Figure 2

Comparison of antisense RNA transcripts generated in vivo from the HIVaINR with HIV-1 sense transcripts in the absence of Tat protein. (A) Map of landmarks in the HIV-1 LTR and the relative location of primers used for RT-PCR (not to scale). The DNA template used to synthesize the HIV-1 sense and antisense RNA internal standard IS+ is also diagrammed. (B) Analysis of in vivo transcripts from U38 cells, which have stably incorporated HIV-1 LTR-CAT sequences, clearly demonstrates intrinsic antisense RNA transcripts. Total RNA was obtained from U38 cells grown with and without transient stimulation with PMA and Ca ionophore (Stim +/-). U38 RNA samples were split and subjected to DNase treatments or DNase and RNase treatments, as indicated. Each sample was then analyzed by non-radioactively labeled RT-PCR for the presence of HIV-1 antisense RNA (expected product 183 bp) and for the presence of HIV-1 sense transcripts (expected size product 285 bp). Each set of RT-PCR reactions also included a titration of the internal standard IS+ RNA subjected to the identical RT-PCR reaction, as well as a primer alone control (pr), in which no RNA template was added. The IS+ titration is indicated above the lanes. Additional band(s) in the HIV antisense RT-PCR with stimulation (+) could represent antisense RNA self-primed with a 5' terminal hairpin and extending to predicted pseudoknot motifs (expected size 312, ~500). M lanes received biotinylated ø-X174 HinfI markers with bands from 24–726 bp in size. Triplet open arrowheads indicate 311, 249, and 200 bp in M, with the single arrow indicating antisense RT-PCR product (labeled as+) and single arrowhead indicating sense RT-PCR product (labeled s+). PCR + and – lanes received pHIV-CAT or no template with the same primers used for the corresponding RT-PCR. This analysis was performed in triplicate with similar results.