Suppression of the antiviral response by an influenza histone mimic

Journal name:
Nature
Volume:
483,
Pages:
428–433
Date published:
DOI:
doi:10.1038/nature10892
Received
Accepted
Published online

Abstract

Viral infection is commonly associated with virus-driven hijacking of host proteins. Here we describe a novel mechanism by which influenza virus affects host cells through the interaction of influenza non-structural protein 1 (NS1) with the infected cell epigenome. We show that the NS1 protein of influenza A H3N2 subtype possesses a histone-like sequence (histone mimic) that is used by the virus to target the human PAF1 transcription elongation complex (hPAF1C). We demonstrate that binding of NS1 to hPAF1C depends on the NS1 histone mimic and results in suppression of hPAF1C-mediated transcriptional elongation. Furthermore, human PAF1 has a crucial role in the antiviral response. Loss of hPAF1C binding by NS1 attenuates influenza infection, whereas hPAF1C deficiency reduces antiviral gene expression and renders cells more susceptible to viruses. We propose that the histone mimic in NS1 enables the influenza virus to affect inducible gene expression selectively, thus contributing to suppression of the antiviral response.

At a glance

Figures

  1. Influenza NS1 contains a histone mimic.
    Figure 1: Influenza NS1 contains a histone mimic.

    a, The homologous carboxy-terminal NS1 and the amino-terminal histone H3 sequences are shown (red letters). The table displays C-terminal NS1 sequences of the influenza A subtypes. b, Methylation or acetylation of the NS1 peptide (top panel), the GST–NS1 protein (middle panel) or of the viral NS1 in A549 infected cells (bottom panel) are shown. KR, NS1 substrates where K229 is replaced by arginine. c, Association of the NS1 histone mimic with the hPAF1C subunits and CHD1 in nuclear extracts. In, input material. d, NS1 histone mimic binds to CHD1. Unmodified or methylated NS1 (K229) or methylated H3 (K4) peptides were incubated with the recombinant CHD1 or the CHD1 double-chromodomain. Binding to NS1 was revealed by silver or Coomassie staining (top and bottom panel, respectively). e, Binding of Flag-tagged hPAF1C subunits to NS1 or histone H3 peptides was assessed by western blotting (left and right panels, respectively). IP, immunoprecipitation.

  2. Functional interaction between NS1 and PAF1 in infected cells.
    Figure 2: Functional interaction between NS1 and PAF1 in infected cells.

    a, The ChIP-seq profiles show the distribution of indicated proteins at inducible genes before (black line) and after (red line) infection. The induced genes were revealed by RNA-seq and ChIP-seq analysis of infected A549 cells (Supplementary Tables 1 and 2). TSS and TES, the transcriptional start and end sites, respectively. b, The NS1 levels at gene promoters in PAF1- or CHD1-deficient cells (blue and green lines, respectively). The scrambled siRNA-treated cells (red line) were used as control. The insert shows knockdown of PAF1 or CHD1 in A549 cells. c, PAF1, RNA Pol II and H3K4me3 levels at the TSS and TES of the induced genes in uninfected (ui) cells, cells infected with the wild-type (WT) or PAF1-binding mutant virus (ΔPAF). Data are representative of three independent experiments; error bars show the s.e.m.

  3. NS1 suppresses antiviral gene transcription in infected cells.
    Figure 3: NS1 suppresses antiviral gene transcription in infected cells.

    a, Left: the GRO-seq profile of inducible RNA transcripts in uninfected (ui) A549 cells (black line) or cells infected with wild-type or ΔPAF virus (green and red lines, respectively). Right: GRO-seq profile of IFIT1 and IFI6 genes in uninfected and infected cells. b, GRO-seq profile of A549-expressed genes that are not affected by virus infection (left panel) or of the HPRT1 gene (right panel). Reads from either DNA strands are indicated as+/−. The y axes display reads per million mapped reads per 25bp.

  4. NS1 inhibits transcriptional elongation in vitro.
    Figure 4: NS1 inhibits transcriptional elongation in vitro.

    a, The full-length NS1 protein (NS1) or NS1 lacking the PAF1-binding sequence (NS1(ΔPAF)) (Supplementary Fig. 5c) was added to the RNA elongation reaction as indicated. b, The amount of the 390-nt RNA elongation product was quantified by ImageJ. The results of two independent experiments are shown.

  5. PAF1 controls antiviral response.
    Figure 5: PAF1 controls antiviral response.

    a, b, The expression levels of mRNAs in influenza infected (a) or IFN-β1-treated (b) control (siCtrl) or PAF1-deficient (siPAF) A549 cells. The tables show the siPAF-affected gene categories. Ut, untreated with siRNA. c, Dynamics of virus replication in control or PAF1-deficient A549 cells. p.f.u. plaque-forming units. Data are representative of three independent experiments. Error bars show the s.e.m.

References

  1. Kornberg, R. D. & Thomas, J. O. Chromatin structure—oligomers of histones. Science 184, 865868 (1974)
  2. Campos, E. I. & Reinberg, D. Histones: annotating chromatin. Ann. Rev. Genet. 43, 559599 (2009)
  3. Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D. & Patel, D. J. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nature Struct. Mol. Biol. 14, 10251040 (2007)
  4. Kouzarides, T. Chromatin modifications and their function. Cell 128, 693705 (2007)
  5. Kelly, A. E. et al. Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B. Science 330, 235239 (2010)
  6. Fernandez-Capetillo, O. et al. DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1. Nature Cell Biol. 4, 993997 (2002)
  7. Li, B., Carey, M. & Workman, J. L. The role of chromatin during transcription. Cell 128, 707719 (2007)
  8. Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 10671073 (2010)
  9. Nicodeme, E. et al. Suppression of inflammation by a synthetic histone mimic. Nature 468, 11191123 (2010)
  10. Nishiyama, A. et al. Intracellular delivery of acetyl-histone peptides inhibits native bromodomain-chromatin interactions and impairs mitotic progression. Febs Lett. 582, 15011507 (2008)
  11. Hargreaves, D. C., Horng, T. & Medzhitov, R. Control of inducible gene expression by signal-dependent transcriptional elongation. Cell 138, 129145 (2009)
  12. Sampath, S. C. et al. Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly. Mol. Cell 27, 596608 (2007)
  13. Elde, N. C. & Malik, H. S. The evolutionary conundrum of pathogen mimicry. Nature Rev. Microbiol. 7, 787797 (2009)
  14. Hale, B. G., Randall, R. E., Ortin, J. & Jackson, D. The multifunctional NS1 protein of influenza A viruses. J. Gen. Virol. 89, 23592376 (2008)
  15. Garcia-Sastre, A. et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252, 324330 (1998)
  16. Lu, Y., Wambach, M., Katze, M. G. & Krug, R. M. Binding of the influenza-virus NS1 protein to double-stranded-RNA inhibits the activation of the protein-kinase that phosphorylates the Elf-2 translation initiation-factor. Virology 214, 222228 (1995)
  17. Gack, M. U. et al. Influenza A Virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I. Cell Host Microbe 5, 439449 (2009)
  18. Pichlmair, A. et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Science 314, 9971001 (2006)
  19. Hale, B. G., Jackson, D., Chen, Y. H., Lamb, R. A. & Randall, R. E. Influenza A virus NS1 protein binds p85b and activates phosphatidylinositol-3-kinase signaling. Proc. Natl Acad. Sci. USA 103, 1419414199 (2006)
  20. Krug, R. M., Yuan, W. M., Noah, D. L. & Latham, A. G. Intracellular warfare between human influenza viruses and human cells: the roles of the viral NS1 protein. Virology 309, 181189 (2003)
  21. Nemeroff, M. E., Barabino, S. M. L., Li, Y. Z., Keller, W. & Krug, R. M. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3' end formation of cellular pre-mRNAs. Mol. Cell 1, 9911000 (1998)
  22. Das, K. et al. Structural basis for suppression of a host antiviral response by influenza A virus. Proc. Natl Acad. Sci. USA 105, 1309313098 (2008)
  23. Satterly, N. et al. Influenza virus targets the mRNA export machinery and the nuclear pore complex. Proc. Natl Acad. Sci. USA 104, 18531858 (2007)
  24. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8Å resolution. Nature 389, 251260 (1997)
  25. Hale, B. G., Barclay, W. S., Randall, R. E. & Russell, R. J. Structure of an avian influenza A virus NS1 protein effector domain. Virology 378, 15 (2008)
  26. Xhemalce, B. & Kouzarides, T. A chromodomain switch mediated by histone H3 Lys 4 acetylation regulates heterochromatin assembly. Genes Dev. 24, 647652 (2010)
  27. Becker, P. B. et al. Site-specific acetylation of ISWI by GCN5. BMC Mol. Biol. 8, (2007)
  28. Ruthenburg, A. J., Allis, C. D. & Wysocka, J. Methylation of lysine 4 on histone H3: Intricacy of writing and reading a single epigenetic mark. Mol. Cell 25, 1530 (2007)
  29. Wang, P. F. et al. Global analysis of H3K4 methylation defines MLL family member targets and points to a role for MLL1-mediated H3K4 methylation in the regulation of transcriptional initiation by RNA polymerase II. Mol. Cell. Biol. 29, 60746085 (2009)
  30. Guillemette, B. et al. H3 lysine 4 is acetylated at active gene promoters and is regulated by H3 lysine 4 methylation. PLoS Genet. 7, (2011)
  31. Lachner, M., O’Carroll, N., Rea, S., Mechtler, K. & Jenuwein, T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116120 (2001)
  32. Shi, X. B. et al. ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature 442, 9699 (2006)
  33. Lan, F. et al. Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression. Nature 448, 718722 (2007)
  34. Wysocka, J. Identifying novel proteins recognizing histone modifications using peptide pull-down assay. Methods 40, 339343 (2006)
  35. Sims, R. J. et al. Human but not yeast CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains. J. Biol. Chem. 280, 4178941792 (2005)
  36. Kim, J., Guermah, M. & Roeder, R. G. The human PAF1 complex acts in chromatin transcription elongation both independently and cooperatively with SII/TFIIS. Cell 140, 491503 (2010)
  37. Ramirez-Carrozzi, V. R. et al. A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell 138, 114128 (2009)
  38. Kim, K. Y. & Levin, D. E. Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination. Cell 144, 745756 (2011)
  39. Chen, Y. X. et al. DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation. Genes Dev. 23, 27652777 (2009)
  40. Jaehning, J. A. The Paf1 complex: platform or player in RNA polymerase II transcription? Biochim. Bioiphys. Acta 1799, 379388 (2010)
  41. Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 18451848 (2008)
  42. Min, I. M. et al. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Gene Dev. 25, 742754 (2011)
  43. Mapendano, C. K., Lykke-Andersen, S., Kjems, J., Bertrand, E. & Jensen, T. H. Crosstalk between mRNA 3′ end processing and transcription initiation. Mol. Cell 40, 410422 (2010)
  44. Loucaides, E. M. et al. Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies. Virology 394, 154163 (2009)
  45. Engelhardt, O. G., Smith, M. & Fodor, E. Association of the influenza a virus RNA-dependent RNA polymerase with cellular RNA polymerase II. J. Virol. 79, 58125818 (2005)
  46. Jackson, D., Hossain, M. J., Hickman, D., Perez, D. R. & Lamb, R. A. A new influenza virus virulence determinant: The NS1 protein four C-terminal residues modulate pathogenicity. Proc. Natl Acad. Sci. USA 105, 43814386 (2008)
  47. Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 4145 (2000)
  48. Turner, B. M. Histone acetylation and an epigenetic code. Bioessays 22, 836845 (2000)
  49. Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 10741080 (2001)
  50. Yang, Y. et al. The transmissibility and control of pandemic influenza A (H1N1) virus. Science 326, 729733 (2009)

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Author information

Affiliations

  1. Laboratory of Immune Cell Epigenetics and Signaling, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA

    • Ivan Marazzi,
    • Jessica S. Y. Ho,
    • Uwe Schaefer,
    • Kate L. Jeffrey &
    • Alexander Tarakhovsky
  2. Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA

    • Jaehoon Kim &
    • Robert G. Roeder
  3. Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1124, New York, New York 10029, USA

    • Balaji Manicassamy,
    • Randy A. Albrecht,
    • Chris W. Seibert &
    • Adolfo García-Sastre
  4. Global Health and Infectious Pathogens Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1124, New York, New York 10029, USA

    • Balaji Manicassamy,
    • Randy A. Albrecht &
    • Adolfo García-Sastre
  5. Genomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA

    • Scott Dewell
  6. Epinova DPU, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK

    • Rab K. Prinjha &
    • Kevin Lee
  7. Department of Medicine, Division of Infectious Diseases, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1124, New York, New York 10029, USA

    • Adolfo García-Sastre

Contributions

I.M. contributed to design, execution, analysis of the experiments and manuscript preparation. J.S.Y.H. studied the role of PAF1 in viral infection and assisted in manuscript preparation. J.K. and R.R. studied the impact of NS1 on hPAF1C and transcriptional elongation. B.M., R.A.A. engineered the recombinant influenza viruses and studied viral infectivity. U.S. was involved in gene expression studies. S.D. performed bioinformatic analysis. C.W.S. generated antibody against viral polymerase. K.L.J. gave technical assistance. R.K.P. and K.L. contributed to manuscript preparation and enabled ChIP-seq and RNA-seq. A.G.-S. supervised and discussed the work with infectious influenza viruses. A.T. conceived and supervised this study and wrote the final manuscript.

Competing financial interests

R.K.P. and K.L. are employees of GlaxoSmithKline. Research support, excluding salaries to the members of The Rockefeller University, was partially provided by GlaxoSmithKline.

Corresponding authors

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Supplementary information

PDF files

  1. Supplementary Information (1.4M)

    This file contains Supplementary Figures 1-12, Supplementary Methods, additional references and full legends for Supplementary Tables 1-8.

Excel files

  1. Supplementary Table 1 (135K)

    This table shows genes affected by Influenza Infection - see Supplementary Information file for full legend.

  2. Supplementary Table 2 (43K)

    This table contains a list of genes used for the integrated ChIP-seq profile - see Supplementary Information file for full legend.

  3. Supplementary Table 3 (249K)

    This table shows siPAF dependent genes in PR8/∆NS1 infected cells - see Supplementary Information file for full legend.

  4. Supplementary Table 4 (656K)

    This table shows siPAF dependent genes in Influenza (H1N1) infected cells - see Supplementary Information file for full legend.

  5. Supplementary Table 5 (596K)

    This table shows siPAF dependent genes in Influenza (H1N1) infected cells - see Supplementary Information file for full legend.

  6. Supplementary Table 6 (888K)

    This table shows siPAF dependent genes in Poly(I:C) transfected cells - see Supplementary Information file for full legend.

  7. Supplementary Table 7 (111K)

    This table shows siPAF dependent genes in IFNβ1 treated cells - see Supplementary Information file for full legend.

  8. Supplementary Table 8 (24K)

    This table shows that expression of housekeeping genes are not affected by siPAF mediated hPAF1 deficiency - see Supplementary Information file for full legend.

Additional data