Fig 7.

AS601245 inhibits TNF-α-stimulated P-TEFb release. To test the ability of AS601245 to act as an inhibitor of HIV-1 reactivation by inhibiting the release of P-TEFb from its inactive complex with HEXIM-1, we performed glycerol gradient analysis to determine the effect of AS601245 on the P-TEFb/HEXIM-1 complex composition in the latently infected CA5 T cells. CA5 T cells were left untreated or treated with AS601245 (10 μM) for 1 h. Cells were then left unstimulated or HIV-1 reactivation was triggered with TNF-α (3 ng/ml). Cell lysates were separated on a glycerol gradient (10 to 45%). Each gradient fraction was separated on a 10% SDS-PAGE gel and transferred by Western blotting. Blots were stained with anti-CDk9 MAb, a component of the P-TEFb complex. (A) Release of P-TEFb from the inactive complex with HEXIM-1 (large complex), which is found in the glycerol gradient fractions with higher glycerol content, is indicated by a shift to a smaller complex (CDK9/CycT1) found in the gradient fractions with lower glycerol content. Changes in the band intensity in the P-TEFb/HEXIM-1 complex composition relative to that of control cells for CA5 T cells stimulated with TNF-α or stimulated with TNF-α following pretreatment with AS601245 are shown. All blots were stained with an anti-CDK9 antibody to reveal drug-induced shifts in the complex composition. Band densities were then determined using ImageJ. (B) CDK9 band intensities of the glycerol fractions for control cells, TNF-α-activated cells, and AS601245 pretreated/TNF-α-activated cells (6 h). The data are representative of results from two independent experiments. (C) Concentration-dependent inhibition of HMBA-induced HIV-1 reactivation by AS601245 in the latently infected CA5, CG3, and EF7 T cells.