HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag

Abstract

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 Pr55Gag . 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 G 2 /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 Pr55Gag 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 Pr55Gag -TC. This TC motif is tethered to the C terminus of Pr55 Gag and does not interfere with Pr55Gag 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 Pr55Gag -TC mutants confirm that the 41 LXXLF domain of Gag-p6 is essential for Pr55 Gag -Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag -Vpr complexes are still formed when Pr55 Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag -Vpr recognition and complex formation occur early during Pr55Gag assembly.

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…Another is the Vpr-Gag interaction. Vpr is incorporated into nascent virions through the binding of Vpr to NC Gag and p6 Gag (315,316,324). A third interaction is the Vpx-p6 Gag interaction.…”

“…During the early assembly of Gag, Vpr is encapsulated into nascent viral particles via its interaction with Gag (312-316). Vpr multimerization is crucial for the Gag-Vpr interaction (315). On the one hand, NC Gag cooperates with p6…”

“…This interaction has been identified by using yeast twohybrid assays, immunoprecipitation assays, maltose-binding protein pulldown assays, nuclear magnetic resonance, fluorescence lifetime imaging microscopy, and plasmon waveguide resonance spectroscopy techniques (314,315,322,328 [327]). The phosphorylation of position S487 in p6…”

“…Data from yeast two-hybrid assays suggest that SIV Vpx does not interact with NC Gag (314), although HIV-1 Vpr interacts with both NC Gag and p6 Gag (315,316,324). …”

A Mutagenesis Approach for the Study of the Structure-Function Relationship of Human Immunodeficiency Virus Type 1 (HIV-1) Vpr

“…Oligomerization seems necessary for other functions, such as nuclear localization and virion incorporation (Fritz et al, 2008;Fritz et al, 2010;Venkatachari et al, 2010). Venkatachari et al (2010) hypothesized from a structural model of the oligomerization of Vpr that residues at the predicted helical interfaces contribute to dimerization.…”