Figure 7

The S79A mutant of Vpr is unable to activate NF-κB and AP-1. (A) Vpr S79A failed to associate with endogenous TAK1. HEK293T cells (4 × 10⁶) were transfected with wild type Flag-Vpr or its mutant S79A. After forty-eight hours, cell lysates were immunoprecipitated with anti-Flag antibody. Samples from both cell lysates and immunoprecipitates were probed with rabbit anti-TAK1 and anti-Flag antibodies. (B) Vpr S79A was unable to enhance the phosphorylation of TAK1. A total of 0.5 × 10⁶ HEK293T cells were transfected with wild type Flag-Vpr or its mutant S79A. After forty-eight hours, cells were pretreated with 20 nM Calyculin A for 5 min. Whole cell lysates were subjected to Western blotting and probed with indicated antibodies. (C) HEK293T cells (4 × 10⁶) were transfected with 1 μg pVSV-G along with 8 μg NLENY1-ES (WT), NLENY1-ΔVpr (ΔVpr), or NLENY1-S79A (S79A) by PEI. The cell lysates and viral supernatants were subjected to Western blotting with the indicated antibodies. (D) Jurkat cells (2 × 10⁶) were infected with VSV-G pseudotyped WT, ΔVpr, or S79A equivalent to 500 ng p24 in the presence of 5 μg/ml polybrene by spinoculation at 300 xg for 30 min. After two hours, cells were pretreated with 15 nM Calyculin A for 5 min. Whole cell lysates were subjected to Western blotting with the indicated antibodies. (E) Ser-79 is required for Vpr-induced NF-κB, AP-1, and HIV-1 LTR activation. HeLa cells (0.1 × 10⁶) were transfected with Flag-Vpr or its S79A mutant along with κB, AP-1, or HIV-1 LTR luciferase reporter plasmid. After forty-eight hours, luciferase activities were measured. The results shown are the averages of three independent experiments. The error bars indicate standard deviations. *P < 0.05, **P < 0.01 (paired t test).