FIGURE 2.

Cyclin K inhibits HIV-1 LTR-driven gene expression and replication in Nef-dependent manner. A, Cyclin K does not modulate LTR-driven luciferase expression in the presence of Tat alone. HEK-293T cells were transfected with HIV-1 LTR-Luc reporter vector along with pc-CycK in increasing amounts (0.3–1.2 μg) and with pcDNA-Tat (0.25 μg) followed by a luciferase assay 36 h post-transfection. The total amount of DNA transfected was equal in all lanes. B, Cyclin K down-regulates Tat-induced LTR-driven luciferase expression in the presence of Nef. HEK-293T cells were transfected with HIV-1 LTR-Luc reporter vector along with pc-CycK in increasing amounts (0.3–1.2 μg) and with pcDNA-Nef (0.5 μg) and pcDNA-Tat (0.25 μg). 36 h post-transfection, cells were lysed in Promega cell lysis reagent, and a luciferase assay was performed. C, Cyclin K inhibits integrated LTR-driven gene expression in T-cells. Jurkat-1G5 cells were transfected with pNL4-3 (1 μg) and increasing amounts of pc-CycK (0.25–0.5 μg). 48 h post-transfection, cells were lysed in Promega cell lysis reagent followed by a luciferase assay. D, Nef is necessary for Cyclin K-mediated inhibition of HIV-1 replication. Jurkat-1G5 cells were transfected with either pNL4-3 (gray bars) or pNL4-3ΔNef (black bars) and with increasing amounts of pc-CycK (0.25–0.5 μg) as presented in the figure. 48 h post-transfection, the culture supernatant was collected, and the amount of virus present in the culture supernatant was estimated by a p24 gag antigen capture ELISA.