Fig. 4

3’-Proximal RNA pseudoknot is essential for activation of PKR and splicing. A Secondary structure of the 123-nucleotide region in the HIV-1 3’-UTR that contains the 3’-terminal TAR element and upstream pseudoknot. Pseudoknot stems P1 and P2 are indicated. Boxed nucleotides in P1 were mutated to the nucleotide sequence in the complementary strand (P1b, UUGCC > AGCGG; P1a, AGCGG > UUGCC). The 3R mutation in TAR was CUAG > UGGC; the 3 mutation in TAR was CUGG > GCCA [18]. B, C Both intact 3′-terminal TAR and pseudoknot stem P1 are required for PKR activation. Activation of PKR was assayed using rPKR (85 ng per lane) in the absence of RNA (-) or in the presence of wild type (wt), TAR3R or P1b mutant transcript at 0.1 μg/ml RNA. Position of phosphorylated rPKR (68 kDa) is indicated. A representative experiment is shown (B). Band intensity was quantitated and is plotted in bar graph (C), subtracting the value in the absence of RNA (error bars, SEM; n = 3). D Mutation of TAR or pseudoknot stem P1 impairs rev/tat splicing efficiency. BHK-21 cells were transfected with 3 µg of pcDNA-3’HIV DNA wt, TAR3R or P1b. Total RNA was isolated at 20 h and spliced and unspliced rev/tat transcripts were determined by qRT-PCR. Splicing efficiency, expressed as mRNA/pre-mRNA ratio, was determined for each DNA construct and corrected for between-session variation [58] (error bars, SD). E Mutation of each strand within pseudoknot stem P1 affects rev/tat splicing efficiency. BHK-21 cells were transfected with 3 µg of pcDNA-3’HIV DNA wild type (wt) or mutant forms P1b, P1a or TAR3R. Total RNA was isolated at the indicated times post-transfection. Spliced and unspliced rev/tat transcripts were determined by qRT-PCR. Splicing efficiency is expressed as mRNA/pre-mRNA ratio (error bars, SEM; n = 3). A representative experiment is shown. F Reduction in rev/tat intron splicing within the cell by TAR3 and TAR3R mutations and partial restoration by double mutation TAR3R3. BHK-21 cells were transfected with 3 µg of pcDNA-3’HIV DNA wild type (wt) or mutant forms P1b, TAR3, TAR3R or TAR3R3. Total RNA was isolated at 18 h post-transfection. Spliced and unspliced rev/tat transcripts were determined by qRT-PCR. Splicing efficiency is expressed as mRNA/pre-mRNA ratio (error bars, SEM; n = 3)