Inhibition or downregulation of proteasome increases Tat-transactivation by stabilizing ELL2 to form more ELL2-SECs.

A. The Jurkat-based 1G5 and 1G5+Tat cells containing an integrated HIV-1 LTR-luciferase reporter construct were treated with the indicated concentrations of bortezomib or 0.1% DMSO for 20 hr. Luciferase activities in cell extracts were measured and shown. B. & C. Results of Western blot analyses of the effects of CRISPRi- or RNAi-induced downregulation of the indicated proteasome subunits in 2D10 cells on levels of the various proteins labeled on the left. D. Nuclear extracts (NE) were prepared from 2D10 cells treated with 10 nM bortezomib or 0.1% DMSO (-) for 18 hr and then subjected to immunoprecipitation (IP) with the anti-CDK9 antibody or rabbit total IgG as a control (ctl.). The NE and IP eluates were examined by Western blotting for the indicated proteins. E. Result of FACS analysis of the effects of treatment with 7.5 nM bortezomib or 0.1% DMSO for 18 hr on HIV-1 activation in three different 2D10-based cell lines: WT, ΔELL2 (ELL2-knockout) and ΔELL2-R2 (ΔELL2 cells containing an integrated vector expressing ELL2-Flag to about the endogenous level). F. A model showing the proposed mechanism by which the inhibition or downregulation of the proteasome stabilizes ELL2 to form more ELL2-SECs for HIV-1 Tat-transactivation and latency reversal. In A and E, error bars represent mean +/- SD from three experimental replicates.