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FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins

Nature Microbiology volume 9, pages 988–1006 (2024)Cite this article

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Abstract

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)—a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT–ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

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Fig. 1: VV A51R interacts with hSpt16 and FACT depletion promotes VV, VSV, YFV and IAV replication but inhibits HSV-1 replication.
Fig. 2: Poxvirus A51R proteins specifically and directly bind the middle domain of a SUMOylated form of hSpt16 using a conserved motif.
Fig. 3: VV A51R blocks hSpt16SUMO nuclear accumulation triggered by VV early gene expression.
Fig. 4: VV A51R outcompetes SSRP1 to inhibit hSpt16SUMO–SSRP1 interaction.
Fig. 5: hSpt16SUMO–SSRP1 (FACT) complexes bind transcriptionally active chromatin during infection to activate ETS-1 expression and VV restriction, which is antagonized by VV A51R.
Fig. 6: hSpt16SUMO is required for ETS-1 expression and VV restriction.

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Data availability

The authors declare that the main data supporting the findings of this study are available within the article, its source data and/or its Supplementary Information. RNA-seq datasets are available under Gene Expression Omnibus accession GSE185829. The human genome assembly (hg38/GRCh38) is available under National Center for Biotechnology Information accession GCF_000001405. Source data are provided with this paper.

Code availability

Custom code used for analysis of the RNA-seq data is available in the Supplementary Information.

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Acknowledgements

This work was supported by: NIH grants 1R21AI144203-01 and 1R35GM137978-01 and Welch Foundation grant I-2062-20210327 (to D.B.G.); NIH grant 1R35GM142689-01 and Cancer Prevention and Research Institute of Texas grant RR 170047 (to D.C.H.); funding from the INC TV Initiative (to F.G.F.); and NIH Training Grant T32 AI007520 (to C.P., E.A.R. and A.E.). We thank the University of Texas Southwestern Medical Center Electron Microscopy Core Facility, funded by NIH grant 1S10OD021685-01A1. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We thank D. Evans (University of Alberta) for the mouse anti-VV F4L and I3L antibodies. We thank C. Mello (University of Massachusetts Medical School) for the ΔA51R, ΔA51RFA51R and VSV-GFP virus strains, as well as the Flag-VV A51R, CPXV FA51R, ECTV FA51R and YLDV FA51R pCDNA3 plasmids. We also thank J. Schoggins (University of Texas Southwestern Medical Center) for the YFV-17D-Venus and HSV-1-GFP virus strains and the MDCK cells. We thank H. W. Virgin (University of Washington School of Medicine) for the IAV strain A/PR/8/34. We thank M. Binder (German Cancer Research Center) for the non-targeting, ΔIRF3 and ΔIFNAR A549 cells. Finally, we thank B. Arif (Natural Resources Canada) for the provision of LD652 cells.

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Authors and Affiliations

  1. Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Emily A. Rex, Dahee Seo, Aaron Embry, David Heisler, Neal M. Alto, Robert Orchard & Don B. Gammon

  2. Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Sruthi Chappidi, Chelsea Pinkham, Moiz Munir, Robert Orchard & Dustin C. Hancks

  3. Laboratório de Virologia Básica e Aplicada, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

    Sabrynna Brito Oliveira & Flávio Guimarães da Fonseca

  4. Department of Biochemistry, University of Colorado Boulder, Boulder, CO, USA

    Yang Liu & Karolin Luger

  5. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Karolin Luger

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Contributions

D.B.G. and E.A.R. conceived of the experiment, devised the methodology, curated the data, wrote the original draft of the manuscript and visualized the data. D.B.G., E.A.R., D.S., S.C., C.P., S.B.O., A.E., D.H., Y.L. and M.M. performed the investigation. D.B.G., E.A.R., K.L., N.M.A., F.G.F., R.O. and D.C.H. reviewed and edited the manuscript. D.B.G., K.L., N.M.A., F.G.F., R.O. and D.C.H. supervised the experiments. D.B.G., F.G.F. and D.C.H. acquired the funding.

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Correspondence to Don B. Gammon.

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Extended data

Extended Data Fig. 1 The ΔA51R strain exhibits normal viral gene expression and genome replication but displays defective virion assembly in the presence of hSpt16.

a, IB of early (F4L, I3L) and late (F9L, A27L) in A549 WCE at indicated times post-infection (MOI = 10). IB is representative of at least two independent experiments. b, VV genome quantification by qPCR in A549 cultures at indicated times post-infection (MOI = 10). Data are means ± SD for n = 3 experiments. Statistical significance was determined by unpaired two-tailed Student’s t-test comparing genome copy number between ΔA51RFA51R and ΔA51R treatments within each time point. Only statistical comparisons with P < 0.05 are shown. c,d Analysis of total mature virus (MV) and immature virus (IV) frequencies (expressed as a percentage of total particles counted) using electron microscopy in A549 cells 24 hpi (MOI = 10) under scrambled (scram.) or hSpt16 RNAi conditions. c, Representative images of MV particles and various IV forms (crescents, spheres, spheres with nucleoid DNA) typically found in ΔA51RFA51R (top image) and ΔA51R-infected (bottom image) cells, respectively under control RNAi conditions. Selected examples of different types of MV and IV particles are indicated. Scale bar, 800 nm. d, Percentage of MV particles counted among total (MV + IV particles) particles in cells counted from each infection treatment. Data are means ± SD for n = 3 biologically independent experiments.

Source data

Extended Data Fig. 2 Conservation of Spt16 SUMOylation among eukaryotes.

a-c, IB of endogenous Spt16 proteins from indicated human (a) and mammalian animal cell lines (b) and from invertebrate cell lines and animal tissues (C. elegans) (c) showing SUMOylated (upper bands) and non-SUMOylated (lower bands) Spt16 forms. Non-human cell lines derive from the following: BSC-40 (Chlorocebus sabaeus); BHK-21 (Mesocricetus auratus); L929 (Mus musculus); SIRC (Oryctolagus cuniculus); R06E (Rousettus aegyptiacus); and LD652 (Lymantria dispar). Images in a-c are representative of at least two independent experiments.

Source data

Extended Data Fig. 3 VV A51R associates with microtubules (MTs) and tethers hSpt16SUMO to MTs.

a, Alignment of VV A51R with Proline Rich (PR) Region and Region 1 of human Tau40 MT-binding domains (MTBD)25. “KXGS” motif conserved in all four MTBDs in Tau is boxed. Residues in red indicate those converted to alanine in the triple A51R mutant. b, immunofluorescence staining of U2OS cells transfected with wild-type or triple mutant FA51R expression constructs after 24 h. Where indicated, 40 μM nocodazole was added to depolymerize MTs 6 h post-transfection MTs. Scale bar, 5 μm. c, Overview of in vitro MT co-sedimentation assay. d-e, Stain-free protein gels showing in vitro MT co-sedimentation assays with purified wild-type and mutant His-A51R. S, supernatant; P, pellet. f, Co-IP of transfected wild-type and mutant FA51R constructs with hSpt16 and tubulin in 293 T WCE. g, Overview of in vitro Flag-hSpt16 Co-IP with tubulin in the absence/presence of His-A51R. h, in vitro Co-IP of purified Flag-hSpt16, His-A51R and tubulin. Images in b, d-f, and h are representative of at least two independent experiments. Images in c and g were created with biorender.com.

Source data

Extended Data Fig. 4 VV A51R does not affect total hSpt16 protein levels.

a, Representative IB of hSpt16 in A549 WCE at indicated times post-infection (MOI = 10). b-c, Densitometric quantification of IB experiments represented in a for total hSpt16SUMO (b) or non-SUMOylated hSpt16 (c) protein levels plotted as fold change relative to levels in mock-infected WCE at each respective time point. Data in b-c are means ± SD for n = 3 biologically independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t-test comparing hSpt16/hSpt16SUMO levels in infected versus mock-infected WCE at each time point. However, no significant differences were detected at any time point in either cell type (not shown). d, Representative IB of hSpt16 in U2OS WCE at indicated times post-infection (MOI = 10). e-f, Densitometric quantification of IB experiments represented in d for total hSpt16SUMO (e) or non-SUMOylated hSpt16 (f) protein levels plotted as fold change relative to levels in mock-infected WCE at each respective time point. Data in e-f are means ± SD for n = 3 biologically independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t-test comparing hSpt16/hSpt16SUMO levels in infected versus mock-infected WCE at each time point. However, no significant differences were detected at any time point in either cell type (not shown). g, Quantification of fractionation experiments of U2OS cells mock- or ΔA51R-infected for 18 h performed as in Fig. 3h,i. Where indicated, proteasome inhibitor [bortezomib, (5 nM)] was added to media 2 hpi. Data are means ± SD; n = 3 biologically independent experiments.

Source data

Extended Data Fig. 5 hSpt16 SUMOylation does not affect histone or nucleosome interactions in vitro.

a, in vitro nickel bead pulldown of His-tagged H3/H4 and H2A/H2B complexes with purified Flag-hSpt16. b, in vitro FACT binding assay with reconstituted (H3-H4)2 tetrasomes or (H2A-H2B)-(H3-H4)2 hexasomes50. Where indicated, purified FACT complexes were treated with ULP-1 to remove hSpt16 SUMOylation prior to being added to binding assays. IBs of FACT treatments are shown below indicating hSpt16 and SSRP1 levels. Images in a,b are representative of two independent experiments.

Source data

Extended Data Fig. 6 ETS-1 induction by VV infection occurs by 4 hpi and requires early VV gene expression but not viral DNA replication.

a, Representative IB of ETS-1 levels in WCE from A549 cells infected with indicated strains for indicated times (MOI = 10). b, Densitometric quantification of n = 3 biologically independent IB experiments represented in a. Fold change in ETS-1 protein levels are plotted relative to levels in mock-infected WCE harvested 1 hpi. Data are means ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test. Only statistical comparisons where ETS-1 levels in infected WCE were significantly different (P < 0.05) from levels in mock-infected WCE are shown. c, IB of ETS-1 in WCE from A549 cells under indicated infection conditions 4 hpi. Where indicated, inoculum was heat-inactivated prior to infection to prevent VV gene expression. d, IB of ETS-1 in WCE from A549 cells under indicated infection conditions. Where indicated, AraC was added to media 1 hpi. VV A27L is a late VV protein, and its expression requires VV DNA replication and thus is inhibited by AraC treatment. Images in c-d are representative of two independent experiments.

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Extended Data Fig. 7 The Type I IFN and FACT-ETS-1-depedent antiviral responses are independent pathways.

a-d, Representative IB (a,c) and quantification (b,d) of ETS-1 expression using IB of WCE from control or IRF3 knockout A549 cells infected with the indicated strains for 4 h (a,b) or 8 h (c,d). e-h, Representative IB (e,g) and quantification (f,h) of ETS-1 expression using IB of WCE from control or IFNAR knockout A549 cells infected with the indicated strains for 4 h (e,f) or 8 h (g,h). i-l, Representative IB (i,k) and quantification (j,l) of ETS-1 expression using IB of WCE from control or STAT1 knockout A549 cells infected with the indicated strains for 4 h (i,j) or 8 h (k,l). m, VV titers 48 hpi (MOI = 0.01) in IRF3 knockout A549 cells after treatment with indicated RNAi. Scram., scrambled. n-o, Representative IB (n) and quantification (o) of ETS-1 expression using IB of WCE from A549 cells treated with indicated doses of recombinant IFN-β for 24 h. IB of two ISGs (IFIT3 and STAT1) is also shown to illustrate ISG expression increases after IFN-β treatment. p-s, Representative IB (p,r) and quantification (q,s) of IFIT3 (ISG) expression using IB of WCE from A549 cells treated with scrambled or ETS-1 RNAi and then mock- or IFN-β (500 U/mL)-treated for 8 h (p-q) or 24 h (r-s). Data in b,d,f,h,j,l,m,o,q,s are means ± SD for n = 3 biologically independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t-test. For brevity, only the most relevant statistical comparisons are shown. ns, not significant.

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Extended Data Fig. 8 A51R-hSpt16SUMO interaction suppresses ETS-1 expression and promotes VV virulence in BALB/c mice.

a, Representative IB of A549 WCE from cells infected with indicated VV (MOI = 10) for 4 h. b, Densitometric quantification of ETS-1 protein levels from a. Data are means ± SD; n = 3 biologically independent experiments. c, VV titers 48 hpi (MOI = 0.01) in A549 cell cultures transfected with indicated RNAi treatments. d, Percent change in body weight of BALB/c male mice still alive at each time point (n = 20 animals over two independent experiments) after intranasal VV infection (102 PFU/animal). Data are means ± SE. e, Percent survival of BALB/c mice from d over time. f, Tissue viral loads in BALB/c mice (n = 5 animals) challenged as in d 8 dpi. Horizontal lines indicate means. g, qRT-PCR analysis of relative Ets1 mRNA levels in lung tissues from BALB/c animals (n = 5) infected with 102 PFU at 8 dpi. Ets1 mRNA levels are plotted as relative to levels in mock (PBS)-treated animals. Horizontal lines indicate means. ns, not significant. Image in a is representative of at three independent experiments. Statistical significance was determined by unpaired two-tailed Student’s t-test in b and c, by Log-Rank (Mantel-Cox) tests in e, and by unpaired two-tailed Mann-Whitney U tests in f and g. Only the most relevant statistical comparisons with P < 0.05 are shown for brevity.

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Extended Data Fig. 9 Additional analyses of ΔA51RFA51R158-162Ala mutant infection in C57BL/6 mice.

a, Percent change in body weight of C57BL/6 male mice (n = 15 animals over two independent experiments) still alive at each time point after intranasal VV infection (105 PFU/animal). Data are means ± SE. b, Percent survival of C57BL/6 mice from a over time. c, Tissue viral loads from C57BL/6 mice (n = 5 animals) after intranasal challenge with 102 PFU at 8 dpi. Horizontal lines indicate means. Statistical significance was determined by Log-Rank (Mantel-Cox) tests in b and by unpaired two-tailed Mann-Whitney U tests and in c.

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Extended Data Fig. 10 Model for FACT-ETS-1 Antiviral Response (FEAR) pathway.

VV early gene expression triggers hSpt16SUMO nuclear accumulation and formation of FACT complexes containing hSpt16SUMO that license FACT to associate with H2BK120ub sites in transcriptionally active chromatin and activate ETS-1 expression. Elevation of ETS-1 expression levels to a “critical threshold” results in ETS-1-dependent antiviral gene expression and VV restriction. VV A51R is expressed early during infection5 and inhibits FEAR pathway by tethering hSpt16SUMO to MTs and blocking its nuclear import. A51R may also prevent FACT complex formation in the cytosol by outcompeting SSRP1 for hSpt16SUMO binding, which would also serve to prevent nuclear accumulation of hSpt16SUMO-containing FACT complexes (not shown). Note: our fraction data in Fig. 3b-c, indicate that there are constant pools of hSpt16SUMO in the nucleus in uninfected cells (not shown) and thus we hypothesize that VV infection triggers hSpt16SUMO nuclear translocation from cytosolic pools to reach a critical level of hSpt16SUMO-containing FACT complexes in the nucleus required to induce ETS-1 levels to a critical threshold required for antiviral gene expression. Image created with biorender.com.

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Rex, E.A., Seo, D., Chappidi, S. et al. FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins. Nat Microbiol 9, 988–1006 (2024). https://doi.org/10.1038/s41564-024-01646-5

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