Figure 3.

Destabilization of the 3' TAR hairpin affects polyadenylation. (A) In the HIV-rtTA-SV40 constructs the SV40 polyadenylation site was placed downstream of the viral genome. The position of the oligonucleotides that were used as primer in the RNA analyses (panels E and F) are indicated. (B) C33A cells were transfected with 5', 3' and 5'+3' mutated constructs and the CA-p24 level in the culture medium was measured after culturing with dox for 48 h. Average values obtained in three transfections are shown, with the error bars indicating the standard deviation. (C) Intracellular RNA was isolated and analyzed by Northern blotting with a probe against the U3/R region of HIV-rtTA. The position of the 18S and 28S rRNA bands, and the unspliced (9 kb), single spliced (4 kb) and double spliced (2 kb) viral transcripts are indicated. (D) The Northern blot was stripped and rehybridized with a probe against the downstream SV40 sequences. Only the extended RNA transcripts observed for the variants with a 3' A or 3' B mutation hybridized with this probe. The residual staining of the normally sized transcripts is due to incomplete stripping of the blot. (E) The isolated RNA was used as template for the production of viral cDNA. The cDNA products were amplified with indicated primers for the unspliced (1+2), single-spliced (3+4) and double-spliced transcripts (3+5). (F) Polyadenylation site usage was analyzed by PCR amplification of the cDNA with primers 6 and 7. Polyadenylation at the 3' LTR results in a 939-bp product, whereas polyadenylation at the SV40 sequence results in a 1215-bp product. For constructs with the A, B and AB deletion in the 3' TAR hairpin, these fragments will be 14, 10 and 24 bp shorter, respectively. The identity of these PCR products was confirmed by sequence analysis. (G) The polyadenylation efficiency at the 3' LTR was calculated by quantification of the 2 kb RNA bands in Fig. 3C.