PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

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Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

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Figure 1: STAT1-CC mice exhibit increased interferon responsiveness and protection against viral infection.
Figure 2: PARP9-DTX3L mediates the control of viral replication by STAT1-CC.
Figure 3: PARP9-DTX3L regulates the control of viral replication by STAT1.
Figure 4: PARP9-DTX3L interacts with STAT1 and enhances translocation of STAT1 to the nucleus and its binding to ISGs and activation of ISG promoters.
Figure 5: PARP9-DTX3L regulates STAT1's control of ISG expression.
Figure 6: Specific protein domains mediate formation of the PARP9-DTX3L complex and binding to STAT1 for antiviral and ISG-expression functions.
Figure 7: DTX3L mediates immunoproteasomal degradation of viral 3C protease.
Figure 8: DTX3L co-localizes with STAT1 and EMCV 3C in situ and directly ubiquitinates viral 3C proteases in vitro.

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We thank A.C. Palmenberg (University of Wisconsin) for mouse monoclonal antibody to EMCV 3D; G. Stark (Cleveland Clinic) for the U3A cell line; A. Winoto (University of California, Berkeley) for plasmid pCI-His-Ub; E. Yeh (University of Texas, Houston) for plasmid pHA-Ubiquitin; J. Gern (University of Wisconsin) for HRV A16; and G. Amarasinghe (Washington University) for respiratory syncytial virus NS1. Supported by the US National Institutes of Health (National Institute of Allergy and Infectious Diseases Asthma and Allergic Diseases Cooperative Research Center U19-AI070489 to M.J.H.; U54-AI05160 to M.J.H.; RO1-AI111605 to M.J.H.; and R15-AI099134 to T.G.L.) and the Martin Schaeffer Fund (M.J.H.).

Author information

Y.Z. organized and performed cell and mouse experiments; D.M. performed cell and mouse experiments; W.T.R. performed immunoblot analyses; X.J. performed confocal microscopy; A.C.P. analyzed gene-expression microarray data; D.A.P. performed nuclear imaging and domain mapping; E.A. prepared viruses; Z.W. performed protein purification; R.M.T. performed knockout mouse breeding; J.J.A. performed mouse experiments; G.H. performed transcription-activity experiments; R.M. performed cell injections and implantations; J.Y. analyzed ubiquitination array data; N.E.Y. and S.P. performed Biosafety Level 3+ experiments; T.G.L. generated antibodies to 3C; N.S.O. performed biolayer interferometry assays; T.J.B. designed mutant PARP-DTX3L; M.J.H. designed experimental plans, analyzed data and wrote the manuscript; and all authors discussed the results and commented on the manuscript.

Correspondence to Michael J Holtzman.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 CAG-STAT1 and CAG-STAT1-CC mice exhibit widespread transgene expression and activation without disease.

(a) Scheme for the expression cassette for CAG-STAT1 and CAG-STAT1-CC transgenic mice using CAG promoter and human STAT1 and STAT1-CC genes. (b) Immunoblots of tissue homogenates from wild-type (WT) mice and CAG-STAT1 and CAG-STAT1-CC transgenic mice using anti-FLAG or anti-β-actin Ab. (c) Hematoxylin-eosin staining of tissue sections from CAG-STAT1-CC transgenic mice at 2 years of age. Bar = 500 µm. (d) Immunostaining for STAT1 and STAT1-CC in myocardial tissue from WT mice and CAG-STAT1 and CAG-STAT1-CC transgenic mice at indicated times after treatment with IFN-γ (20,000 U intraperitoneally). Sections were stained using anti-FLAG Ab and an alkaline phosphatase system and then counterstained with hematoxylin. Control staining with non-immune IgG gave no signal above background (data not shown). Scale bar, 40 µm. Analysis of pancreas tissue gave similar results (data not shown). (e) For treatment conditions in (c), immunostaining of myocardial tissue with anti-phospho-STAT1 or anti-FLAG Ab and then secondary Ab conjugated to cyanine or FITC, respectively. Scale bar, 40 µm. Data are representative of two independent experiments (b,c,d,e).

Supplementary Figure 2 CAG-STAT1-CC mice show protection against infection with IAV and VEEV.

(a) Survival rates of WT and CAG-STAT1-CC transgenic mice inoculated with IAV-A/WS/33 at 25 PFU (n=15 mice per group). * P < 0.01. (b) Corresponding lung levels of IAV polymerase 3 (P3) RNA (n = 5-8 mice per group). (c) Corresponding hematoxyline-eosin staining of lung tissue. Scale bar, 40 µm. (d) Survival rates of WT and CAG-STAT1-CC transgenic mice inoculated with IAV-Vietnam/1203/04 at 1 x 105 TCID50 (n = 16 mice per group). * P < 0.01. (e) Corresponding IAV levels based on plaque-forming assay of samples from lung and brain at 6 days after infection. Values were normalized per gm of tissue. * P < 0.01. (f) Corresponding immunostaining for IAV with DAPI counterstaining of lung and brain tissue at 6 days after infection. Bar = 40 µm. (g) Survival rates of CAG-STAT1 and CAG-STAT1-CC transgenic mice infected with VEEV-ZPC738 (16 PFU given subcutaneously) (n = 11 mice per CAG-STAT1 and 16 mice per CAG-STAT1-CC group). * P < 0.01. (h) Corresponding viral levels based on plaque-forming assay of samples from lung 5 days after infection. * P < 0.01. Significance was determined with unpaired t-test (a,d,e,g,h).

Supplementary Figure 3 STAT1-CC expression enhances ISG expression, translocation of STAT and viral control in U3A cells.

(a) Immunostaining for STAT2 from high-throughput nuclear translocation assay in U3A-STAT1 and U3A-STAT1-CC cells at indicated times after treatment with IFN-β (1000 U/ml). Bar = 40 µm. (b) For the assay in (a), quantification of nuclear translocation of STAT1 and STAT2 after treatment with IFN-β (1000 U/ml) or IFN-γ (100 U/ml) in U3A-STAT1 and U3A-STAT1-CC cells for the indicated times. Values represent the difference in fluorescence intensity between nucleus and cytoplasm (mean ± SEM for 3 wells, 500 cells/well). Initial negative values indicate that STAT1 is predominantly cytoplasmic. * P < 0.01. (c) Target mRNA levels in indicated U3A cell lines with and without IFN-β (1000 U/ml) for 1 day. * P < 0.01. (d) Viral titers in indicated U3A cell lines at 1 day after infection with EMCV or 2 days after infection with IAV-A/WS/33 or SINV. * P < 0.01. (e) Viral titers in indicated U3A cell lines at 1 day after infection with EMCV (MOI 1), 2 days after infection with IAV-A/WS/33 (MOI 1) or SINV (MOI 10), or 1 day after infection with IAV-Vietnam/1203/04 (MOI 1) with or without pretreatment with the indicated concentrations of IFN-γ or IFN-β for 6 hours. * P < 0.01. Significance was determined with unpaired t-test (b,c,d,e). Data are representative of two independent experiments (a-e).

Supplementary Figure 4 STAT1-CC enhances PARP9-DTX3L expression in tissue and cells.

(a) Levels of PARP9 and DTX3L mRNA at baseline and after IFN-γ (1 U/ml) or IFN-β (10 U/ml) for 1 day in indicated U3A cell lines. (b) Levels of PARP9 and DTX3L mRNA at baseline and after IFN-γ (100 U/ml) or IFN-β (1000 U/ml) treatment for 1 day in indicated U3A cell lines. (c) For conditions in (a), corresponding levels of PARP9, DTX3L, STAT1, and β-actin protein in U3A cell lines and 2fTGH cells. (d) Parp9 and Dtx3l mRNA levels in pancreas tissue from WT mice and CAG-STAT1 and CAG-STAT1-CC transgenic mice treated with or without IFN-β (200,000 U i.p.) for 1 day. * P < 0.01. Analysis of lung tissue from these mice showed similar results (data not shown). Significance was determined using unpaired t-test (a,b,d). Data are representative of three independent experiments (a-e).

Supplementary Figure 5 Knockdown of PARP9 and DTX3L regulates expression and activation of PARP9-DTX3L but not of STAT1.

(a) Levels of PARP9 and DTX3L mRNA in indicated U3A cell lines stably transduced with lentivirus encoding PARP9 or control shRNA. (b) Corresponding analysis for lentivirus encoding DTX3L shRNA. For (a,b), * P < 0.01 for a significant decrease from corresponding control shRNA level. (c) Levels of PARP9 and DTX3L in indicated U3A cell lines stably transduced with lentivirus encoding PARP9 or control shRNA. (d) Levels of PARP9 and DTX3L for conditions in (c) but using DTX3L shRNA. (e) Immunoblot for the indicated proteins in U3A-STAT1 and U3A-STAT1-CC cells that were subjected to DTX3L gene knockdown and then treated with IFN-γ (100 U/ml) or IFN-β (1000 U/ml) for the indicated times with subsequent treatment with staurosporine to block JAK activity. (f) Immunoblot for the indicated proteins in indicated U3A cell lines that were subjected to PARP9 gene knockdown and reconstitution and infected with EMCV (MOI 1 for 6 hours). (g) Corresponding levels of EMCV-3D RNA for conditions in (f). (h) Levels of PARP9 mRNA in indicated U3A cell lines stably transduced with lentivirus encoding PARP9 or control shRNA as in (a) but including IFN treatment for 1 day. Significance was determined using unpaired t-test (a,b). Data are representative of three independent experiments (a-h).

Supplementary Figure 6 PARP9-DTX3L interacts with STAT1 without Tyr701 phosphorylation and does not enhance STAT1 phosphorylation, p300 recruitment, or histone H4 ubiquitination.

(a) Co-IP of STAT1 with PARP9 using U3A cells that express WT or mutant (Tyr701 to Phe701) STAT1 and PARP9 and were treated with IFN as indicated. (b) Immunoblot of STAT1 phosphorylation in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L cells that were treated with IFN-β (1000 U/ml) or IFN-γ (100 U/ml) plus staurosporine at indicated times. (c) Co-IP of STAT1 with p300 in U3A-STAT1 and U3A-STAT1-PARP9-DTX3L versus STAT1-CC in U3A-STAT1-CC cells with or without IFN-β (1000 U/ml) or IFN-γ (100 U/ml) for 0.5 hours. (d) Immunoblot with anti-histone H4 Ab and anti-phospho-STAT1 Ab in indicated U3A cell lines with and without IFN-β (1000 U/ml) treatment. Predicted size of undetectable histone H4-Ub is also indicated. Data are representative of three independent experiments (a-d).

Supplementary Figure 7 Structure-based mutations inactivate PARP9-DTX3L function.

(a) Diagram of lentiviral vector constructs for WT and mutant PARP9 and DTX3L expression with sequence for double-glycine to glutamic acid mutations in the PARP9 macrodomains and double-cysteine to serine mutations in the DTX3L RING domain. (b) Levels of poly(ADP-ribose) (PAR) binding to PARP9 or PARP9 macrodomain mutant (PARP9M) in indicated U3A-STAT1 cell lines determined with a PARP9-PAR binding assay captured by anti-PAR Ab. (c) PAR binding assay for the indicated U3A-STAT1 cell lysates incubated with anti-FLAG M2 affinity gel for 6 h at 4 °C and then PAR-PARP1 (PAR-PARP) for 16 hours at 4 °C. The PAR-binding complex was eluted using FLAG peptide competition and immunoblotted with anti-PAR Ab. (d) Levels of ubiquitination of DTX3L or DTX3L ring domain mutant (DTX3LM) in HEK293T cells that were transiently transfected with the indicated vectors and then treated with MG-132 (20 µM) for 14 hours before immunoblotting with anti-c-Myc Ab. (e) Co-IP of DTX3L with STAT1 from indicated U3A-STAT1 cell lines using anti-c-Myc Ab and then immunoblot for the indicated proteins. (f) Co-IP of PARP9 with DTX3L from indicated cell lines using anti-FLAG or anti-cMyc Ab. (g) Immunoblot for STAT1 phosphorylation in U3A-STAT1 cell lines that were treated with or without IFN as indicated. (h) Diagram of the chromatin accessibility by real-time PCR (CHART) assay using Avr II endonuclease digestion of the human IFIT1 promoter region. Accessibility was assessed using IFIT1 promoter-specific PCR primers directed to nucleotides -135 to -50 relative to the transcription start site (TSS). (i) CHART assays for U3A-STAT1, U3A-STAT1-PARP9-DTX3L, and U3A-STAT1-PARP9M-DTX3LM cells under the indicated IFN-β treatment conditions. Values are expressed as the percentage of the accessibility observed in unstimulated digested DNA sample, i.e., % Accessibility = (1- (+)IFN/(-)IFN) x 100). (j) Co-IP of histone HA-H2BJ, FLAG-PARP9, c-Myc–DTX3L, and STAT1 in indicated HEK293T cell transfections. (k) Immunoblot levels of H2B-Ub and H2A-Ub detected and quantified with an infrared imaging system in indicated U3A-STAT1 cell lines (V, vectors alone; WT, PARP9-DTX3L; M, PARP9M-DTX3LM) with and without EMCV infection (MOI 1, 6 h). Significance was determined using unpaired t-test (b). Data are representative of three independent experiments (a-k).

Supplementary Figure 8 Formation of the PARP9-DTX3L-STAT1 complex depends on specific domain-mediated interactions.

Scheme for PARP9-DTX3L-STAT1 domain interactions based on co-ip assays of HEK293T cells transfected with individual domain components. Thin dashed lines indicate weak interactions, and thick lines indicate strong interactions in this assay system.

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Zhang, Y., Mao, D., Roswit, W. et al. PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection. Nat Immunol 16, 1215–1227 (2015) doi:10.1038/ni.3279

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