Primary CD4+T cells treated with Velcade produce fewer, less infectious virions. A. Schematic of Gn construct used to produce replication-competent virus for this experiment. The patterned box indicates GFP reporter gene insertion and the yellow box specifies a T2A sequence, which directs bicistronic expression [88, 89]. B. CD4+ T cells isolated from healthy donor PBMCs were activated and infected with Gn virus. Six hours post-infection, cells were either treated with 10 nM Velcade or left untreated as a positive control. Seventy-two hours post-infection, virus-containing supernatants were collected and p24 levels were analyzed via p24 ELISA. Values shown represent p24 levels (pg/mL) calculated using standard curve values. C. Untreated and Velcade-treated virus containing supernatants were used to infect HeLaT4 cells. Untreated supernatants were also used to infect HeLaT4 cells in the presence of 10nM Velcade [PI (target)] to control for effects that may arise from residual Velcade in the inoculum collected from Velcade-treated virus producing cells [PI (virus)]. Forty-eight hours post-infection, GFP+ cell numbers were analyzed via flow cytometry. Values shown indicate percent GFP+ cells normalized to the p24 (ng/ml) concentration of the inoculating viral supernatant. Error bars indicate SEM. Asterisks indicate significant differences (** p<0.01; *** p<0.001) between Velcade treatments and untreated (positive control) cells. P-values calculated using one-tailed Student’s t test. The figure represents average values from three independent experiments, each of which utilized primary cells isolated from different healthy donors.