HIV-1 Vpr oligomerization but not that of gag directs the interaction between Vpr and Gag

During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55^Gag. Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G₂/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55^Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55^Gag-TC. This TC motif is tethered to the C terminus of Pr55^Gag and does not interfere with Pr55^Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55^Gag-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55^Gag-TC mutants confirm that the ⁴¹LXXLF domain of Gag-p6 is essential for Pr55 ^Gag-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55^Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55^Gag-Vpr complexes are still formed when Pr55^Gag carries mutations impairing its multimerization. These findings suggest that Pr55^Gag-Vpr recognition and complex formation occur early during Pr55^Gag assembly.