Figure 3

PIs activate latent HIV-1 gene expression in primary CD4⁺T cell models. A. Schematic of gGnΔ construct used to establish latently infected T_CM-like and BCL2-transduced cells. Black box represents a deletion in env, which renders this construct replication-incompetent. Patterned boxes indicate GLUC and GFP reporter gene insertions. The yellow box specifies a T2A sequence, which directs bicistronic expression [88, 89]. B. T_CM-like cells and C. BCL2-transduced primary CD4⁺ T cells were treated for 48 hours with PIs as indicated. Flow cytometry was performed and the values shown indicate GFP mean channel fluorescence (MCF). All cells treated with PIs were simultaneously treated with Raltegravir to prevent the integration of as yet unintegrated viral genomes. MG-132 and Velcade treatments significantly activated latent virus in both T_CM-like cells (p<0.05; p<0.001, respectively) and in BCL2-transduced cells (p<0.01; p<0.001, respectively) in comparison to untreated (negative control) cells. P-values calculated using one-tailed Student’s t-test. D. Resting CD4⁺ T cells isolated from healthy donor PBMCs were treated with 10 nM Velcade or 500 nM MG-132 for 48 hours and then incubated with FITC-conjugated CD25 antibody. The percentage of CD25⁺ cells in each sample was determined via flow cytometry. In all experiments, the vertical open bars represent average values from all healthy donors while symbols represent individual donor results; each donor result represents a singlicate experiment. Cells treated with CD3/CD28 activating beads served as positive controls.