Figure 4

ABX464, influences REV-mediated HIV RNA biogenesis and export, and interacts with the CBC complex. a Schematic representation of the HIV-1 reporter gene. b Visualization of GFP-MS2-Bound RNA in HeLa cells expressing Tat, MS2-GFP and HIV-reporter transcripts in non-transfected or transfected cells with Rev. Rev-transfected cells were untreated (DMSO) or treated with ABX464 (ABX464). Arrow indicates transcription site. c Quantification of images corresponding to untreated (DMSO) or treated (ABX464) in (b) of total GFP (upper panel), in the nucleoplasm (left panel) or at the start site (right panel). Box-plots show the average GFP intensity (foci numbers or starting transcription point numbers) of Rev-transfected HeLa cells for each condition. Whiskers correspond to the minimum and maximum, boxes, to the 25–75 percentiles and the band inside the box, to the median. Statistical analysis was performed on at least 15 nuclei of Rev positive cells using an unpaired /t/-test (**: p < 0.005 and ****: p < 0.0001). d Purified recombinant CBC20 and CBC80 proteins were incubated with increasing concentrations of ABX464 (left panel) or ABX464-N-glucuronide (right panel, Gluc) and treated for 30 minutes with UV light. The proteins were revealed by Western Blotting using CBC20 and CBC80 antibodies. e Unlike m7GpppG cap structure, neither ABX464 nor ABX464-N-glucuronide interferes with the binding of capped RNA to CBC complex. Recombinant human CBC was incubated with a capped RNA substrate and analysed by native gel electrophoresis in order to resolve the different RNA and RNA-protein complexes: free RNA (lane 1), and CBC-RNA complexes (lanes 2–10) in the presence of 12 mM of m7GppG (lanes 10) or 5 μM, 10 μM or 50 μM of ABX464 (lanes 2–4, respectively) or 5 μM, 10 μM, 50 μM or 100 μM of ABX464-N-glucuronide (lanes 5–9, respectively).