(A) Intracellular expression of two types of hemaglutinin (HA)-tagged Vpr proteins in HEK 293T cells. Whole cell lysates of untransfected 293T cells or 293 T cells transfected with either an empty vector (pcDNA) or with selected Vpr-containing plasmids were separated using 10% SDS-PAGE and probed with anti-HA antibody. In lanes 3 and 4, pcDNA-3HA-Vpr or the faster-migrating constructs (6His-HA-Vpr) have been transfected, respectively. As a loading control, membrane was stripped and probed with anti-β-actin antibody. (B) Expression of p24 capsid protein of virions collected 48 h post-transfection from the supernatant of 293T cells transfected with the constructs reported below. (C) After ultracentrifugation at 28,000 rpm, cell-free, virus-free supernatant was collected and precipitated with acetone. Subsequently, immunoprecipitation (with anti-HA antibody) was performed followed by western immunoblotting. Only the samples cotransfected with Vpr and the other viral proteins display Vpr in the CFVF. *Possible presence of a Vpr dimer. 6His: 6-histidine