Fig 6. Validation of ING3 in combination with AZD5582 as a HIV-1 latency maintenance factor.

(A) HIV-1 reverse transcriptase activity (y-axis) of the viral supernatant of NTC sgRNA transduction or ING3 knockout in J-Lat 10.6 and 5A8 treated with 10 nM AZD5582 or an equivalent volume of DMSO. The ICE knockout score for the J-Lat 5A8 ING3 knockout cell line is 72 and the ICE knockout score for the J-Lat 10.6 ING3 knockout cell is 66. ING3 knockout and AZD5582 treatment combine to result in a significant increase in viral reactivation. Paired t-test, p-value = <0.05 = *, = <0.01 = **. (B) Representative flow cytometry plots of primary CD4+ T cell HIV-1 latency model cells that are AAVS1 or ING3 knockouts treated with DMSO or 1 μM AZD5582 treatment. Thy1.2-, GFP- cells (quadrant 4) are uninfected; Thy1.2+, GFP- (quadrant 3) cells are infected with the dual reporter HIV-1 virus and latent; Thy1.2+, GFP+ cells (quadrant 2) are infected and reactivated. (C) Independent knockouts of AAVS1 and ING3 in primary CD4+ T cell HIV-1 latency model cells were performed in three healthy donors and each pool of knockout cells were treated with DMSO or 1 μM AZD5582. Reactivation fold change is calculated based on percent Thy1.2+, GFP+ cells. The quantified percent Thy1.2+, GFP+ cells for each condition (knockout of AAVS1 or ING3 and treatment of DMSO or 1 μM AZD5582) was then normalized to the AAVS1 knockout with DMSO treatment (negative control) condition. Knockout of ING3 and AZD5582 treatment combined resulted in significant reactivation compared to knockout of AAVS1. Paired t-test, p-value = <0.05 = *. (D) Western blot showing similar p52 levels are detected in the control and ING3 knockout J-Lat 10.6 and 5A8 cell lines upon treatment of 10 nM AZD5582. Activation of the non-canonical NFκB (NFκB2) pathway is marked by a decrease in p100 and an increase in the cleaved product of p52.