Figure 5. Rev-RRE Electrophoretic Mobility Shift Assays and Functional Studies.
(A) EMSA assays. Rev-binding reactions were loaded onto 6% or 10% nondenaturing TBE gels depending on the size of the RRE mutant (domain II-III-IV: 119 nt; domain I-V: 108 nt; 1 turn: 253 nt; 1.5 turn: 263 nt; 2 turn: 273 nt). The first eight lanes contain 0.5 pmol RRE, nonspecific competitor RNA (RiboA) at 20-fold mass excess, and titrating amounts of Rev at the molar ratio indicated above each lane. The last 2 lanes contain only competitor RNA, either in the absence of Rev or with Rev added to the same level as in the highest Rev:RRE stoichiometry. White lines indicate cropping of irrelevant lanes.
(B) Fractions of WT, truncated or mutant RRE RNAs engaged in high-order complexes are plotted as a function of Rev:RRE molar ratio. Data from three independent EMSA experiments are shown as mean ± SD.
(C) Plasmid pCMVgagpol-RRE (Srinivasakumar et al., 1997) was modified to simplify the RRE reporter assay. First, the pol gene was deleted; removal of the protease domain within pol prevents Gag cleavage, thus confining Gag to one band on a western blot. Second, the 233-nt “core” RRE was rendered identical in sequence to the NL4.3 RRE analyzed by SAXS. Third, the N-terminal glycine codon of gag was replaced by an alanine codon, preventing Gag myristylation, and thus, virion release.
(D) Western analysis of cell lysates using anti-p24CA and anti-β-actin antibodies. Top: pcDNA: cells transfected with neither pCMVRev nor reporter plasmid; Rev, cells transfected with pCMVRev alone; and remaining lanes contain reporter plasmids as indicated. WT: wild-type RRE; I-V: domains I-V (domains II-III-IV have been deleted); II-III-IV: domains II-III-IV (domains I-V have been deleted); 1 turn, 1.5 turns, and 2 turns: insertion mutants with one, one and a half and two turns of a duplex, respectively. Bottom: Quantification of the western analysis. Gag synthesis relative to WT (set at 100%): Gag levels were normalized to actin levels and to transfection efficiency, as measured by G. Luciferase assays. Gag synthesis is shown as the mean ± SD from three to five independent transfections.