Oncogenic human T-cell lymphotropic virus type 1 tax suppression of primary innate immune signaling pathways

Abstract

Human T-cell lymphotropic virus type I (HTLV-1) is an oncogenic retrovirus considered to be the etiological agent of adult T-cell leukemia (ATL). The viral transactivator Tax is regarded as the oncoprotein responsible for contributing toward the transformation process. Here, we demonstrate that Tax potently inhibits the activity of DEx(D/H) box helicases RIG-I and MDA5 as well as Toll-dependent TIR-domain-containing adapter-inducing interferon-β (TRIF), which function as cellular sensors or mediators of viral RNA and facilitate innate immune responses, including the production of type I IFN. Tax manifested this function by binding to the RIP homotypic interaction motif (RHIM) domains of TRIF and RIP1 to disrupt interferon regulatory factor 7 (IRF7) activity, a critical type I IFN transcription factor. These data provide further mechanistic insight into HTLV-1-mediated subversion of cellular host defense responses, which may help explain HTLV-1-related pathogenesis and oncogenesis.

Importance: It is predicted that up to 15% of all human cancers may involve virus infection. For example, human T-cell lymphotropic virus type 1 (HTLV-1) has been reported to infect up to 25 million people worldwide and is the causative agent of adult T-cell leukemia (ATL). We show here that HTLV-1 may be able to successfully infect the T cells and remain latent due to the virally encoded product Tax inhibiting a key host defense pathway. Understanding the mechanisms by which Tax subverts the immune system may lead to the development of a therapeutic treatment for HTLV-1-mediated disease.

FIG 1

HTLV-1 Tax inhibits RIG-I and MDA-5 signaling. (A) An IFN-β-Luc reporter plasmid was transfected in HeLa cells with or without increasing amounts of an expression vector encoding Tax for 24 h. The cells were transfected with poly(I·C) (5 mg/ml), and luciferase activity was measured. The expression of endogenous MDA-5 and ectopic Tax were detected by immunoblotting (IB) analysis. (B) IFN-β-Luc reporter plasmids were transfected in 293T cells with expression vectors encoding either FLAG-RIG-I or FLAG-MDA-5 and with empty vector or Tax. Cells were then treated as described for panel A. (C) Luciferase reporter assays were performed in 293T cells as described for panel B using FLAG-RIG-I variants (aa 1 to 284) or FLAG-MDA-5 variants (aa 1 to 349), and increasing amounts of Tax. (D) ELISAs were performed to detect production of endogenous IFN-β protein in 293T cells after transfecting with FLAG-RIG-I (1-284) and Tax. (E) Luciferase reporter assays were performed in Jurkat T cells after transfecting IFN-β-luc, Tax, and poly(I·C) (5 mg/ml). (F) Viral replication of VSV-GFP was detected in primary C57BL/6 MEFs after transfecting with either an empty vector or Tax for 24 h. Photomicrographs were taken 24 h postinfection. (G) Standard plaque assays for viral progeny output were performed from the supernatants from cells treated as described for panel F. (H) Real-time PCR analysis of ifnβ mRNA level in primary C57BL/6 MEFs that were transfected with either an empty control vector or Tax for 24 h and treated with poly(I·C) for the indicated periods. (I) Immunofluorescence (IF) assays were used to detect expression of Tax (red) in Tax-inducible Jurkat T cells after treating with doxycycline (1 μg/ml) for 24 h. (J) Real-time PCR analysis for ifnβ mRNA transcription in Tax-inducible Jurkat T cells after the infection of VSVΔM for 6 h. (K) Real-time PCR analysis was performed to evaluate mRNA levels of ifnβ in MT-4 cells or ATLL-84c cells that were treated with VSVΔM (MOIs = 1) for the indicated times. Tax expression was also tested in those cell lines by immunoblotting using anti-Tax antibody. Error bars indicate ± standard deviations (SD).

FIG 2

HTLV-1 Tax selectively inhibits IRF3/7 signaling. (A) Luciferase reporter assays were performed in 293T cells with IFN-β-luc, FLAG-RIG-I(1-284), full-length IPS-1, TRIF, TBK1, IRF3 (superactive mutant), or IRF7 (superactive mutant), with increasing amounts of Tax. (B) Luciferase reporter assays were performed in 293T cells after transfecting IRF3/7-responsive PRDIII-I-luciferase plasmid, expression vectors encoding either FLAG-RIG-I(1-284), FLAG-MDA-5(1-349), or full-length TBK1, and increasing amounts of Tax. (C) The luciferase reporter assays illustrated in panel B were performed in 293T cells with NF-κB-responsive PRDII-luciferase plasmid. Error bars, ±SD.

FIG 3

HTLV-1 Tax interacts with RIP1. (A) S. cerevisiae strain AH109 was cotransformed with Tax, together with the indicated prey plasmids. Only cells coexpressing Tax and RIP1 grew under high-stringency selection conditions, indicating interaction. (B) Schematic diagram of RIP1 and RIP1 deletion mutants. (C) Coimmunoprecipitation (Co-IP) analyses were performed in 293T cells after cotransfecting Tax and FLAG-tagged RIP1 mutants using anti-FLAG antibody. IB analysis detected the indicated proteins. (D) IF assays detected expression of Tax (red) and RIP1(green) in Tax-inducible Jurkat T cells or primary ATL cells after treating with doxycycline (1 μg/ml) for 24 h. (E) Luciferase reporter assays were performed in 293T cells after transfecting IFN-β-luc, PRD-III luciferase, or PRD-II luciferase reporter plasmids, expression vectors encoding FLAG-TRIF, and increasing amounts of Tax. (F) Co-IP analyses were performed in 293T cells after cotransfecting expression plasmids encoding Tax and the FLAG-TRIF or FLAG-RIP1 variant (aa 301 to 588). After 36 h, the cell lysates were pulled down using anti-FLAG antibody and IB analysis was performed using anti-Tax antibody. Error bars, ±SD.

FIG 4

RIP1 associates with RIG-I in the presence of dsRNA. (A) HeLa cells were treated with or without IFN-β for 18 h and poly(I·C) for 3 h, and the lysates were subjected to poly(I·C) to dsRNA affinity chromatography. Precipitated poly(I·C) agarose was resolved by SDS electrophoresis analysis. Indicated antibodies were used for IB. (B) Co-IP analyses were performed in 293T cells transfected with HA-RIP1, either FLAG-RIG-I variants (full length, aa 1 to 284, or aa 219 to 925) or FLAG-MDA5 variants (full length, aa 1 to 357, or aa 236 to 1026). The lysates were immunoprecipitated (IP) with anti-FLAG antibody, and immunoblot analysis was performed with anti-HA antibody. (C) Yeast two-hybrid assays were used to detect the GAL4 binding domain (BD)-conjugated full-length RIP1, which strongly interacts with full-length FADD in yeast. Weak interaction of RIP1 and RIG-I (aa 1-200) or MDA-5 (aa 101-200) was also detected in the yeast two-hybrid system. The GAL4 activation domain was fused to full-length FADD, RIG-I variants (top left, aa 1-200; top right, aa 1-100; bottom left, aa 101-200), and MDA-5 variants (top left, aa 1-200; top right, 1-100; bottom left, aa 101-200). (D) Co-IP analyses were performed in 293T cells after overexpressing plasmids encoding HA-RIG-I (aa 1 to 284) and FLAG-RIP1 deletion variants. The lysates were immunoprecipitated with-anti FLAG antibody and IB analysis was performed with anti-HA antibody. (E) 293T cells were transfected with HA-RIG-I(1-284) and the FLAG-intermediate domain of RIP1(301-588), either with or without the expression vector encoding Tax. The cell lysates were immunoprecipitated, and IB analysis was performed with the indicated antibodies. (F) Luciferase reporter assays were performed in 293T cells after transfecting IFN-β-luc expression vectors, plasmids encoding either FLAG-RIG-I(1-284) or FLAG-MDA-5(1-357), and increasing amounts of an expression vector encoding full-length RIP1 or an intermediate domain of RIP1(301-588) for 36 h. (G) Luciferase reporter assays were performed in 293T cells after transfecting IFN-β-luciferase expression vectors, plasmids encoding either FLAG-RIG-I(1-284) or FLAG-MDA-5(1-357), and increasing amounts of plasmid encoding FADD or the death effector domain of FADD (aa 1 to 97) for 36 h. (H, I) IFN-β-Luciferase reporter plasmids were transfected in 293T cells with expression vectors encoding either RIG-I(1-284) along with expression vectors encoding full-length RIP1 or FADD and increasing amounts of plasmid encoding Tax. Thirty-six hours later, the cells were subjected to luciferase reporter assay. Error bars, ±SD.

FIG 5

Tax abrogates RIP1 and IRF7 interaction. (A) Co-IP analyses were performed in 293T cells after overexpressing Tax and FLAG-RIP1, FLAG-intermediate domain of RIP1 (aa 301 to 588), FLAG-TRAF3, or FLAG-TANK, using anti-FLAG antibody. IB analyses were done with the indicated antibody. (B) Co-IP analysis was done in 293T cells using expression plasmids encoding FLAG-RIP1 or HA-TRAF3 and either with or without Tax. (C) Co-IP analyses were performed as described for panel A using expression vectors encoding FLAG-RIP1, TBK-GFP, and Tax. (D, E) Co-IP analyses were performed as described for panel A using expression vectors encoding FLAG-IRF3 or FLAG-IRF7, TBK-GFP, and Tax. (F) Co-IP analyses were done in 293T cells as described for panel A using plasmids encoding Myc-RIP1 and FLAG-IRF3 or FLAG-IRF7. (G) Co-IP analysis in 293T cells as described for panel A using plasmids encoding full-length Myc-IRF7 and FLAG-RIP1 mutants: kinase domain (aa 1 to 300), intermediate domain (aa 301 to 588), or death domain (aa 589 to 671). (H) Native PAGE gel analysis for IRF3 dimerization in 293T cells with transfection of an empty vector or expression vector encoding HA-RIG-I (aa 1 to 284), either with or without plasmid encoding Tax. The cells were stimulated with poly(I·C) (1 μg/ml) for 4 h, and the lysates were subjected to IB with the indicated antibodies. (I) Luciferase reporter assays were performed in 293T cells after transfecting with GAL4 binding luciferase plasmid and an expression vector encoding GAL4-conjugated full-length IRF3 and full-length IRF7 with increasing amounts of Tax. (J) Co-IP analysis in 293T cells after overexpressing plasmids encoding Myc-RIP1 and FLAG-IRF7 with an increasing amount of Tax. IB analysis was done with the indicated antibodies. (K) Depletion of Tax with shRNAs restores RIP1-IRF7 interaction in HTLV-1-transformed cells. Co-IP analysis was done in C8166 cells after lentiviral transduction of control or Tax short hairpin RNAS (shRNAs) (Tax#3 and Tax#5) for 5 days. IB was performed after IgG control or RIP1 IP with the indicated antibodies. Error bars, ±SD.

FIG 6

Tax preferentially inhibits activity of IRF7. (A, B) Schematic figures of intermediate domain of RIP1 variants (A) and IFN-β-Luc (B) reporter assays examined in 293T cells using expression vector encoding FLAG-RIG-I(1-284), FLAG-RIP1 (ID, aa 301 to 588), FLAG RIP1(ID12, aa 291 to 490), or FLAG-RIP1(ID23, aa 391 to 588), either with or without Tax. (C, D, E) Co-IP analysis in 293T cells after expressing FLAG-RIP1 (ID, aa 301 to 588), FLAG-RIP1 (ID12, aa 291 to 490), or FLAG-RIP1(ID23, aa 391 to 588), and either Myc-IRF7, Tax, or HA-RIG-I (aa 1 to 284). Thirty-six hours later, cell lysates were immunoprecipitated, and IB analysis was performed with the indicated antibodies. (F) Structure of dID2 (deletion, aa 391 to 490) of RIP1. (G) Co-IP assay revealed that intermediate domain aa 391 to 490 of RIP1 was essential for Tax association to RIP1 but not for HA-RIG-I(1-284). 293T cells were transfected with ID2 of FLAG-RIP1 (deletion, aa 391 to 490), and either Tax or HA-RIG-I(1-284) for 36 h. The lysates were pulled down with anti-FLAG antibody, and IB was performed with anti-Tax antibody. (H) Schematic model of RIP1-interacting proteins and Tax. Error bars, ±SD.

FIG 7

RIP1 is involved in the induction of IFN-α. (A, B) ELISA measurements of IFN-β and IFN-α with supernatants of RIP1^+/+ and RIP1^−/− MEFs after stimulation with poly(I·C), VSV, or SeV at MOIs of 1 and 10. Error bars, SD. (C) Microarray analysis of dsRNA-inducible genes were performed in RIP1^+/+ and RIP1^−/− MEFs after treatment with poly(I·C) for 12 h. (D) A model of Tax suppression of innate immune signaling. RNA sensor RIG-I, MDA-5, or TLR3 recognizes and activates IPS-1 or TRIF, forming a complex with molecules TRAF3, FADD, and RIP1. This leads to phosphorylation of TBK1, which phosphorylates the transcription factor IRF3 for primary IFN-β induction. Secreted IFN-β binds to IFN receptors and induces ifn αand irf7 through JAK/STAT signaling. HTLV-1 viral protein Tax strongly binds to RIP1 to impede IRF7 function and type I IFN signaling.