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. 2016 Mar 28;90(8):3902-3912.
doi: 10.1128/JVI.00129-16. Print 2016 Apr.

Suppression of Type I Interferon Production by Human T-Cell Leukemia Virus Type 1 Oncoprotein Tax through Inhibition of IRF3 Phosphorylation

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Suppression of Type I Interferon Production by Human T-Cell Leukemia Virus Type 1 Oncoprotein Tax through Inhibition of IRF3 Phosphorylation

Chun-Kit Yuen et al. J Virol. .

Abstract

Infection with human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and tropical spastic paraparesis. Type I interferons (IFNs) are key effectors of the innate antiviral response, and IFN-α combined with the nucleoside reverse transcriptase inhibitor zidovudine is considered the standard first-line therapy for ATL. HTLV-1 oncoprotein Tax is known to suppress innate IFN production and response but the underlying mechanisms remain to be fully established. In this study, we report on the suppression of type I IFN production by HTLV-1 Tax through interaction with and inhibition of TBK1 kinase that phosphorylates IRF3. Induced transcription of IFN-β was severely impaired in HTLV-1-transformed ATL cells and freshly infected T lymphocytes. The ability to suppress IRF3 activation was ascribed to Tax. The expression of Tax alone sufficiently repressed the induction of IFN production by RIG-I plus PACT, cGAMP synthase plus STING, TBK1, IKKε, IRF3, and IRF7, but not by IRF3-5D, a dominant-active phosphomimetic mutant. This suggests that Tax perturbs IFN production at the step of IRF3 phosphorylation. Tax mutants deficient for CREB or NF-κB activation were fully competent in the suppression of IFN production. Coimmunoprecipitation experiments confirmed the association of Tax with TBK1, IKKε, STING, and IRF3.In vitrokinase assay indicated an inhibitory effect of Tax on TBK1-mediated phosphorylation of IRF3. Taken together, our findings suggested a new mechanism by which HTLV-1 oncoprotein Tax circumvents the production of type I IFNs in infected cells. Our findings have implications in therapeutic intervention of ATL.

Importance: Human T-cell leukemia virus type 1 (HTLV-1) is the cause of adult T-cell leukemia (ATL), an aggressive and fatal blood cancer, as well as another chronic disabling disease of the spinal cord. Treatments are unsatisfactory, and options are limited. A combination of antiviral cellular protein alpha interferon and zidovudine, which is an inhibitor of a viral enzyme called reverse transcriptase, has been recommended as the standard first-line therapy for ATL. Exactly how HTLV-1 interacts with the cellular machinery for interferon production and action is not well understood. Our work sheds light on the mechanism of action for the inhibition of interferon production by an HTLV-1 oncogenic protein called Tax. Our findings might help to improve interferon-based anti-HTLV-1 and anti-ATL therapy.

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Figures

FIG 1
FIG 1
Loss of IFN-β induction in HTLV-1-transformed ATL cells. (A and B) Expression of viral and IFN-β transcripts. HTLV-1 Jurkat and CEMT4 cells, as well as HTLV-1+ MT2, MT4, and C8166, were mock infected or infected with Sendai virus (SeV). RNAs were extracted at 24 h postinfection. The expression of Tax and IFN-β (IFNB) transcripts was analyzed by quantitative RT-PCR and normalized to endogenous expression of β-actin (ACTB) mRNA. Results represent the means ± the SD of three independent experiments. ND, not detected. (C) RT-PCR analysis of Sendai virus DI RNA (SeV-DI). The band represents a PCR product of 546 bp. (D and E) No induction of IFN-β mRNA by VSV-GFP in HTLV-1-transformed cells. Cells were infected with VSV-GFP at a multiplicity of infection of 0.1. Cells were harvested for quantitative RT-PCR and flow cytometric analysis at 24 h postinfection. The GFP intensity of VSV-GFP-infected cells was determined by flow cytometry. Cells were fixed with freshly prepared 4% paraformaldehyde. The GFP signal of individual cells was analyzed by BD Biosciences LSRFortessa cell analyzer. Mock-infected cells were used to set the gates for GFP-negative cells. The mean fluorescence intensity of infected cells with GFP signal was calculated using FlowJo software package. ***, P < 0.001 (Student t test).
FIG 2
FIG 2
Suppression of IFN-β induction in Jurkat cells freshly infected with HTLV-1. (A) Activation of IRF3 reporter by RIG-IN in Jurkat cells. Cells were cotransfected with pIRF3-Luc and escalating amounts of RIG-IN plasmid. Cells were harvested for dual-luciferase assays after 48 h. Only 5 to 10% of Jurkat cells were transfected, as verified by confocal microscopy. (B) Verification of LTR activation. Jurkat cells were transfected with pLTR-Luc. After 24 h, escalating numbers (2 × 105 and 4 × 105) of CEMT4 or MT2 cells were cocultured with the transfected Jurkat cells. Cells were harvested and assayed for dual-luciferase activity after another 24 h. (C) Analysis of IRF3 reporter activity. Jurkat cells were transfected with pIRF3-Luc reporter and RIG-IN expression plasmid. Coculture and a dual-luciferase assay were carried out essentially as described for panel B.
FIG 3
FIG 3
Suppression of IRF3 activation by Tax. (A) Reporter assay. Jurkat cells were cotransfected with pIRF3-Luc, RIG-IN plasmid and escalating doses of Tax expression construct. (B) Native gel analysis of IRF3 dimerization. HEK293 cells were transfected with expression plasmids for V5-tagged IRF3 (V5-IRF3), RIG-IN, and Tax. After 24 h, the cells were lysed, and the dimerization of IRF3 was analyzed using native gel electrophoresis. Total IRF3 and β-tubulin were resolved by SDS-PAGE as internal controls.
FIG 4
FIG 4
Inhibition of IRF3 activation by Tax. (A to L) HEK293 cells were transfected with the indicated reporters, together with a fixed amount (100 ng) of expression plasmids for activator proteins and increasing doses of Tax plasmid (1, 10, and 100 ng). At 30 h posttransfection, the cells were harvested for dual-luciferase assays. The results were statistically analyzed by Student t test. (C) Representative Western blot indicating target protein expression. ***, P < 0.001; N.S., not significant.
FIG 5
FIG 5
(A to C) Inhibition of IRF3 and cGAS activity by Tax. HEK293 cells were transfected, and a luciferase assay was performed as in Fig. 4. ***, P < 0.001; **, P < 0.01; N.S., not significant.
FIG 6
FIG 6
Tax inhibition of IFN-β production does not require CREB or NF-κB activation. (A) Activity of RIG-IN; (B) activity of cGAS; (C and D) verification of the activity of Tax mutants on LTR and κB promoters. HEK293 cells were transfected, and luciferase assays were performed as in Fig. 4.
FIG 7
FIG 7
(A to E) Association of Tax with TBK1, IKKε, STING, and IRF3. Coimmunoprecipitation was performed reciprocally using the indicated antibodies. Tax was not detected in the TBK1 complex immunoprecipitated from Jurkat or CEMT4 cells (data not shown). In panels C to E, HEK293 cells were transfected with plasmids expressing the indicated proteins. IP, immunoprecipitation; β-tub, β-tubulin; IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain.
FIG 8
FIG 8
Inhibition of TBK1 kinase activity by Tax. (A) Verification of the purity of recombinant proteins. (B) Verification of TBK1 in the immunoprecipitate. (C) Kinase assay was performed with purified GST-IRF3, TBK1, and MBP-Tax proteins in the presence of [γ-32P]ATP. Phosphorylated IRF3 (IRF3P) was separated by SDS-PAGE and visualized by autoradiography. (D) Densitometric analysis of phosphorylated IRF3. (E) Influence of Tax on RIG-IN-induced phosphorylation of TBK1 in HEK293 cells. (F) Schematic diagram illustrating Tax suppression of IFN production and signaling.

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