Direct Vpr-Vpr Interaction in Cells monitored by two photon fluorescence correlation spectroscopy and fluorescence lifetime imaging

Joëlle V. Fritz, Pascal Didier, Jean Pierre Clamme, Emmanuel Schaub, Delphine Muriaux, Charlotte Cabanne, Nelly Morellet, Serge Bouaziz, Jean Luc Darlix, Yves Mély, Hugues de Rocquigny*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

39 Citations (Scopus)

Abstract

Background: The human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Results: 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. Conclusion: 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.

Original languageEnglish
Article number87
JournalRetrovirology
Volume5
DOIs
Publication statusPublished - 22 Sep 2008
Externally publishedYes

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