Tat and nicotinamide cause T-cell hyperactivation
(A) Western blot analysis of Tat protein in Jurkat T cells infected with lentiviral vectors expressing LTR-GFP and LTR-Tat-GFP. Infected cultures were activated for 2 h and 8 h with α-CD3/28 antibodies.
(B) Real-time RT-PCR analysis of IL-2 mRNA levels in infected cultures 2 h after activation with α-CD3/28. Results are expressed as fold induction by α-CD3/28 treatment.
(C) IL-2 protein levels measured by ELISA in the supernatant of infected cultures 8 h after α-CD3/28 treatment.
(D) Real-time RT-PCR analysis of IL-2 mRNA levels in noninfected Jurkat T cells after treatment with nicotinamide and activation with α-CD3/28.
(E) Real-time RT-PCR analysis of IL-2 mRNA levels in Jurkat T cells infected with lentiviral vectors after treatment with nicotinamide and activation with α-CD3/28 antibodies.
(F) Western blot analysis of Tat protein in Jurkat T cells infected with lentiviral vectors after treatment with nicotinamide and activation with α-CD3/28. In B–E averages of three independent experiments (±SEM) are shown.

Tat neutralizes the inhibitory effect of SIRT1 on NF-κB-responsive promoters
Promoter reporter assays using (A) the IκBα, (B) the E-selectin, and (C) the 3X-κB luciferase reporter construct. Luciferase constructs (0.2 μg) were transiently cotransfected with expression vectors for p65 or p65 K310R (1 ng), SIRT1 or SIRT1 P447E (0.2 μg), and Tat (50 ng) into HeLa cells. The mean of three independent experiments (±SEM) is shown.

Tat inhibits the SIRT1 deacetylase activity
(A) In vitro deacetylation assay of recombinant SIRT1 in the presence of synthetic Tat (aa 1–72). The rate of 1 μM SIRT1 was calculated for 200 μM fluorogenic deacetylase substrate (aa 379–382 of p53) in the presence of twelve concentrations of synthetic Tat. The data were plotted in Prism with X equal to Log₁₀[Tat(μM)] and Y equal to rate. A nonlinear regression was done using the one-site competitive binding equation to determine the IC₅₀.
(B) In vitro deacetylase assay of recombinant SIRT1 and recombinant acetylated p65 in the presence of synthetic Tat or nicotinamide. Western blotting was performed with α-AcK310 p65 and α-p65 antibodies.
(C) In vivo acetylation assay of overexpressed p65. Expression vectors encoding T7-tagged p65 (0.5 μg), p300 (2 μg), SIRT1 (1 μg), and Tat (0.1, 0.25, and 0.5 μg) were transiently cotransfected in 293 cells as indicated. Total cell lysates were analyzed by western blotting with α-AcK310 p65, α-T7, and α-FLAG antibodies. p300 was immunoprecipitated and subjected to western blotting with α-HA antibody.
(D) In vivo acetylation assay of endogenous p65. 293 cells were cotransfected with expression vectors encoding Tat (0.5 μg) and p300 (10 μg) as indicated and treated with TNFα (20 ng/ml) and trichostatin (TSA; 400 nM) over night. TSA is an inhibitor of class I and II HDACs, but not sirtuins and was added to prolong nuclear retention of p65 through hyperacetylation of K218 and K220. Endogenous p65 was immunoprecipitated using α-p65 antibody and subjected to western blotting with α-AcK310 p65 or α-p65 antibodies. Western blotting for p300 was performed with α-HA antibody.

Tat blocks the SIRT1 deacetylase activity and superinduces T-cell activation and HIV transcription via NF-κB. See text for details.

Tat-mediated superinduction of NF-κB-responsive genes requires SIRT1
(A) Western blot analysis of SIRT1, Tat and β-actin in SIRT1−/− MEF cells infected with MSCV-based retroviral particles expressing SIRT1 or Tat.
(B–D) Real-time RT-PCR analysis of IκBα (B), E-selectin (C), and GAPDH (D) mRNA levels in the four polyclonal MEF populations described in (A) after incubation with TNFα (20 ng/ml) for 2 h and 8 h. Data are presented relative to values obtained in cells lacking Tat (100%). The mean of three independent experiments (±SEM) is shown.

Tat binds to the acetyl lysine-binding site in SIRT1
(A) Coimmunoprecipitation assay of Tat and SIRT1 deletion mutants. Expression vectors for Myc-tagged murine Sir2α deletion mutants or full length human SIRT1 (each 1 μg) were transiently cotransfected with constructs expressing FLAG-tagged Tat (1 μg) in 293 cells. Immunoprecipitations were performed with α-FLAG agarose and western blotting with α-Myc and α-FLAG antibodies.
(B) Schematic summary of Tat binding to murine Sir2α deletion mutants. The relative localization of the human SIRT1 HDAC domain is shown at the bottom.
(C) Schematic representation of the human SIRT1 HDAC domain with point mutations.
(D) In vitro HDAC assays of immunoprecipitated human SIRT1 mutants with and without NAD⁺.
(E) Coimmunoprecipitation of SIRT1 mutants and Tat in 293 cells. Cell extracts were immunoprecipitated with α-T7 agarose followed by western blotting with α-FLAG and α-T7 antibodies.

Hyperacetylation of p65 by Tat contributes to T-cell hyperactivation
(A) Induction of p65 hyperacetylation by wild type and mutant Tat. Expression vectors encoding T7-tagged p65 (0.5 μg), p300 (2 μg), wild type and mutant Tat (1 μg each) were transiently cotransfected in 293 cells as indicated. Immunoprecipitations were performed with α-T7 agarose or α-FLAG agarose to isolate p65 or Tat followed by western blotting with α-AcK310 p65, α-T7, and α-FLAG antibodies. p300 was immunoprecipitated and subjected to western blotting with α-HA antibody.
(B) Flow cytometry of Jurkat T cells infected with lentiviral vectors expressing wild type or mutant Tat and GFP. Data are representative of three independent experiments in which infection efficiencies ranged from 75–85% GFP⁺ cells.
(C) Real-time RT-PCR analysis of IL-2 mRNA levels induced in infected Jurkat T cells after activation with α-CD3/28 antibodies. Data are presented as fold induction by α-CD3/28 treatment relative to vector (EF-1α-GFP)-infected cells (100%). The average (mean±SEM) of three independent experiments is shown.