Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat

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.Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.Human Immunodeficiency Virus type 1 (HIV-1) infection is characterized by a massive depletion of CD4+ T cells that leads to the loss of immune competence [1,2]. This is in part mediated by the HIV-1 Tat protein, which is produced by HIV-infected cells and is efficiently taken up by the neighboring cells [3-5]. Tat is an 86 to 106-amino acid-long protein whose primary role is to transactivate the transcription of the HIV-1 proviral DNA from the long terminal repeat (LTR) by binding to the nascent TAR (Trans-Acting Responsive element) RNA sequence [6-8].In addition, extracellular Tat shows many additional functions, which contribute to the AIDS syndrome. In particular, Tat induces the apoptosis of macrophages and cytotoxic T-lymphocytes by several mechanisms [9]. These different pathways include the up-regulation of Fas ligand [10], the down-regulation of cellular genes encoding for superoxide-dismutase [11] and manganese-dependent superoxide dismutase [12], and the activation of