Vpr enhances the phosphorylation of TAK1 following HIV-1 infection. (A) Schematic representation of NLENY1-ES-IRES and NLENY1-ΔVpr viruses. A mutated start codon of vpr gene was introduced in NLENY1-ES to generate NLENY1-ΔVpr. (B-D) Virion-associated Vpr enhances the phosphorylation of TAK1 on Thr-187 in HIV-1 permissive cells. A total of 2 × 106 Jurkat cells (B), THP-1 cells (C), and THP-1 differentiated macrophage-like cells (D) were infected with VSV-G pseudotyped WT or ΔVpr viruses equivalent to 500 ng p24 in the presence of 5 μg/ml polybrene by spinoculation at 300 xg for 30 min. After another 1.5 hours, cells were pretreated with 15 nM Calyculin A for 5 min. Then cells were harvested by centrifugation at 1000 × g for 3 min at 4°C, washed twice with ice-cold 1xphosphate-buffered saline, lysed in 70 μl lysis buffer, and chilled on ice for 30 min with frequent agitation. Whole cell lysates were subjected to Western blotting and probed with anti-phospho-TAK1 (Thr-187), rabbit anti-TAK1, anti-p24, anti-Vpr, and anti-Tubulin antibodies. (E) Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors. PBMCs were activated with phytohemagglutinin (PHA; 5 mg/ml) and IL-2 (20 U/ml) for 24 h, followed by infection with VSV-G pseudotyped WT or ΔVpr (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 examined in Western blotting with the indicated antibodies.