Figure 1

Identification of an antisense transcript originating from the HIV-1 antisense INR (HIVaINR). (A) The TAR sequence in HIV-1 DNA from the cap site (start of sense transcription) to +59 is shown. Sequence comprising the HIVaINR is indicated with a heavy overbar and reverse arrow, and this region of sequence (reversed) is compared to the terminal deoxynucleotidyl transferase (TdT) INR element [14] and the consensus INR[15]. Underlined sites in the HIV-1 TAR DNA indicate location of putative sense INR elements [19]. (B) Gel-purified DNA templates used for the generation of transcripts from the HIV-1 promoter and the HIVaINR. The original template is labeled "O" (650 bp) and contains the entire LTR from U3 through the PBS. Two additional truncated templates are designated "A"(270 bp) and "B"(350 bp). (Not to scale.) The primers U3, AvaII, and AvaI (forward arrows) and 510–530 (reverse arrow) are indicated above the original template (O). (C) Primer extension of purified RNA generated in vitro using the above HIV-1 LTR templates and Drosophila embryo nuclear extracts. cDNA products were produced from an antisense RNA annealing to a biotinylated sense 5'AvaI primer (213 bp, lanes 1 and 2) or biotinylated sense 5'AvaII primers (274 bp, lanes 5 and 6), but not with a biotinylated sense 5'U3 primer (lane 3), as demonstrated by denaturing PAGE, Southern transfer and colorimetric detection (method adapted from U.S. patents 5,900,358; 5,919,677; 6,392,029). The same RNA samples were analyzed for the presence of HIV-1 sense transcripts using a biotinylated antisense primer (3'510–530) (lanes 8–14 correspond to lanes 1–7, respectively), but yielded no transcripts of the expected size (97 bp). This experiment was performed three times with the same results. M lanes received biotinylated ¡-X174 markers with bands from 24–460 bp shown. NT = no template control.