Research

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

Joëlle V Fritz¹ , Pascal Didier¹ , Jean-Pierre Clamme² , Emmanuel Schaub¹ , Delphine Muriaux³ , Charlotte Cabanne⁴ , Nelly Morellet⁵ , Serge Bouaziz⁵ , Jean-Luc Darlix³ , Yves Mély¹ and Hugues de Rocquigny¹

¹  Département de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR 7175 CNRS, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg 1, 74, Route du Rhin, 67401 Illkirch Cedex, France

²  Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

³  LaboRétro Unité de Virologie Humaine INSERM 758, IFR 128 Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364 Lyon, France

⁴  Ecole Supérieure de Technologie des Biomolécules de Bordeaux, Université V Ségalen, Bordeaux 2, 146, rue Léo Saignat, 33076 Bordeaux Cedex, France

⁵  Unité de Pharmacologie Chimique et Génétique, Inserm U640 CNRS UMR8151 UFR des Sciences Pharmaceutiques et Biologiques 4, Avenue de L'observatoire, 75006 Paris, France

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Retrovirology 2008, 5:87doi:10.1186/1742-4690-5-87

Published:	22 September 2008

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.