Vpr incorporation into HIV-1 particles. (A) Immunoblot analysis of Vpr incorporation. Virions were collected from culture media of HEK 293 T cells transfected with the respective proviral plasmids by ultracentrifugation through a sucrose cushion. NL4-3 Vpr(−) (generated by transferring a SpeI – AgeI fragment from pNL43_E-_R-_Luc3/p6:M1A () into pNL4-3) and NL4-3 ΔVpr were used as negative controls. Samples were separated by SDS-PAGE, and viral particles were analyzed by quantitative immunoblot (LiCor) using antisera against recombinant HIV-1 CA (top) and Vpr (bottom). The positions of molecular mass standards are shown on the left and proteins are indicated on the right. (B) Quantification of Vpr incorporation. The ratio of Vpr to CA represents relative signal intensities from quantitative immunoblots, as shown in panel A. Mean values and SD from three independent experiments performed in duplicates are shown. p-values were calculated using an unpaired student’s t-test (n.s. > 0.05, *** < 0.001).