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. Author manuscript; available in PMC: 2021 Mar 9.
Published in final edited form as: Cell Rep. 2020 Feb 25;30(8):2472–2480.e4. doi: 10.1016/j.celrep.2020.02.004

Figure 3.

Figure 3.

Probing the conformational equilibrium of HIV-1 RRE in vitro and in vivo. (A) HIV-1 RRE RNA has two ESs that reshuffle the non-canonical base pairs at the purine-rich region (blue), U72 (yellow), and A68 (green) bulge. (B) Example of the stabilize-and-rescue approach for trapping RRE ES2 using an ES-stabilizing mutant A68C (orange) and corresponding rescue mutations G50A and C69U (blue). For A and B, weak base pairs (open dot) and non-Watson-Crick base pairs (red dot) are indicated. (C) Summary of RRE mutants with mutated base pairs highlighted (red box). The extent of stabilization was estimated based on NMR line broadening (Figure S2). (++) minimal line broadening, (+) partial line broadening, and (−) extensive line broadening. (D) Rev-RRE dependent RNA export assays of RRE mutants determined to be primarily in the GS conformation (black labels) or ES conformation (orange labels) based on NMR. 293T cell were transfected with pFLuc-RRE reporter plasmid and an RLuc internal control in the presence (+ Rev) or absence (-Rev) of a Rev expression plasmid. Reported values are the quotient of FLuc and RLuc activities with values normalized to WT RRE for every independent replicate reported as the mean ± SD (n=3). MP-1 is the control plasmid (no RRE). Statistical significance of the difference in +Rev and -Rev activity between wtRRE and each mutant was determined after log-transformation of the data; P-values between RRE-variants are shown (two-sided ANOVA). (E) Cell-based trans-activation assays to detect Rev-RRE binding. Tat-Rev fusion protein dependent trans-activation assays of RRE and mutants. HeLa cells were transfected with pFLuc-TAR/RRE reporter plasmid and an RLuc internal control in the presence of (+ Tat/Rev) or (+Tat) expression plasmid. At 48 hr post transfection, cell lysates were tested for luciferase activity. Reported values are the quotient of FLuc and RLuc activities with values normalized to WT RREII for every independent replicate reported as the mean ± SD (n=3). P-values between RRE variants are shown (two-sided ANOVA).