Turbidity time course of the in vitro microtubule assembly. (A) Tubulin in presence of various ligands. Samples with 15 μM tubulin are maintained at 4°C. After addition of 8 μM Tat (line 4), 4 μM Tat (line 2) from HIV-1 HxB2 strain or 15 μM paclitaxel (line 3), the assembly reaction was started by warming the samples at 37°C (time 0, arrow) and compared to tubulin alone (line1). The ΔOD350 nm is measured every 30 sec. After 30 min the temperature was lowered to 10°C (arrow). (B) Electron microscopy of microtubules formed in the presence of Tat. Aliquot from samples reaching the ΔDO350 nm plateau at 37°C were adsorbed on coated Formvar films on copper grids. Electron micrographs of microtubules formed without Tat (Control) or with indicated concentrations of Tat and paclitaxel are presented with 4000-fold magnification. Microtubules formed with Tat at 8 μM are also presented at 40000-fold magnification. (C) Production of microtubules in the presence of Tat. Samples of 15 μM tubulin alone (Control) and with 8 μM Tat (Tat 8), 16 μM Tat (Tat 16) or 15 μM paclitaxel (Paclitaxel) at the time they reach the plateau at 37°C were ultracentrifuged and supernatant (S) and pellets (P) were analyzed on SDS-PAGE. (Tat 8) × 5 indicates a fivefold increase in the quantity of the sample loaded. The mass of tubulin (Tub 55 kDa) and Tat (Tat 10 kDa) are indicated.

Sequences of HIV-1 Tat strains. These three Tat variants and the peptides were obtained by solid phase synthesis with a procedure previously described [43, 50]. Sequences correspond to the viral strains HIV-1 Eli [30], HIV-1 HxB2 [29] and HIV-1 Oyi [30].

Effect of different Tat variants on cell cycle progression, apoptosis and microtubule network in lymphocytes. (A) Table showing data from fluorescence microscopy after 20 hours treatment with indicated concentration of Tat or paclitaxel. Percentage of apoptotic cells are determined after DAPI staining and the differences in apoptosis between treated and untreated cells used as control obtained in three independent experiments are presented. (B) Jurkat cells were treated with indicated concentrations of paclitaxel and various Tat or were untreated (control). After 20 hours, cells were stained with PI and analysed by flow cytometry. Percentage of cells in G2/M (G2 / M) or apoptotic cells (H) with hypodiploid DNA are indicated in the upper corner of each cell (C) Jurkat cells were treated with 10 μM Tat Eli or 1 μM paclitaxel and processed for immunofluorescence labeling with anti-alpha tubulin antibody as described in materials and methods. Control corresponds to untreated cells.

Tat induces cytochrome c release from isolated mitochondria. After 2 hour treatment with 10 μM (P10) of peptide 73–99 or 0.2 μM (T0.2), 2 μM (T2) and 10 μM (T10) of Tat Eli or without peptide (T-), samples were centrifuged and supernatants and pellets were separated. (A). Western blots of the supernatant with antibodies against cytochrome c (Cyt C) and with antibodies against VDAC. (B) Western blots of Mitochondria Pellets. Data are representative of four independent experiments.