Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging.
Fiche publication
Date publication
septembre 2008
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MELY Yves
Tous les auteurs :
Fritz JV, Didier P, Clamme JP, Schaub E, Muriaux D, Cabanne C, Morellet N, Bouaziz S, Darlix JL, Mely Y, de Rocquigny H
Lien Pubmed
Résumé
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 a 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 alpha-helices could perturb the leucine zipper like motifs. The Delta Q44 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 a helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.
Référence
Retrovirology. 2008 Sep 22;5:87