Fig. 2.

Construction of CRF07_BC Rev expression plasmids and evaluation of their expression levels and Rev-RRE activities. A BC WT Rev-His: clade BC wild type Rev gene sequence; BC Rev Stop101Q-His: full length Rev gene in which the stop codon at residue 101 was replaced by Q. These two inserts were cloned into pcDNA3.1-TOPO-V5-His vector in frame with V5-His tag, separately. B Analysis of expression levels of Rev constructs with His Tag using Western blotting. C Relative expression levels of Rev mutant to wild type. Each bar represents the average and standard deviation of at least three replicate experiments. D BC WT Rev: clade BC wild type Rev gene sequence carrying a premature stop codon; BC Rev Stop101Q: full length Rev gene in which the premature stop codon at residue 101 was replaced by Q, and a TAG stop codon was contained at the end of sequence. These two fragments were separately cloned into pcDNA3.1-TOPO-V5-His vector to produce proteins without V5-His tag. E Measurement of Rev-RRE activity of WT and mutated CRF07_BC Rev protein. HEK 293T cells were transfected with clade BC Gag-Pol-RRE plasmid plus Rev expression plasmid (BC WT Rev or BC Rev Stop101Q). CMV-EGFP plasmid was co-transfected as control. Supernatants were harvested 48 h post transfection for p24 quantification using ELISA. The p24 antigen was normalized to transfection efficiency as determined by percentage of EGFP expressing cells measured using flow cytometry. Data is representative of at least three independent experiments. ns: no significance.