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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.

Abstract Background During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. Results To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20°C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37°C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. Conclusion Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.