Small-Molecule Inhibition of HIV pre-mRNA Splicing as a Novel Antiretroviral Therapy to Overcome Drug Resistance
Figure 5
Identification of the Step in HIV Life Cycle Inhibited by IDC16
HOS-CD4+-CCR5+ cells were exposed to various concentrations of IDC16 and infected with Δenv-defective virions containing the luciferase gene (A). The efficiency of the reverse transcription and of the integration was evaluated by quantifying early and late reverse transcriptase products and the number of copies of integrated HIV DNA, respectively (B). Forty eight hours later, intracellular luciferase activity was measured as a marker of spliced viral RNA expression (C). The same infected cells treated with 0.1 μM, 0.5 μM, or 1 μM of IDC16 were used to examine the relative amount of unspliced precursor and multiply spliced HIV-1 RNA. Real-time RT-PCR was used to rigorously quantify the changes in unspliced (D, left panel) and multiply spliced (D, right panel) HIV-1 RNA levels after IDC16 treatment. The values are the average of two independent experiments and the level of expression is normalized with that of the internal control gene (GAPDH). The error bars indicate standard deviations. Arrows in (A) indicate the position of primers to amplify unspliced precursors (La 8.1 and La 9) and multiply spliced (P659 and P413MOD) HIV RNA species according to Brussel and Sonigo [48].