Figure 3

RTEm26-CTE replaces Rev/RRE of HIV. A) Structure of rev and RRE-minus HIV-1 containing RTEm26-CTE. Multiple point mutations inactivate both rev and RRE. CTE, RTE or RTEm26-CTE were inserted between env and the 3'LTR, rendering these clones nef-minus. B) Human 293 cells were transiently transfected with the indicated plasmids. Two days later, cell extracts were analyzed on Western immunoblots using HIV patient sera. Total intra- and extracellular Gag production was measured using commercial HIV p24 antigen capture assays and GFP production was quantitated. Normalized values (total gag in pg/total GFP units) are shown. C) HIV propagation in Jurkat cells. Transfected 293 cells were cocultivated with Jurkat cells: wild type NL4-3 (filled triangle), the Rev-independent HIV containing RTEm26-CTE (two clones filled diamond, open circle), CTE (open triangle), RTE (filled circle), and no insert (open diamond). Virus production was monitored over time using a commercial HIV p24gag antigen capture assay. Similarly, upon cell-free infection (not shown), the RTEm26-CTE replicates to a similar extent like the RTE- or CTE-containing Rev-independent HIV viruses.