In vivo antagonistic role of the Human T-Cell Leukemia Virus Type 1 regulatory proteins Tax and HBZ

Abstract

Adult T cell leukemia (ATL) is an aggressive malignancy secondary to chronic infection by the human T-cell leukemia virus type 1 (HTLV-1) infection. Two viral proteins, Tax and HBZ, play central roles in ATL leukemogenesis. Tax expression transforms T cells in vitro and induces ATL-like disease in mice. Tax also induces a rough eye phenotype and increases hemocyte count in Drosophila melanogaster, indicative of transformation. Among multiple functions, Tax modulates the expression of the enhancer of zeste homolog 2 (EZH2), a methyltransferase of the Polycomb Repressive Complex 2 (PRC2), leading to H3K27me3-dependent reprogramming of around half of cellular genes. HBZ is a negative regulator of Tax-mediated viral transcription. HBZ effects on epigenetic signatures are underexplored. Here, we established an hbz transgenic fly model, and demonstrated that, unlike Tax, which induces NF-κB activation and enhanced PRC2 activity creating an activation loop, HBZ neither induces transformation nor NF-κB activation in vivo. However, overexpression of Tax or HBZ increases the PRC2 activity and both proteins directly interact with PRC2 complex core components. Importantly, overexpression of HBZ in tax transgenic flies prevents Tax-induced NF-κB or PRC2 activation and totally rescues Tax-induced transformation and senescence. Our results establish the in vivo antagonistic effect of HBZ on Tax-induced transformation and cellular effects. This study helps understanding long-term HTLV-1 persistence and cellular transformation and opens perspectives for new therapeutic strategies targeting the epigenetic machinery in ATL.

Fig 1. Overexpression of HBZ in vivo activates the PRC2 complex but does not induce transformation.

Representative light microscopy images (Scale bar 100μm) and scanning electron microscopy images (Scale bar 100μm and 50μm) of adult eyes from transgenic flies expressing Tax (GMR-Gal4>UAS-Tax) (Tax Tg), or HBZ (GMR-Gal4>UAS-HBZ) (HBZ Tg), under the control of the eye-specific GMR promoter (GMR-GAL4). GMR-GAL4>w1118 was used as control. (B) Relative roughness was quantified based on the number of ommatidial fusions and the extent of bristle organization (n = 37, from three independent crosses). p<0.001 (***) and p<0.05 (*). (C) Cell lysates (150 μg) from control, Tax Tg and HBZ-Tg transgenic adult flies heads were analyzed by western blotting and probed with anti-Myc or anti-actin antibodies. Genotypes indicated are under the control of eye specific promoter GMR-GAL4. (D) Levels of expression of D-jun in the transgenic adult flies heads as indicated. Transcript levels were normalized to Rp49. Reported values are the average of three independent experiments and error bars represent SD of triplicates. (ns = not significant), p<0.01 (**). (E) Cell lysates (300 μg) from control (HMLΔ-Gal4>w1118), Tax Tg (HMLΔ-Gal4>UAS-Tax) and HBZ-Tg (HMLΔ-Gal4>UAS-HBZ) transgenic larvae were analyzed by western blotting confirming the expression of Tax and HBZ transgene in larval hemocytes. Genotypes indicated are under the control of the hemocyte-specific promoter (HMLΔ-GAL4). (F) Circulating haemocytes were counted in control larvae (HMLΔ-Gal4>w1118), larvae expressing Tax (HMLΔ-Gal4>UAS-Tax) (Tax Tg) or HBZ (HMLΔ-Gal4>UAS-HBZ) (HBZ Tg) (n = 30, from three independent crosses). Data represent mean ± standard error of the mean. p<0.01 (**), p< 0.001 (***). (G) Cell lysates (150 μg) from control (GMR-GAL4>w1118), Tax Tg (GMR-Gal4>UAS-Tax), or HBZ Tg (GMR-Gal4>UAS-HBZ) transgenic adult flies heads were analyzed by western blotting and probed with indicated antibodies. Densitometry histograms represent an average of 3 independent experiments.

Fig 2. Tax-induced transformation in vivo is NF-κB and PRC2 dependent.

(A) Relish, E(z), and SUZ12 expression was inhibited by RNAi in flies overexpressing Tax in the compound eye. Representative scanning electron microscopy images of adult eyes are shown. Scale bar 100μm. (B) Relative roughness was quantified based on the number of ommatidial fusions and the extent of bristle organization (n = 30, from three independent crosses). (ns = not significant), p<0.01 (**), p<0.001 (***). (C) Relish, E(z), and SUZ12 expression was inhibited by RNAi in flies overexpressing Tax in the hemocytes. Circulating hemocytes were counted in control larvae (HMLΔ-Gal4> mCherry RNAi), (HMLΔ-Gal4; UAS-Tax>mCherry RNAi), (HMLΔ-Gal4;UAS Tax>Relish RNAi), (HMLΔ-Gal4;UAS-Tax>E(z) RNAi) and (HMLΔ-Gal4;UAS-Tax>Suz12 RNAi) (n = 30, from three independent crosses). Data represent mean ± standard error of the mean. p<0.0001 (****). (D) Levels of expression of Relish in the control (GMR-GAL4>w1118), Tax Tg (GMR-Gal4>UAS-Tax), or HBZ Tg (GMR-Gal4>UAS-HBZ) transgenic adult flies heads. The values were normalized to Rp49. Reported values are the average of three independent experiments and error bars represent SD of triplicates. p<0.001 (***). (E) Levels of expression of Diptericin, a Relish target gene, in the control (GMR-GAL4>w1118), Tax Tg (GMR-Gal4>UAS-Tax), or HBZ Tg (GMR-Gal4>UAS-HBZ) transgenic adult flies heads. The values were normalized to Rp49. Reported values are the average of three independent experiments and error bars represent SD of triplicates p<0.001 (***). (F) Cell lysates (150 μg) from control (GMR-GAL4>w1118), Tax Tg (GMR-Gal4>UAS-Tax), or HBZ Tg (GMR-Gal4>UAS-HBZ), transgenic adult flies heads were analyzed by western blotting and probed with anti-Relish or anti-actin antibody. (G) Transcript expression levels of Relish and Diptericin in the control (GMR-Gal4>mCherry RNAi), Tax control (GMR-Gal4;UAS-Tax>mCherry RNAi), Tax/Relish RNAi (GMR-Gal4;UAS-Tax>Relish RNAi), Tax/E(z) RNAi (GMR-Gal4;UAS-Tax>E(z) RNAi) and Tax/Suz12 RNAi (GMR-Gal4;UAS-Tax>Suz12 RNAi) transgenic flies as indicated. Transcript levels were normalized to Rp49. Reported values are the average of three independent experiments and error bars represent SD of triplicates. p<0.01 (**), p< 0.0001 (****). (H) Transcript levels of expression of E(z) and Suz12 in in the control (GMR-Gal4>mCherry RNAi), Tax control (GMR-Gal4;UAS-Tax>mCherry RNAi), Tax/Relish RNAi (GMR-Gal4;UAS-Tax>Relish RNAi), Tax/E(z) RNAi (GMR-Gal4;UAS-Tax>E(z) RNAi) and Tax/Suz12 RNAi (GMR-Gal4;UAS-Tax>Suz12 RNAi) transgenic flies as indicated. Transcript levels were normalized to Rp49. Reported values are the average of three independent experiments and error bars represent SD of triplicates. p<0.01 (**), p< 0.001 (***). (I) Cell lysates (150 μg) from the control (GMR-Gal4>mCherry RNAi), Tax control (GMR-Gal4;UAS-Tax>mCherry RNAi), Tax/Relish RNAi (GMR-Gal4;UAS-Tax>Relish RNAi), Tax/E(z) RNAi (GMR-Gal4;UAS-Tax>E(z) RNAi) and Tax/Suz12 RNAi (GMR-Gal4;UAS-Tax>Suz12 RNAi) transgenic adult flies heads were analyzed by western blotting and probed with H3K27me3 antibody. Densitometry histograms represent an average of 3 independent experiments. (ns = not significant), p<0.05 (*), p<0.001 (***), p<0.0001 (****).

Fig 3. HBZ overexpression abrogates Tax-induced NF-kB, PRC2 activation and in vivo transformation.

(A) Cell lysates (150 μg) from control (GMR-GAL4>w1118), Tax Tg (GMR-Gal4>UAS Tax), HBZ Tg (GMR-Gal4>UAS-HBZ) or Tax/HBZ Tg (GMR-Gal4; UAS-Tax>UAS-HBZ), transgenic adult flies heads were analyzed by western blot confirming the expression of tax and hbz transgenes. (B) Representative light microscopy images (Scale bar 100μm) and scanning electron microscopy images (Scale bar 100μm and 50μm) of adult eyes are shown. (C) Relative roughness was quantified based on the number of ommatidial fusions and the extent of bristle organization (n = 30, from three independent crosses). p<0.001 (***). (D) Circulating hemocytes counted, in control larvae (HMLΔ-Gal4>w1118), larvae expressing transgenic Tax alone (HMLΔ-Gal4>UAS-Tax), HBZ alone (HMLΔ-Gal4>UAS-HBZ) or both HBZ and Tax (HMLΔ-Gal4; UAS-Tax>UAS-HBZ) (n = 30, from three independent crosses). Data represent mean ± standard error of the mean. p< 0.0001 (****). (E, F) Levels of expression of Relish and Diptericin in the control (GMR-GAL4>w1118), Tax Tg (GMR-Gal4>UAS-Tax), HBZ Tg (GMR-Gal4>UAS-HBZ), or Tax/HBZ Tg (GMR-Gal4;UAS-Tax>UAS-HBZ) transgenic adult flies heads. The values were normalized to Rp49. Reported values are the average of three independent experiments and error bars represent SD of triplicates p<0.01 (**) p<0.001 (***). (G) Cell lysates (150 μg) from control (GMR-GAL4>w1118), Tax Tg (GMR-Gal4>UAS-Tax), HBZ Tg (GMR-Gal4>UAS-HBZ) or Tax/HBZ Tg (GMR-Gal4;UAS-Tax>UAS-HBZ), transgenic adult flies heads were analyzed by western blotting and probed with indicated antibodies.

Fig 4. HBZ overexpression alleviates Tax-induced senescence in vivo.

(A) SA-β-gal expression in larval eye-imaginal discs from control (GMR-GAL4>w1118), Tax Tg (GMR-Gal4>UAS-Tax), HBZ Tg (GMR-Gal4>UAS-HBZ) or Tax/HBZ Tg (GMR-Gal4; UAS-Tax>UAS-HBZ). (B) Levels of expression of Dacapo (p21/p27) in the transgenic adult flies heads as indicated. The values were normalized to Rp49. Reported values are the average of three independent experiments and error bars represent SD of triplicates p<0.01 (**) p<0.001 (***). (C) Levels of expression of Tax and HBZ transcript levels in control larvae (HMLΔ-Gal4>w1118), larvae expressing transgenic Tax alone (HMLΔ-Gal4>UAS-Tax), HBZ alone (HMLΔ-Gal4>UAS-HBZ) or both HBZ and Tax (HMLΔ-Gal4; UAS-Tax>UAS-HBZ). (D) SA-β-gal expression in circulating hemocytes in transgenic larvae as indicated. (E) Quantification of senescent hemocytes. Results are expressed as percentage of control. p<0.001 (***). (F) SA-β-gal expression in circulating hemocytes in larvae from the control mcherry RNAi (HMLΔ-Gal4> mCherry RNAi), control Tax/mCherry RNAi (HMLΔ-Gal4;UAS-Tax>mCherry RNAi), Tax/Relish RNAi (HMLΔ-Gal4;UAS Tax>Relish RNAi), Tax/E(z) RNAi (HMLΔ-Gal4;UAS-Tax>E(z) RNAi) and Tax/Suz12 RNAi (HMLΔ-Gal4;UAS-Tax>Suz12 RNAi) as indicated. Results are expressed as percentage of control. (ns = not significant), p<0.0001 (****).

Fig 5. HBZ overexpression prevents Tax-induced PRC2 activation in human cells.

(A) HEK293T cells were transiently co-transfected with His-Tax and increasing amount Myc-HBZ. Western blot was performed with indicated antibodies. Asterisk denotes to the band corresponding to EZH2. (B) Jurkat cells were transiently co-transfected with an empty vector (control), Tax, HBZ, or Tax and HBZ expression plasmids. Western blot was performed with indicated antibodies. Asterisk denotes to the band. (C) Chromatin immunoprecipitation (ChIP)-qPCR analyses were performed on HEK293T cells co-transfected with either an empty vector or HIS-Tax alone or together with Myc-HBZ. 48 hours post-transfection, genomic DNA was fixed with 1% formaldehyde and sheared using Bioruptor. Sheared chromatin was diluted and immunoprecipitated using anti-H3k27me3 antibody. GAPDH was used as a negative control and α-satellite as a positive control. The immunoprecipitated material was quantified by qPCR. Results were normalized to inputs and expressed as %DNA input. (ns = not significant), p<0.001 (***) and p<0.01 (**). (D) Transcript levels of CDKN1A, NDRG2 and BIM. Reported values are the average of three independent experiments, and the error bars represent SD of the triplicates. (ns = not significant), p<0.01 (**).), p<0.001 (***), p<0.0001 (****). (E) MT-1 cells were transduced with non-targeting control shRNA (sh scrambled) or shRNA against HBZ (shHBZ). qRT-PCR of HBZ confirming HBZ knockdown in MT-1 cells. Reported values are the average of three independent experiments, and the error bars represent SD of the triplicates. p<0.01 (**). (F) Western blot analysis was performed with indicated antibodies in MT-1 cells transduced with shHBZ or sh Scrambled. Asterisk denotes to the band corresponding to EZH2. (G) HEK293T cells transiently transfected with an empty vector (control), Tax, HBZ, or Tax and HBZ expression plasmids. EZH2 immunoprecipitates (IP: EZH2) were blotted against EZH2 and Tax (Left panel).–AB represents a control with no antibody. Corresponding control cell lysates are shown as indicated (Right panel).

Fig 6. HBZ interacts with PRC2 components EZH2 and SUZ12 and is associated with H3K27me3 accumulation in human cells.

(A) HEK293T cells transiently transfected with Myc-HBZ. Western blot was performed with indicated antibodies. (B) HEK293T cells transiently transfected with Myc-HBZ. Myc immunoprecipitates (IP: Myc) were blotted against EZH2, SUZ12 and Tax (Left panel).–AB represents a control with no antibody. Corresponding control cell lysates are shown as indicated (Right panel). (C) HBZ-EZH2 and HBZ-SUZ-12 interactions detected by Duolink Proximity Ligation [73] assay in HeLa cells transfected with HBZ (Scale bar 10 μm), or MT-1 expressing endogenous HBZ. Nuclei were stained with 4,6 diamidino-2-phenylindole (DAPI) (blue). (Scale bar 5 μm). (D) Confocal microscopy (Z-Stacks) of HeLa cells transiently expressing Myc-HBZ, and showing partial co-localization of HBZ-EZH2 (Upper panel) and HBZ-SUZ12 (Lower panel). Nuclei were stained with 4,6 diamidino-2-phenylindole (DAPI) (blue) (Scale bars 10μm and 5μm respectively).

Fig 7. Proposed model.