5 mutant or both (TARm/B5), and cultured with dox for 48 h. The intracellular and virion RNA was isolated from equal amounts of cells and culture supernatant that contained a similar amount of virus particles (based on the CA-p24 level), respectively, and used as template for cDNA synthesis. The cDNA products were amplified with primers that specifically detect unspliced (primers 1wt/B and 2+348), single spliced (1wt/B and 4) and double spliced transcripts (1wt/B and 5). The PCR product was digested with NarI and the fragment containing the TAR sequence, which is 10 nt smaller for the B variant, is indicated. When necessary, the number of PCR cycles was increased to visualize all small DNA products, which obscured some of the quantitative differences between the variants as seen in Figure 3. (B) Viral RNAs produced upon transfection of cells with the HIV-rtTA variants A5, AB5 or A5 plus AB5 (A5/AB5) were analyzed as described for panel A, but PCR primer 1wt/B was replaced by primer 1A/AB. This primer anneals to the 5′ end of the A5 and AB5 transcripts and the AB5 products are 10 nt smaller than the A5 products.