Abstract

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses1. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses2. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones3. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles4,5, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses6,7. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signalling.

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References

  1. 1.

    & The evolutionary conundrum of pathogen mimicry. Nature Rev. Microbiol. 7, 787–797 (2009)

  2. 2.

    , & Chromatin contributions to the regulation of innate immunity. Annu. Rev. Immunol. 32, 489–511 (2014)

  3. 3.

    & A histone-like protein from adenovirus chromatin. Nature 267, 552–554 (1977)

  4. 4.

    , & Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection. EMBO J. 5, 1633–1644 (1986)

  5. 5.

    , & The structure of nucleoprotein cores released from adenovirions. Nucleic Acids Res. 11, 441–460 (1983)

  6. 6.

    et al. HMGB1 in health and disease. Mol. Aspects Med. 40, 1–116 (2014)

  7. 7.

    & High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nature Rev. Immunol. 5, 331–342 (2005)

  8. 8.

    & Epigenetic reprogramming of host genes in viral and microbial pathogenesis. Trends Microbiol. 18, 439–447 (2010)

  9. 9.

    et al. Suppression of the antiviral response by an influenza histone mimic. Nature 483, 428–433 (2012)

  10. 10.

    , & Viral manipulation of the host epigenome for oncogenic transformation. Nature Rev. Genet. 10, 290–294 (2009)

  11. 11.

    et al. Snapshots: chromatin control of viral infection. Virology 435, 141–156 (2013)

  12. 12.

    et al. Adenovirus small E1A employs the lysine acetylases p300/CBP and tumor suppressor Rb to repress select host genes and promote productive virus infection. Cell Host Microbe 16, 663–676 (2014)

  13. 13.

    & The structural organization of sperm chromatin. J. Biol. Chem. 278, 29471–29477 (2003)

  14. 14.

    & Examining histone posttranslational modification patterns by high-resolution mass spectrometry. Methods Enzymol. 512, 3–28 (2012)

  15. 15.

    , , & Extraction, purification and analysis of histones. Nature Protocols 2, 1445–1457 (2007)

  16. 16.

    & Salt fractionation of nucleosomes for genome-wide profiling. Methods Mol. Biol. 833, 421–432 (2012)

  17. 17.

    et al. Chromosomes. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere. Science 348, 699–703 (2015)

  18. 18.

    , & A quantitative investigation of linker histone interactions with nucleosomes and chromatin. Sci. Rep. 6, 19122 (2016)

  19. 19.

    Chromatin modifications and their function. Cell 128, 693–705 (2007)

  20. 20.

    & Acetylation of histone-like proteins of adenovirus type 5. J. Virol. 35, 637–643 (1980)

  21. 21.

    et al. Molecular evolution of human adenoviruses. Sci. Rep. 3, 1812 (2013)

  22. 22.

    , , , & Binding modes of the precursor of adenovirus major core protein VII to DNA and template activating factor I: implication for the mechanism of remodeling of the adenovirus chromatin. Biochemistry 45, 303–313 (2006)

  23. 23.

    , , , & Involvement of template-activating factor I/SET in transcription of adenovirus early genes as a positive-acting factor. J. Virol. 80, 794–801 (2006)

  24. 24.

    , & Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418, 191–195 (2002)

  25. 25.

    et al. Biochemical observation of the rapid mobility of nuclear HMGB1. Biochim. Biophys. Acta 1729, 57–63 (2005)

  26. 26.

    et al. Novel role of PKR in inflammasome activation and HMGB1 release. Nature 488, 670–674 (2012)

  27. 27.

    , & Targeting airway smooth muscle in airways diseases: an old concept with new twists. Expert Rev. Respir. Med. 5, 767–777 (2011)

  28. 28.

    et al. Contributions of high mobility group box protein in experimental and clinical acute lung injury. Am. J. Respir. Crit. Care Med. 170, 1310–1316 (2004)

  29. 29.

    et al. Adenovirus protein VII condenses DNA, represses transcription, and associates with transcriptional activator E1A. J. Virol. 78, 6459–6468 (2004)

  30. 30.

    & Adenovirus core protein VII protects the viral genome from a DNA damage response at early times after infection. J. Virol. 85, 4135–4142 (2011)

  31. 31.

    , & Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells. Proc. Natl Acad. Sci. USA 108, 12799–12804 (2011)

  32. 32.

    , , , & Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene. Nature Genet. 13, 54–62 (1996)

  33. 33.

    , , & The adenovirus E1b55K/E4orf6 complex induces degradation of the Bloom helicase during infection. J. Virol. 85, 1887–1892 (2011)

  34. 34.

    et al. Core labeling of adenovirus with EGFP. Virology 351, 291–302 (2006)

  35. 35.

    , , & Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein. Virology 128, 480–484 (1983)

  36. 36.

    , , , & The intrinsic antiviral defense to incoming HSV-1 genomes includes specific DNA repair proteins and is counteracted by the viral protein ICP0. PLoS Pathog. 7, e1002084 (2011)

  37. 37.

    , , & A simple method of mouse lung intubation. J. Vis. Exp . 73, e50318 (2013)

  38. 38.

    , , & Transcriptional profiling of lipopolysaccharide-induced acute lung injury. Infect. Immun. 72, 7247–7256 (2004)

  39. 39.

    et al. Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation. J. Immunol. 164, 2151–2159 (2000)

  40. 40.

    Micrococcal nuclease analysis of chromatin structure. Curr. Protoc. Mol. Biol. Chapter 21, Unit 21.1 (2005)

  41. 41.

    & A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984)

  42. 42.

    & MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnol. 26, 1367–1372 (2008)

  43. 43.

    et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011)

  44. 44.

    et al. Expression and purification of recombinant human histones. Methods 33, 3–11 (2004)

  45. 45.

    , , , & Characterization of nucleosome core particles containing histone proteins made in bacteria. J. Mol. Biol. 272, 301–311 (1997)

  46. 46.

    et al. The octamer is the major form of CENP-A nucleosomes at human centromeres. Nature Struct. Mol. Biol . 20, 687–695 (2013)

  47. 47.

    , , & The structure of (CENP-A–H4)2 reveals physical features that mark centromeres. Nature 467, 347–351 (2010)

  48. 48.

    , , & Characterization of histone post-translational modifications during virus infection using mass spectrometry-based proteomics. Methods 90, 8–20 (2015)

  49. 49.

    & Steroids completely reverse albuterol-induced β2-adrenergic receptor tolerance in human small airways. J. Allergy Clin. Immunol. 122, 734–740 (2008)

  50. 50.

    , , & Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002)

  51. 51.

    , , & Universal approach to FRAP analysis of arbitrary bleaching patterns. Sci. Rep. 5, 11655 (2015)

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Acknowledgements

We thank members of the Weitzman laboratory for insightful discussions and input, especially R. Dilley and B. Simpson for generating reagents. We also thank R. Panetierri and C. Koziol-White for providing precision-cut lung slices. We are grateful to D. Curiel for sharing recombinant protein-VII–GFP vectors and L. Gerace for anti-protein-VII antibodies. We thank the Penn Vector Core for assistance in purifying recombinant vectors, the Penn CDB Microscopy Core for imaging and FRAP assistance, and the CHOP Pathology core for immunostaining of mouse lungs. We thank members of the Black, Garcia and Worthen laboratories for technical help. We thank C. Bassing, I. Brodsky, J. Henao-Mejia, R. Kohli, C. Lopez, A. Resnick, S. Shin, K. Spindler and J. Weitzman for advice and critical reading of the manuscript. D.C.A. was supported in part by T32 CA115299 and F32 GM112414. N.J.P. was supported in part by T32 NS007180. N.S. was supported in part by funding from the American Cancer Society. Research was supported by grants from the National Institutes of Health (CA097093 to M.D.W., AI102577 and CA122677 to P.H., AI118891 and GM110174 to B.A.G., and GM082989 to B.E.B.), the Institute for Immunology of the University of Pennsylvania, and funds from the Children’s Hospital of Philadelphia (M.D.W.).

Author information

Author notes

    • Nikolina Sekulic

    Present address: Biotechnology Centre of Oslo and Department of Chemistry, University of Oslo, Oslo 0316, Norway.

Affiliations

  1. Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA

    • Daphne C. Avgousti
    • , Katarzyna Kulej
    • , Emigdio D. Reyes
    •  & Matthew D. Weitzman
  2. Division of Cancer Pathobiology, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA

    • Daphne C. Avgousti
    • , Christin Herrmann
    • , Katarzyna Kulej
    • , Neha J. Pancholi
    • , Joana Petrescu
    • , Emigdio D. Reyes
    •  & Matthew D. Weitzman
  3. Cell and Molecular Biology Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA

    • Christin Herrmann
    •  & Neha J. Pancholi
  4. Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA

    • Nikolina Sekulic
    • , Ben E. Black
    •  & Benjamin A. Garcia
  5. Epigenetics Program, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA

    • Nikolina Sekulic
    • , Rosalynn C. Molden
    • , Ben E. Black
    •  & Benjamin A. Garcia
  6. Villanova University, Villanova, Pennsylvania 19085, USA

    • Joana Petrescu
  7. Division of Cell Pathology, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA

    • Daniel Blumenthal
  8. Division of Pulmonary, Allergy, and Critical Care Medicine, Hospital of the University of Pennsylvania, and the Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA

    • Andrew J. Paris
  9. Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York 11794, USA

    • Philomena Ostapchuk
    •  & Patrick Hearing
  10. Protein and Proteomics Core, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA

    • Steven H. Seeholzer
  11. Division of Neonatology, Children’s Hospital of Philadelphia, Philadelphia, and Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA

    • G. Scott Worthen

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Contributions

D.C.A. and M.D.W. conceived the project and designed experiments; D.C.A., C.H., N.S., J.P., N.J.P. and E.D.R. performed the experiments; D.C.A., C.H. and J.P. generated constructs and cell lines; K.K., R.C.M., S.H.S. and B.A.G. performed MS analysis; P.O. and P.H. generated Ad5-flox-VII virus and provided 293-Cre cell line; D.C.A. and D.B. performed the FRAP experiments; A.J.P. and G.S.W. conducted all mouse experiments; B.E.B. and B.A.G. designed experiments and interpreted the data; D.C.A. and M.D.W. interpreted the data and wrote the manuscript and all authors were involved in editing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Matthew D. Weitzman.

All proteomic raw files have been deposited in the Chorus database under project number 1047 (https://chorusproject.org/).

Reviewer Information Nature thanks M. Bianchi and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figures

    This file contains the Source data gels for Figures 1d, e, f, 2a, 3b, c, j, k, 4b and Extended Data Figures 2a, b, c, d, e, f, g, 4a, b, c, 8a, g, 9c, f.

Excel files

  1. 1.

    Supplementary Table 1

    Summary of post-translational modifications found on histones upon expression of protein VII.

  2. 2.

    Supplementary Table 2

    Total list of proteins significantly changed upon protein VII expression identified by mass spectrometry analysis of high salt fractions. Table includes the log2 fold change of MaxQuant-derived iBAQ values obtained for protein VII-HA induced and uninduced highest salt fractions (600mM). Proteins with homoscedastic two-tailed t-test p-value smaller than 0.05 were considered as significantly altered between the two tested conditions. N/A defines not assigned t-test p-values; this is either due to the presence of the protein in only one condition, or if in one condition the protein was quantified in only one replicate. The number of peptides used for quantification was also highlighted. Commonly occurring contaminants, such as human keratins or trypsin, were removed from the final list.

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https://doi.org/10.1038/nature18317

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