Vpr is presented in the MAM.

<p><b>A</b>, Using Percoll self-generating gradient fractionation, Vpr-GFP was localized in the ER, MAM, and mitochondria. Cyto: cytosolic fraction, LM: microsomal fraction, Mito: mitochondrial fraction. <b>B</b>, Likewise, Vpr_52–96-GFP was located in the ER, MAM, and mitochondria. <b>C</b>, Relative expression levels of Vpr-GFP and Vpr_52–96-GFP in different subcellular fractions are similar. <b>D</b>, Immunofluorescent confocal microscopy showed that Vpr was co-localized with the MAM marker, PSS-1 protein. Bulged MAM became evident in Vpr_52–96-GFP expressing cells. Yellow fluorescence (white arrow) indicates an overlap between Vpr (green fluorescence) and MAM (red fluorescence). Bar: 10 µm. <b>E</b>, Colocalization of Vpr_52–96-GFP or Vpr-GFP with the marker of ER, MAM and mitochondria was assessed based on confocal images, and 100–120 cells were scored in three independent experiments. The results showed that both proteins were present in the three organelles. <b>F,</b> HEK293 cells were transfected with the plasmid encoding HA-Vpr for 48 hours. Cells were harvested and fractionated on a self-generated Percoll gradient. HA-Vpr was localized in the ER, MAM, and mitochondria. Cyto: cytosolic fraction, LM: microsomal fraction, Mito: mitochondrial fraction. <b>G,</b> HEK293 cells were either transfected with the plasmid encoding Vpr-GFP, Vpr_52–96-GFP, or HA-Vpr for 48 hours, or infected with Lenti-Vpr (48 and 72 hours). Cells were harvested and cell cycle was analyzed by flow cytometry. Vpr-induced G2 arrest was observed in Vpr-expressing cells.