Figure 7.
G I3 -2 is specifically bound by hnRNP F/H and A2/B1. (A) Schematic illustration of the RNA pull-down experiment and binding site of locked nucleic acids directed against GI3-2 within HIV-1 intron 3. (B) RNA pull-down assay using HeLa nuclear extract. Substrate RNAs containing a MS2 sequence and the wild type or mutant G runs sequence were covalently linked to adipic acid dihydrazide-agarose beads and incubated with HeLa cell nuclear protein extract. MS2-proteins were added to monitor the RNA input. For interference with protein:RNA interaction, wt RNA was pre-incubated with the GI3-2 LNA in a ratio of either 1:5 or 1:1 relative to the amount of RNA substrate. The precipitated proteins were resolved by SDS-PAGE (16%) and detected by immunoblot analysis using anti hnRNP F/H and A2/B1 antibodies. MS2 specific antibodies were used as a loading control. (C) HeLa cells were co-transfected with pNL4-3 and locked nucleic acids (LNAs) masking GI3-2 or the respective mismatch control. Total RNA was isolated 24 h post transfection and subjected to Northern blotting using a HIV-1 specific probe. (D) Immunoblot analysis of p24-CA using cellular lysates (cell) and pelleted virions from the supernatant (sn) of co-transfected cells from (C).