The bZIP transcription factor ATFx binds human T-cell leukemia virus type 1 (HTLV-1) Tax and represses HTLV-1 long terminal repeat-mediated transcription

Abstract

The human T-cell leukemia virus type 1 (HTLV-1) viral protein Tax is a transactivator of transcription driven by the cognate viral long terminal repeat (LTR). Tax exerts its effect through three nonidentical copies of the Tax-responsive element (TxRE), a member of the asymmetric cyclic AMP response element (CRE) family of enhancer sequences. Transactivation is mediated via interaction of Tax with members of the CREB/ATF family bound to TxRE. We have identified a cellular repressor of transcription, activating transcription factor x (ATFx), as a novel Tax-binding protein. In addition to binding directly to Tax we show by electrophoretic mobility shift assay that ATFx binds to the TxRE enhancer element via the bZIP domain. The functional impact of this bridging interaction results in repression of both basal and Tax-induced transcription from the HTLV-1 LTR. ATFx is unique among ATF family of proteins in that it is cell cycle regulated and exerts a tight repressive control over apoptotic signaling. We propose that recruitment of ATFx to the HTLV-1 LTR serves to link viral transcription with critical events in cellular homeostasis.

FIG. 1.

Comparison of structure of the CREB, CREB2/ATF-4 and ATFx proteins, and ATFx expression constructs. (A) Alignment and domain organization of CREB, CREB2/ATF4, and ATFx proteins. The protein domains were predicted by scanning the protein sequences using the Simple Modular Architecture Research Tool (SMART) program, available at http://us.expasy.org/tools/. The N-terminal region of ATFx contains a negatively charged and proline-rich domain. (B) Shown are the different ATFx proteins used in these studies; full-length ATFx, N-terminal deletion of ATFx with intact b-zip domain (ATFx-ΔN), and C-terminal deletion of ATFx with the b-zip domain deleted (ATFx-ΔC).

FIG. 2.

Coimmunoprecipitation of Tax and ATFx. Whole-cell lysates were prepared from cells transfected with plasmids expressing GFP-Tax and GST-ATFx-ΔN (lanes 1 to 3), GFP and GSTATFx-ΔN (lanes 4 and 5) or mock transfected (lane 6). The isolated cell lysates were either subjected to coimmumoprecipitation with either anti-GST (lanes 1 and 4) or anti-Xpress tag antibody (lane 2) or used without immunoprecipitation (lanes 3 and 4). The resulting precipitates were subjected to SDS-PAGE Western blot analysis using anti-GFP antibody for detection. For each lane, proteins present in whole-cell lysates and antibodies used for immunoprecipitation are labeled. The bands corresponding to GFP-Tax and GFP are shown by arrows.

FIG. 3.

Expression of ATFx in normal human tissue. Human multiple tissue RNA blots were hybridized with ³²P-labeled cDNA corresponding to the entire coding sequence of ATFx. The ≈2.2-kb message is shown by an arrow. It appears to be abundant in testis, prostate and liver. An alternative splice variant of ≈1kb is present in skeletal muscle.

FIG. 4.

Western analysis of Tax and ATFx-coexpressing cells. (A) Whole-cell lysates were prepared from 293T cells transiently transfected with plasmids expressing Tax (lane 1), Tax and ATFx-ΔN (lane 2), Tax and Chk2 (lane 3), and mock transfected (lane 4). Lysates were subjected to immunoblotting as described in the methods section and probed with anti-Tax antibody. (B) Cell lysate examined in lane 2 was also probed with anti-X-press tag antibody to detect ATFx-ΔN expression (lane 5), and lane 6 is a mock-transfected negative control. For each lane, proteins present in whole-cell lysates are labeled. Bands corresponding to Tax and ATFx are shown by arrows.

FIG. 5.

Effect of ATFx expression on the activation of the HTLV-1 LTR. (A) ATFx represses transcription from the HTLV-1-LTR. Activity was assessed by CAT reporter assays as described. Shown are the actelyated % conversions.HTLV-1-LTR-CAT (lanes 1 to 4) and CMV-CAT (lanes 5 and 6) were used as reporter plasmids. Plasmids expressing ATFx (lanes 1, 3, and 5) and HTLV-1 Tax (3 and 4) were used as shown. (B) Expression of CREB or CREB2 reverses ATFx repression of Tax-mediated activation of the HTLV-1-LTR. The effect of CREB and CREB2/ATF4 expression on ATFx-mediated repression of HTLV-1 LTR activation was assessed by luciferase assays. Increasing amounts of the respective plasmids, CREB and CREB2/ATF4, are indicated. Shown is fold activation of the resulting luciferase activity over the basal activity of HTLV-1-LTR-luciferase construct alone. The results shown are representative of three experimental replicates.

FIG. 6.

Electrophoretic mobility shift assay to assess interaction between ATFx and the HTLV-1 LTR containing the TRE. Interaction of ATFx with the HTLV-1 LTR was examined by electrophoretic mobility shift assay. The probe used was a 71-bp region spanning TRE's A+B corresponding to the HTLV-1 LTR. Shown is an autoradiograph of EMSA with full-length ATFx (lane 1), b-zip domain deleted ATFx-ΔC (lane 2), and N-terminally deleted ATFx-ΔN (lane 3). Bands corresponding to the probe and ATFx complex are indicated.