Fig. 5

DNA-PK on the HIV-1 genome following Cas9+TAR3/6 transfection and alterations in cutting following F07#13 treatment. a Schematic of the HIV-1 proviral genome, which highlights the 5′ LTR of HIV-1. A series of gRNAs was designed to target the essential TAR loop required for Tat binding and proviral reactivation. b Three infected cell types (J1.1, CHME5/HIV, and U1) were grown in the presence of cART for 1 week prior to transfection. Cells were electroporated with three constructs at a 1:10 ratio (0.1 µg/1 µg of Cas9+TAR3/6) and kept in culture for 5 days. Approximately 1 × 10⁷ cells were used for ChIP assay using antibodies (10 µg) against Pol II large subunit, Cdk9 (T186), p-H2AX, DNA-PK, and ARIDA. Following DNA purification, samples were PCR amplified using LTR primers and run on a 2% Agarose gel. c Similar to panel b except cells were treated with two inhibitors after 5 days. Both inhibitors, DNA-PK inhibitor (Nu 7441, 0.2 µM) and ATM inhibitor (KU 55933, 1 µM), were used for a 2 day treatment of either uninfected (Jurkat) or infected (J1.1) cells prior to CellTiter-Glo. Positive control Fas antibody was used for apoptosis on both cell types. d Similar experimental design to panel b, except J1.1, CHEM5/HIV, and U1 cells were treated with 100 nM TSA after 5 days of transfection. Viruses were isolated from the supernatants with NT086 particles and added to TZM-bl-Luc cells. e A similar experiment as outlined in panel d; however, U1 and ACH2 cells were treated 1 day prior to PHA/PMA treatment with either F07#13 (Day 4), Cas9+TAR3/6, or both together and analyzed by RT-qPCR for the presence of TAR RNA. *p value ≤ 0.05; ***p value ≤ 0.001. f Latent PBMCs (3 independent donors) were created as described previously [7]. After cART/IL-7 addition, samples were divided into 4 sections; two were electroporated (210 V) with TAR3/6 DNA ± F07#13 and kept in culture for 4 days. They were then treated with PMA/PHA for 2 days prior to p24 Western blot