HIV-1 Tat interacts with NFAT1 and upregulates NFAT1-mediated transactivation. (A) Jurkat cells were transfected with 2 μg of the reporter plasmid NFAT3×-Luc and expression plasmids for Tat (0.5 μg) and/or NFAT1 (5 μg). Cells were stimulated for 8 h with 10 nM PMA and 2 μM ionomycin. (B) Similar experiments were carried out with the GAL4-Luc reporter plasmid (2 μg) and pGAL4-NFAT1(1–415) (2.5 μg), which expresses a fusion between the GAL4-DBD and the terminal domain of NFAT1. Total amounts of DNA were adjusted by using the appropriate empty vector. Results are shown as percentages of the luciferase activity of the NFAT1- or GAL4-NFAT1(1–415)-transfected cells. Values are the means + standard errors of results from three independent experiments. ∗, P < 0.02. (C and D) GST-Tat one-exon or Tat two-exon recombinant proteins (6 μg) were assayed for their ability to bind recombinant NFAT1(1–415) (C) or the NFAT1 DBD (D). Binding reaction mixtures were run on SDS-polyacrylamide gels and blotted. The antibodies anti-67.1 against the amino-terminal domain of NFAT1 and R59 against the NFAT1 DBD were used to detect bound NFAT1 proteins. Recombinant GST protein was used as a negative control. (E) Nuclear extracts from ionomycin-stimulated Cl.7W2 cells (N.E.) were incubated, in the presence (lane 2) or absence (lane 1) of purified GST-Tat protein, with radiolabeled oligonucleotides containing the adjacent κB and Sp1 sites of the HIV-1 LTR. Antibodies raised against NFAT1 (lane 4) or HIV-1 Tat (lane 5) were used to check the presence of those proteins in the EMSA bands. Lane 3 contains a binding reaction mixture with radiolabeled probe and GST-Tat without nuclear extract. N.S., nonspecific complex. (F) Total cellular extracts of PMA- and ionomycin-stimulated HEK293T cells expressing HA-tagged NFAT1 alone or coexpressing HIV-1 Tat were immunoprecipitated (IP) with anti HIV-1 Tat. Immunoprecipitates were assayed by Western blotting to detect coimmunoprecipitation of NFAT1. (G) Total cellular extracts of PMA- and ionomycin-stimulated HEK293T cells expressing HIV-1 Tat alone or coexpressing HA-tagged NFAT1 were immunoprecipitated with anti-HA. Immunoprecipitates were assayed by Western blotting to detect coimmunoprecipitation of HIV-1 Tat.

HIV-1 Tat enhancement of NFAT1 transactivation requires a transcriptionally active Tat protein and is mediated by the first 26 amino acids of Tat. (A) The binding affinities of two mutated Tat proteins (TatC22G and TatΔ2–26) for the amino-terminal domain of NFAT1 were assayed. Binding reaction mixtures were resolved on an SDS-acrylamide gel and blotted. The bound products were detected with an antibody against an amino-terminal peptide of NFAT1 (67.1). A Ponceau red staining of the blot showing the amounts of Tat proteins used in the binding reaction mixtures is shown below the immunoblot. (B) An inactive Tat protein does not stimulate NFAT1-mediated transactivation. Jurkat cells were transfected with a GAL4-Luc reporter plasmid (2 μg) and pGAL4-NFAT1(1–415) (2.5 μg), with or without a plasmid expressing a mutant (Cys22-to-Gly) Tat protein (0.5 μg). Cells were stimulated for 8 h with 10 nM PMA and 2 μM ionomycin. Total amounts of DNA were adjusted with the appropriate empty vector. Values are means + standard errors of results from three independent experiments. (C) Expression of a GFP-Tat(1–27) fusion protein inhibits Tat-mediated upregulation of NFAT1 transactivation. Jurkat cells were transfected with a GAL4-Luc reporter plasmid (2 μg), pGAL4-NFAT1(1–415) (2.5 μg), pcTat (0.5 μg), and pEGFP or pEGFPTat(1–27) (2 μg). Cells were stimulated for 8 h with 10 nM PMA and 2 μM ionomycin. Values are the means of results from two independent experiments.

NFAT1 binding to the κB site of the HIV-1 LTR is necessary for NFAT1-mediated downregulation of Tat transactivation. (A) Nuclear extracts from ionomycin-stimulated Cl.7W2 cells (lanes 1 to 4) or the purified NFAT1 DBD (lanes 5 to 8) were incubated with radiolabeled oligonucleotides containing the adjacent κB and Sp1 sites of the HIV-1 LTR (wild type, lanes 1 and 5) or the indicated 5′ and 3′ mutations (Mut). Arrows indicate the positions of the different complexes. Sequences of the oligonucleotides used in the binding assays are shown below. Sequences in boldface type indicate the actual κB and Sp1 sites on the probe. Mutated bases in the oligonucleotides with 5′ or 3′ mutations are underlined. (B) Jurkat cells were transfected with Tat (0.25 μg) and/or NFAT1 (10 μg) expression plasmids and with luciferase reporter vectors containing a mutated LTR in which both κB binding sites were made unable to bind NFAT1 by mutating their 3′ halves from TTCC to AGTT. The effect of NFAT1 on the transactivation caused by Tat was assayed. Results are shown as percentages of the luciferase activity of the pcTat-transfected cells. Values are the means of results from two independent experiments. In all the experiments, the total amounts of DNA were maintained at a constant level by cotransfecting balancing amounts of empty pEFTag plasmid.

The transactivation domain of NFAT1 interacts with HIV-1 Tat. (A) Different recombinant fragments from the amino-terminal domain of NFAT1 (amino acids 1 to 415, lanes 1 to 3; amino acids 67 to 415, lanes 4 to 6; amino acids 140 to 415, lanes 7 to 10; amino acids 1 to 96, lanes 10 to 12) were assayed for binding to the GST–HIV-1 Tat two-exon protein (6 μg) (lanes 3, 6, 9, and 12). Control reaction mixtures with only glutathione-Sepharose beads were included as negative controls (lanes 2, 5, 8, and 11). Anti-72 antibody was used to detect NFAT1 fragments in lanes 1 to 9, and anti-67.1 antibody was used in lanes 10 to 12. TAD, transactivation domain. (B) Jurkat cells were transfected with a GAL4-Luc reporter plasmid (2 μg); pGAL4-NFAT1ΔSP2 (0.25 μg), which expresses a fusion between GAL4-DBD and the amino-terminal domain of NFAT1 with a deletion spanning amino acids 145 to 387; and/or pcTat (0.5 μg). Cells were stimulated for 8 h with 10 nM PMA and 2 μM ionomycin. Total amounts of DNA were adjusted with the appropriate empty vector. Results are shown as percentages of the luciferase activity of the GAL4-NFAT1ΔSP2-transfected cells. Values are the means + standard errors of results from three independent experiments. ∗, P < 0.05.

Effect of NFAT1 on Tat-mediated activation of the HIV-1 LTR. (A) Jurkat cells were transfected with 1 μg of the reporter plasmid HIV-1 LTR CAT and expression plasmids for Tat (0.25 μg) and/or NFAT1 (10 μg). Cells were stimulated for 12 h with 10 nM PMA and 2 μM ionomycin. Results are shown as percentages of the CAT activity of the Tat-transfected cells. Values are means + standard errors of results from three independent experiments. ∗, P < 0.01. (B) Jurkat cells were cotransfected with 1 μg of HIV-1 LTR-Luc, 0.25 μg of pcTat, and increasing amounts of the NFAT1 expression plasmid. Twenty-four hours after transfection, cells were stimulated for 12 h with 10 nM PMA and 2 μM ionomycin. The results of a representative experiment are shown. In all experiments the total amounts of DNA were maintained at a constant level by cotransfecting balancing amounts of empty pEFTag plasmid.

NFAT1 competes with NF-κB1 for binding to the κB site and downregulates NF-κB-mediated activation of the HIV-1 LTR. (A) RelA-mediated activation. In the left graph, Jurkat cells were cotransfected with 1 μg of the reporter plasmid HIV-1 LTR CAT and expression plasmids for NFAT1 (10 μg) and/or RelA (3 μg). Cells were stimulated for 12 h with 10 nM PMA and 2 μM ionomycin. Results are shown as percentages of the CAT activity of the RelA-transfected cells. Values are means + standard errors of results from six independent experiments. ∗∗, P < 0.01. In the right graph, Jurkat cells were cotransfected with 1 μg of the reporter plasmid HIV-1 LTR CAT, 3 μg of a RelA expression plasmid, and increasing amounts of an NFAT1 expression plasmid. Twenty-four hours after the transfection, cells were stimulated for 12 h with 10 nM PMA and 2 μM ionomycin. A representative experiment is shown. In all the experiments the total amounts of DNA were maintained at a constant level by cotransfecting balancing amounts of empty pEFTag plasmid. (B) A labeled oligonucleotide containing the adjacent κB and Sp1 sites of the HIV-1 LTR was incubated with 2 ng of NF-κB p50 in the presence of increasing amounts of the purified recombinant NFAT1 DBD. Arrows indicate the positions of the different complexes.