HIV-1 Tat primes quiescent T cells for productive HIV-1 infection (17). (a) Primary T cells were treated with Tat (12 pmol ml⁻¹) for 48 h or 72 h before infection by HIV-1_NL4-3. Levels of p24 antigen in the culture supernatant were determined on day 3 after infection. (b) Antibodies against Tat inhibit productive infection by HIV-1. A total of 24 pmol of Tat protein (12 pmol μl⁻¹) was incubated on ice for 20 min with 10 μg of anti-Tat 1 [purified monoclonal IgG1 against HIV-1 Tat (Intracel)], or 5 μl of anti-Tat 2 (anti-serum to Tat), or 5 μl of anti-serum to rev before addition to cultures. Cells were primed with Tat protein for 48 h prior to infection. p24 levels in the culture supernatant were determined on day 3 (▪) or 6 (▥) Data in a and b represent averages of triplicates or duplicates in one of three independent experiments.

Aberrant activation of lymphocytes by extracellular Tat is associated with G₀ to G₁ progression but not entry into S phase. (a) Primary lymphocytes remained quiescent after 72 h in culture. (b) Cells activated by extracellular Tat did not enter S phase, unlike (c) cells activated by phytohemagglutinin (PHA-C; Boehringer Mannheim). (d) Primary lymphocytes treated with Tat protein for 72 h displayed activation of cyclin E associated cdk2, a late G₁ kinase. Lanes: 1, control cells; 2, cells treated with Tat (24 pmol ml⁻¹); 3, cells treated with PHA (20 μg ml⁻¹). In a–c, PBMCs were treated with Tat protein (24 pmol ml⁻¹), or PHA (20 μg ml⁻¹) plus 20 units ml⁻¹ recombinant human interleukin 2 (rIL-2; Boehringer Mannheim) for 72 h. Similar results were observed in purified T cells. In d, PBMCs were similarly treated as in b and c.

Aberrant activation of primary T cells by Tat protein. (a and b) Fas (CD95) expression on quiescent T cells was induced by Tat protein. (a) Control T cells; (b) T cells treated with 12 pmol ml⁻¹ Tat for 48 h. ——, Cells were stained with secondary antibody only; ——, cells were stained with anti-Fas followed by secondary antibody. T cells or PBMCs from nine blood donors were tested in independent experiments. (c) Induction of CD95 mRNA by Tat protein. Lanes 1, 3, and 5, mRNA was prepared from untreated T cells; lanes 2, 4, and 6, mRNA was prepared from Tat treated T cells. mRNA was determined by reverse transcription–PCR. From lanes 5 to 1, and, from lanes 6 to 2, cDNA was serially diluted. This is one of three independent experiments. (d) Up-regulation of CD25 expression on PBMCs by extracellular Tat. The expression of CD25 on untreated cells as measured by the mean immunofluorescent intensity was taken as 100, and CD25 expression on other samples was expressed by comparison with that of the untreated group. PBMCs were treated with Tat (12 pmol ml⁻¹) for 48 h. T cells or PBMCs from five different donors were tested. A similar pattern of induction was observed in all experiments, although there was some variation in the absolute values in samples from different individuals. Data are mean values from three independent experiments. (e) Induction of HLA-DR1 and Fas (CD95) expression by Tat protein in mice. Data represent average of four measurements of T cells from two mice in each group. Naive T cells were purified as described, and were infused into athymic mice. After 16 h, mice were treated with Tat protein (250 μg/kg, i.v.). The control group received an equal amount of BSA in PBS. T cells were harvested from peripheral blood after 24 h.

Activation of T cells by Tat protein is inhibited by antibodies against integrins (a) and is associated with activation of MAP kinase (b). (a) T cells were incubated with monoclonal antibodies to integrins β₅, α₃, or α₅ for 1 h at 37°C. Tat protein (12 pmol/ml) was added. After 48 h, expression of CD25 was assayed. Data represent average of triplicates in one of four independent experiments. (b) Primary T cells treated with Tat protein displayed activation of MAP kinase. Lanes: 1, control T cells; 2–4, cells were treated with Tat (24 pmol ml⁻¹) for 2 h (lane 2), 8 h (lane 3), and 24 h (lane 4), respectively. (c and d) Activation of JNK kinase (c) and MAP kinase (d) by Tat protein in Jurkat cells. Lanes: 1, control cells; 2–4, cells were treated with Tat protein at 12, 24, and 48 pmol ml⁻¹, respectively, for 2 h; 5, control IgG for JNK antibody. GST-Jun, glutathione S-transferase–Jun. (d) Jurkat cells were treated with Tat protein for 0, 10, 30, or 60 min, or with phorbol 12-myristate 13-acetate (PMA) for 30 min, respectively. Arbitrary units represent quantitative analysis of myelin basic protein phosphorylation determined by an imaging densitometer (model GS-700; Bio-Rad).