Fig. 1

Production of HIV-1 mRNA species is sensitive to PKR antagonists. A Proviral DNA genome maps of HIV-1 LAI (TAR^wt), HIV-rtTA (TAR^m) and HIV-rtTA-ΔTAR (ΔTAR), with the long terminal repeat (LTR) subdivided into U3, R, and U5 domains. B TAR secondary structures followed by the first five nucleotides of the adjacent poly(A) hairpin (light gray). The Tat/TAR axis of transcriptional regulation was inactivated in TAR^m by nucleotide substitutions in the bulge and loop. The HIV-rtTA variant lacking TAR (ΔTAR) has a new transcription start site at the second nucleotide of the poly(A) hairpin [22]. C Human HEK-293 T cells were transfected with a vector carrying the HIV-1, TAR^m or ΔTAR genome, in the absence or presence of the indicated concentrations of PKR inhibitor (PKRi). Total RNA was isolated 48 h after transfection and analyzed by northern blot using a probe that detects all HIV-1 RNA variants. Unspliced (9 kb), singly spliced (4 kb) and multiply spliced (2 kb) RNA size classes [30] and a band resulting from transcriptional read-through on the vector (*)[57] are indicated. The transcripts observed for the different virus constructs vary in size due to the mutations introduced to generate the HIV-rtTA variants, TAR^m and ΔTAR, including deletions and insertions (rtTA replaced Nef, different 3’UTR). Bottom panel shows 18S and 28S ribosomal RNA loading controls (ethidium bromide staining). A representative of 3 experiments is shown. D HEK-293 T cells were transfected with vector carrying the HIV-1 genome, in the absence or presence of the indicated amounts of Vaccinia E3L expression vector (E3L, ng DNA per transfection). Total RNA was isolated and analyzed by Northern blotting as in C. A representative of 3 experiments is shown