Activation of nuclear factor of activated T cells by human T-lymphotropic virus type 1 accessory protein p12(I)

Abstract

Human T-lymphotropic virus type 1 (HTLV-1) is the agent of an aggressive malignancy of CD4(+) T lymphocytes, called adult T-cell lymphoma/leukemia, and is associated with numerous immune-mediated diseases. To establish infection, HTLV-1 must activate targeted T cells during early stages of infection. We recently demonstrated that the HTLV-1 accessory protein p12(I) is critical for persistent infection in vivo and for viral infectivity in quiescent primary lymphocytes, suggesting a role for p12(I) in lymphocyte activation. To test whether p12(I) modulates signaling pathways required for T-lymphocyte activation, we examined AP-1-, NF-kappaB-, and nuclear factor of activated T cells (NFAT)-driven reporter gene activity in p12(I)-expressing Jurkat T cells compared to vector-transfected control cells. HTLV-1 p12(I) specifically induced NFAT-mediated transcription approximately 20-fold in synergy with the Ras/mitogen-activated protein kinase pathway, but did not influence AP-1- or NF-kappaB-dependent gene expression. Inhibition of calcium-dependent signals by cyclosporin A, BAPTA-AM [glycine, N,N'-1,2-ethanediylbis(oxy-2,1-phenylene)-bis-N-2-(acetyloxy)methoxy-2-oxoethyl]-[bis(acetyloxy)methyl ester], and a dominant negative mutant of NFAT2 abolished the p12(I)-mediated activation of NFAT-dependent transcription. In contrast, inhibition of phospholipase C-gamma and LAT (linker for activation of T cells) did not affect p12(I)-induced NFAT activity. Importantly, p12(I) functionally substituted for thapsigargin, which selectively depletes intracellular calcium stores. Our data are the first to demonstrate a role for HTLV-1 p12(I) in calcium-dependent activation of NFAT-mediated transcription in lymphoid cells. We propose a novel mechanism by which HTLV-1, a virus associated with lymphoproliferative disease, dysregulates common T-cell activation pathways critical for the virus to establish persistent infection.

FIG. 1.

Specific activation of NFAT transcriptional activity in lymphoid cells by HTLV-1 p12^I and the Ras/MAPK pathway. (A) Expression of HA₁-tagged HTLV-1 p12^I in Jurkat T cells and the LAT-deficient Jurkat T-cell line J.Cam2.5, as analyzed by immunoprecipitation and immunoblot. (B) Jurkat T cells transfected with pME or pME-p12 and corresponding reporter plasmids were either left untreated (U) or treated with PMA (P), ionomycin (I), or a combination of PMA and ionomycin (PI). Graphs depict the fold induction of NFAT-luciferase activity in cells expressing p12^I (pME-p12) over those transfected with empty vector (pME). Data points are the means of triplicate samples and at least four independent experiments. Statistical significance of NFAT-luciferase induction was analyzed by Student's t test. (C) Transfected Jurkat T cells were treated with anti-CD3 or anti-CD28 or both antibodies. Data points are the mean of triplicate samples and two independent experiments.

FIG. 2.

HTLV-1 p12^I-induced NFAT-dependent gene expression is inhibited by dominant-negative NFAT2. (A) A dominant negative NFAT2 (dn-NFAT2) inhibits NFAT-dependent gene expression induced in Jurkat T cells by PMA-ionomycin costimulation. Values represent the means of triplicate samples and two independent experiments. Cells were transfected with NFAT-luciferase reporter but no dominant-negative NFAT2 and left unstimulated to obtain a value for basal reporter gene activity (0/−). An additional control included mock-transfected cells without the luciferase reporter and left unstimulated (m). (B) Expression of dn-NFAT2 in HTLV-1 p12^I-transfected Jurkat T cells (+ column as shown above) results in complete abrogation of p12^I-mediated NFAT activation. Only values for PMA-treated cells are shown. dn-NFAT2 or empty vector (20 μg) was cotransfected with pME-p12. Data points are the means of quadruplicate samples and two independent experiments. Statistical significance was analyzed by Student's t test. Inset shows detection of dn-NFAT2 by immunoprecipitation with monoclonal antibody 7A6, followed by immunoblotting with an anti-HA₁ monoclonal antibody.

FIG. 3.

HTLV-1 p12^I-mediated increase in NFAT activity is sensitive to inhibition of calcineurin by the immunosuppressant cyclosporin A (CsA) (A) and the reduction of intracellular calcium concentration by BAPTA-AM (B). Only values for PMA-treated cells are shown. Values are the means of quadruplicate samples and three independent experiments. Statistical significance was analyzed by Student's t test.

FIG. 4.

Inhibition of PLC-γ1 and LAT or PI₃K does not affect p12^I-mediated induction of NFAT activity. (A) A lipase-inactive mutant of PLC-γ1 (PLC-γ-LI) potently inhibits CD3- and CD28-mediated T-cell stimulation. Control (0/−), 0 μg of PLC-γ-LI, no stimulation; m, mock transfection. (B) PLC-γ-LI (10 μg of pc-AUPLC-LI) does not inhibit HTLV-1 p12^I-mediated activation of NFAT. Only values for PMA-treated cells are shown. Values represent the means of quadruplicate samples and two independent experiments. Statistical significance was analyzed by Student's t test. Expression of PLC-γ-LI was determined by AU₁-specific immunoblot and is shown in the graph inset (lower band). (C) HTLV-1 p12^I activates NFAT-dependent gene expression in the LAT-deficient Jurkat line J.CaM2.5 to levels comparable to those in wild-type Jurkat T cells. Values represent the means of quadruplicate samples and two independent experiments. Statistical significance was analyzed by Student's t test. (D) Inhibition of PI₃K does not abolish p12^I-mediated NFAT activation. Jurkat T cells transfected with pME or pME-p12 and pNFAT-luc were treated with wortmannin as described. Values represent the means of quadruplicate samples and two independent experiments

FIG. 5.

HTLV-1 p12^I localizes to the ER in lymphoid cells and functionally substitutes for thapsiargin. (A) Jurkat T cells transfected with pME (not shown) or pME-p12 were fixed and stained for p12^I, endogenously expressed calreticulin, or the nuclear transcriptional activator p300. HTLV-1 p12^I colocalizes with the ER-resident protein calreticulin (upper panel), while no colocalization was observed with p300 (lower panel). (B) Jurkat T cells transfected with pME or pME-p12 were stimulated with PMA (20 ng/ml) and the indicated amounts of thapsigargin. NFAT-luciferase activity was measured as described and plotted on a logarithmic scale as a percentage of maximal activation induced by the addition of thapsigargin. (C) To confirm that the increased NFAT-luc activity in B was due to the expression of HTLV-1p12^I, increasing amounts of pME and pME-p12^I were transfected and cells were stimulated with 0.5 nM thapsigargin and PMA (20 ng/ml). The highest luciferase value obtained with 10 μg of pME-p12^I was set at 100%.

FIG. 6.

HTLV-1 p12^I-mediated activation of NFAT is sensitive to inhibition of the Ras/MAPK pathway. (A) Treatment of pME- and pME-12-transfected Jurkat T cells with U0126 results in complete abrogation of p12^I-mediated NFAT activity. Only values for PMA-treated cells are shown. Values represent the means of quadruplicate samples and two independent experiments. Statistical significance was analyzed by Student's t test. (B) The expression of a dominant negative mutant of AP-1 (A-Fos) (+ column) completely abrogates the p12^I-mediated induction of NFAT activity. Jurkat T cells were cotransfected with pME or pME-p12 and 10 μg of pCMV500-A-Fos or the corresponding empty vector pCMV500. Only values for PMA-treated cells are shown. Values represent the means of quadruplicate samples and two independent experiments. Statistical significance was analyzed by Student's t test. Expression of A-Fos was examined by immunoblot (inset) using a monoclonal antibody directed against the A-Fos N-terminal Flag tag. (C) A-Fos potently inhibits PMA- and ionomycin-stimulated NFAT activity. Jurkat T cells were cotransfected with pNFAT-luc and the indicated amounts of pCMV500-A-Fos and treated with PMA plus ionomycin.

FIG. 7.

Model for HTLV-1 p12^I-mediated activation of NFAT in infected T lymphocytes. HTLV-1 p12^I causes an increase in the release of calcium from the ER, possibly through interaction with calcium-binding proteins such as calreticulin or calnexin. Increased levels of cytosolic calcium lead to activation of the phosphatase calcineurin (CN) and to dephosphorylation of cytosolically retained NFAT. Upon dephosphorylation, NFAT translocates to the nucleus and synergizes with AP-1 at NFAT:AP1 composite promoter elements to activate NFAT-dependent gene expression. Boxes indicate membrane-localized and membrane-activated proteins. NF-κB and NF-AT response elements (re) are indicated.