Human T cell lymphotropic virus 1 manipulates interferon regulatory signals by controlling the TAK1-IRF3 and IRF4 pathways

Abstract

We previously reported that human T cell lymphotropic virus 1 (HTLV-1) Tax oncoprotein constitutively activates transforming growth factor-beta-activated kinase 1 (TAK1). Here, we established Tax-positive HuT-102 cells stably transfected with a short hairpin RNA vector (HuT-shTAK1 cells) and investigated the physiological function of TAK1. Microarray analysis demonstrated that several interferon (IFN)-inducible genes, including chemokines such as CXCL10 and CCL5, were significantly down-regulated in HuT-shTAK1 cells. In contrast, Tax-mediated constitutive activation of nuclear factor-kappaB (NF-kappaB) was intact in HuT-shTAK1 cells. IFN-regulatory factor 3 (IRF3), a critical transcription factor in innate immunity to viral infection, was constitutively activated in a Tax-dependent manner. Activation of IRF3 and IRF3-dependent gene expressions was dependent on TAK1 and TANK-binding kinase 1 (TBK1). On the other hand, IRF4, another member in the IRF family of transcription factors overexpressed in a Tax-independent manner, negatively regulated TAK1-dependent IRF3 transcriptional activity. Together, HTLV-1 manipulates IFN signaling by regulating both positive and negative IRFs.

FIGURE 1.

Establishment of HuT-102 cells stably down-regulating TAK1 expression by shRNA. A, HuT-102 cells were stably transfected with shRNA vectors for TAK1 or luciferase. Protein expressions of TAK1, TAB1, TAB2, and Tax in shRNA-transfected and parent HuT-102 cells were analyzed by immunoblotting. GFP, green fluorescent protein. B, TAK1, TAB1, and TAB2 mRNA expressions in HuT-shTAK1 and HuT-shLuc cells were analyzed by real time RT-PCR. C, cells were treated with or without a proteasome inhibitor, ALLN (100 μm), for 4 h. Whole cells lysates were immunoprecipitated with anti-Tax antibody and then immunoblotted with anti-ubiquitin (Ub) antibody. D, phosphorylation and protein expression of JNK and p38 in shRNA-transfected were analyzed by immunoblotting. E, phosphorylation and protein expression of IKKα/β/γ and NF-κB p65 in shRNA-transfected were analyzed by immunoblotting. F, cells (2 × 10⁶ cells) were seeded in a 75-cm² flask, and the cell number was counted 24 and 48 h after seeding.

FIGURE 2.

Regulation of IFN-inducible genes by TAK1 and Tax. A, microarray analysis was carried out using HuT-shTAK1 and HuT-shLuc cells. Ratios (shTAK1/shLuc) of fluorescence intensities of the representative genes are shown. A total of four arrays were used: two for HuT-shLuc cells and two for HuT-shTAK1 cells. B, CXCL10, IFIT1, and CCL5 mRNA expressions in HuT-shTAK1 and HuT-shLuc cells were analyzed by real time RT-PCR. C, HuT-102 cells were transfected with siRNAs against Tax and Luc. At 60 h after transfection, the expressions of IFN-inducible genes were examined by RT-PCR. D, role of TAK1 and Tax in the expression of viral gag and tax mRNAs was examined by RT-PCR. Data are the mean ± S.D. of triplicate determinations (error bars). The statistical significance of differences between groups was calculated by applying Student's two-tailed t test. *, p < 0.01.

FIGURE 3.

Effects of IRF3 siRNA on CXCL10 and gag expression. A, HuT-102 cells were transfected with IRF3 and Luc siRNAs. At 60 h after transfection, protein expression was analyzed by immunoblotting with antibodies against IRF3, IRF4, TBK1, and actin. B, role of IRF3 in the expression of CXCL10 and gag mRNAs was investigated by RT-PCR.

FIGURE 4.

Tax-dependent constitutive activation of IRF3 via TAK1. A, nuclear extracts from Jurkat and HTLV-1-transformed cells were immunoblotted with anti-phospho-IRF3 (Ser-396), anti-IRF3, and anti-Tax antibodies. B, HuT-102 cells were transfected with Tax and Luc siRNA and then analyzed by immunoblotting as described in A. C, cytoplasmic (Cyto. Ext.) and nuclear extracts (Nuc. Ext.) were prepared from HuT-shTAK1, HuT-shLuc, and parent HuT-102 cells and subjected to immunoblot analysis by using antibodies against phospho-IRF3, IRF3, α-tubulin, and lamin B. D, JPX-9 cells were treated with 120 μm zinc chloride for 9 h and then analyzed by immunoblotting with antibodies against phospho-IRF3, IRF3, phospho-TAK1, TAK1, and Tax. WCE, whole cell extracts.

FIGURE 5.

Role of TBK1 in Tax-mediated IRF3 activation. A, HuT-102 cells were transfected with TBK1 and Luc siRNAs. At 60 h after transfection, protein expression was analyzed by immunoblotting with antibodies against TBK1, phospho-IRF3, and IRF3. B, role of TBK1 in the expression of CXCL10 and CCL5 mRNAs was investigated by RT-PCR.

FIGURE 6.

Suppression of IFN-inducible genes by IRF4. A, total cellular RNAs were extracted from Jurkat and HTLV-1-transformed cells and subjected to RT-PCR using a set of IRF4 primers. B, whole cell lysates were extracted from Jurkat and HTLV-1-transformed cells and subjected to immunoblotting with anti-IRF4 and proliferating cell nuclear antigen (PCNA) antibodies. C, HuT-102 cells were transfected with Tax and Luc siRNA. At 60 h after transfection, IRF4 mRNA expression was examined by RT-PCR. D, HuT-102 cells were transfected with IRF4 and Luc siRNAs. The expressions of CXCL10, IFIT1, IFN-β, and IRF4 mRNA were examined by RT-PCR. E, Tax-negative ED40515(−) cells were transfected with IRF4 and Luc siRNA. At 60 h after transfection, CXCL10 mRNA expression was examined by RT-PCR.

FIGURE 7.

Suppression of TAK1-TBK1-IRF3 pathway by IRF4. A, HuT-shTAK1 and HuT-shLuc cells were transfected with IRF4 and Luc siRNAs. At 60 h after transfection, the expressions of CXCL10 and IRF4 mRNA were examined by RT-PCR. B, HuT-102 cells were transfected with IRF4 and Luc siRNA in combination with IRF3 and TBK1 siRNA, and then CXCL10 mRNA expression was amplified by RT-PCR.