Opening of the 5′ TAR hairpin affects packaging of viral RNAs. (A) The position of the primers used in the RT/PCR analyses is indicated. (B) C33A cells were transfected with the 5′ + 3′ and 3′ mutated HIV-rtTA constructs and RNA from cells and culture supernatant was isolated after culturing with dox for 48 h. The TAR mutations did not affect virus particle production  and the culture supernatant samples contained similar amounts of virus particles (based on the CA-p24 level). RNA isolated from equal amounts of cells (left panels) or virus particles (right panels) was used as template for cDNA synthesis and the cDNA products were amplified with primers that specifically detect unspliced (primers 1 + 2) and double spliced transcripts (3 + 5). The identity of the PCR fragments was confirmed by sequence analysis. The band intensity corresponds with the relative amount of unspliced (upper panels) or spliced (lower panels) viral RNA present in the cells (left panels) or virions (right panels) and this assay thus allows a comparison of the level of these RNAs produced by the wild-type and TAR-mutated constructs. Because the primers, size and sequence of the PCR fragments differ for the unspliced and spliced RNAs, the band intensity of the unspliced RNA cannot be compared with that of the spliced RNAs and this RT-PCR technique does not show the absolute amount of unspliced and spliced RNAs.