Figure 4

RTEm26-CTE replaces rev/RRE of SIV. A) Structure of the rev- and RRE-minus SIVmac239 containing RTEm26-CTE. Multiple point mutations inactivate both rev and RRE of SIVmac239. CTE or RTEm26-CTE was inserted between env and the 3'LTR. B) Human 293 cells were transiently transfected with the indicated plasmids. Two days later, cells and supernatant were analyzed for gag and env expression. Intracellular (1/10 of cell extract) and extracellular (1/150 of supernatant) were analyzed on Western immunoblots using a serum pool from SIV-infected monkeys. Total intra- and extracellular Gag production was measured using commercial SIV p27gag antigen capture assay and GFP production was quantitated. Normalized values (total gag in pg/total GFP units) are shown. C) SIV propagation in CEMx174 cells. Virus stock were generated upon cocultivation of transfected 293 cells with CEMx174 cells and then used to infect fresh CEMx174 cells: wild type SIVmac239 (filled triangle), two stocks containing the Rev-independent SIV containing CTE (filled circle and X, respectively), and two stocks containing the RTEm26-CTE (filled diamond and open square, respectively). Virus production was monitored over time using a commercial SIV p27gag antigen capture assay.