Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging

Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization.We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.As for any replication competent retrovirus, the human immunodeficiency virus type 1 (HIV-1) encodes the precursors to the major structural proteins, enzymes and envelope glycoproteins of the viral particle. In addition, HIV-1 codes for essential regulatory factors, notably Tat, Rev and Vpr. Over the past decade, Vpr has been the subject of many studies because it was suspected to play a direct role in the physiopathology of the viral infection. In fact, Vpr was found to interact with the C-terminus of Gag, causing its virion incorporation [1-4], and with cellular proteins in infected cells. Due to these interactions Vpr promotes the transactivation of HIV-1 long terminal repeat (LTR) and can cause a G2/M arrest and apoptosis of cells, but the relationship between these two roles of Vpr is still a matter of debate (reviewed in [5-7]). Also Vpr appears to contribute to the nuclear import of the pre-integration complex (PIC) and thus of the viral DNA [8,9]. This last function is supported by the nuclear envelope (NE) localization of Vpr, which is medi