FIG. 3.

(A) Dimerization level and thermal stability of the viral RNA isolated from BH10, Δ271–273, and Δ247 viruses, respectively, containing 1.5 × 10¹², 4.5 × 10¹², and 4.5 × 10¹² CAp24. Samples were dissolved in 8 μl of buffer S (10 mM Tris [pH 7.5], 100 mM NaCl, 1 mM EDTA, and 1% sodium dodecyl sulfate) and incubated at the temperatures indicated for 10 min (samples in lanes 1 and 5 were left on ice for 10 min). After the incubations, all samples were loaded without delay and with the voltage on. After electrophoresis (70 V; 4 h; 1% agarose in TBE₂ [89 mM Tris, 89 mM borate, and 2 mM EDTA] at 4°C), the samples were Northern blotted, hybridized, and autoradiographed for 4 h. (B) Lanes 1 to 5: dimerization level of viral RNA isolated from BH10, Δ274–277, Δ243–246, and Δ248–256 viruses, respectively, containing 3.6 × 10¹², 7.1 × 10¹², 7.1 × 10¹², 8.6 × 10¹², and 4 × 10¹² CAp24; experimental conditions were unchanged except for 0.8% agarose in lanes 1 to 3, and autoradiographic exposures of, respectively, 1 h (lanes 1 to 3) and 2 h 10 min (lanes 4 to 5). Lane 6: BH10 viral RNA electrophoresed (45 V, 3 h 10 min, 1% agarose in TBE₂) in a room maintained at 27°C, i.e., under conditions where only tight dimers of partial HIV-1 RNA transcripts remain dimeric. Each lane represents a different transfection. Densitometry of lanes 1, 5, and 9 of panel A, lanes 1 to 5 of panel B, and equivalent lanes from other Northern blots indicate that Δ271–273, Δ247, Δ274–277, Δ243–246, and Δ248–256 viruses package genomic RNA, respectively, 44% ± 4%, 65% ± 11%, 40%± 6%, 25% ± 4%, and 40% ± 6% as well as BH10. Abbreviations: d, dimeric RNA; m, monomer.