Figure 4

The effect of the combination of LEDGF/p75 and Rev on IN enzymatic activity in vitro and in LEDGF/p75-knockdown cells. (A) IN (390 nM) was added to the substrate DNA and then either LEDGF/p75, Rev-GFP or both, in the order indicated by arrows, were added, or, as control, none of them were added (no addition) and IN enzymatic activity was determined as described in Materials and Methods. (B) An ELISA system was used to determine binding of biotinilated DNA to plate-bound IN (), IN + LEDGF/p75 (), IN + Rev-GFP (), IN + Rev-GFP + LEDGF/p75 (), and IN + GFP () used as a control. Binding was estimated as described in Materials and methods. (C) Apparent K_d as calculated for the results presented in (B). (D) Hela P4 (), LEDGF/p75-knockdown () and LEDGF/p75-knockdown-Rev-expressing () cells were infected by the indicated viruses (at a MOI of 1) and integration events per cell were determined at 24 h PI. (E) LEDGF/p75-knockdown cells were infected with ΔRev HIV-1, and IN (red), DNA (blue), Rev (green) and LEDGF/p75 (gray) were immunostained as described in Materials and Methods. Scale bar represents 10 µm. (F) Same as (D) except that p24 production was determined at 72 h PI. (G) Same as (D) except that viral cDNA was determined at 12 h PI. AZT was added at 2 µM.