Figure 5

Anti-stem B oligonucleotide-mediated activation of HIV-2, SIV and HIV-1 RNA tight dimerization. A. Schematic representation of HIV-2, SIV, and HIV-1 SL1 domains. The black bars represent the binding sites of antisense oligonucleotides used in this experiment (Table 2). B and C. The time-dependent dimerization of 1–444 HIV-2, 1–441 SIV and 1–277 HIV-1 RNAs were measured at 55°C as described in Figure 4, without (B) or with (C) a 20-fold excess of antisense oligonucleotides directed against the sequence upstream of SL1 including the 5' side of stem B (A and Table 2). Typical kinetics experiments with 1–444 HIV-2 RNA (B and C, left panels), 1–441 SIV SL1 (B and C, middle panels), and 1–277 HIV-1 RNA (B and C, right panels) are shown. Lanes 1 (B and C), 11 and 20 (B) represent monomeric RNA that was denatured at 90°C, then quenched on ice immediately prior to loading. D. Plots of the kinetic data for RNAs incubated without (open circle) or with (closed diamonds) antisense oligonucleotides. The dimerization rate was extrapolated as described in Figure 4. For 1–444 HIV-2 RNA incubating without or with oligonucleotide, k_dim equals 0.11 ± 0.024 and 0.3 ± 0.039 μM^-1min^-1, respectively (duplicate experiments). For 1–441 SIV RNA incubating without or with oligonucleotide, k_dim equals 0.013 ± 0.007 and 0.24 ± 0.007 μM^-1min^-1, respectively (duplicate experiments). For 1–277 HIV-1 RNA incubating without or with oligonucleotide, k_dim equals 0.055 ± 0.016 and 0.30 ± 0.033 μM^-1min^-1, respectively (duplicate experiments).