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Influenza virus infection causes global RNAPII termination defects

Nature Structural & Molecular Biology volume 25, pages 885–893 (2018)Cite this article

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Abstract

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3′ ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3′ extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

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Fig. 1: NS1 from 1918 pandemic influenza virus is SUMOylated in its unique C-terminal domain.
Fig. 2: 1918 NS1 WT and mutant viruses.
Fig. 3: SUMOylation of NS1 induces assembly and increases pervasive RNAPII termination defects.
Fig. 4: Relationship between RNAPII run-through and splicing defects.

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Data availability

RNA-seq data associated with this study are available through the Gene Expression Omnibus (GEO) data repository, accession number GSE103604. Source data for Fig. 2a,c and 3h,j are available in Supplementary Dataset 2. Data underlying the analysis in Fig. 4f are available in Supplementary Dataset 3. All other data are available upon reasonable request.

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Acknowledgements

We thank all members of the laboratories of I.M. and A.G.-S., and J. Bloom and A. Kornblihtt for valuable discussions and suggestions on the manuscript. We thank the Medicinal Chemistry Core, Integrated Screening Core, Microscopy CoRE,, and Global Health and Emerging Pathogens Institute (GHEPI) at the Icahn School of Medicine at Mount Sinai. H.v.B., I.M., and A.G.-S. are partially supported by HHSN272201400008C–Center for Research on Influenza Pathogenesis (CRIP), a NIAID-funded Center of Excellence for Influenza Research and Surveillance (CEIRS). I.M. is supported in part by the Department of Defense W911NF-14-1-0353. I.M. and H.v.B. are supported by NIH grant 1R01AN3663134. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. This work was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai.

Author information

Author notes

  1. These authors contributed equally to this work: Nan Zhao, Vittorio Sebastiano, Natasha Moshkina.

  2. These authors jointly directed this work: Adolfo García-Sastre, Ivan Marazzi.

Authors and Affiliations

  1. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Nan Zhao, Natasha Moshkina, Nacho Mena, Yixuan Ma, Alex Rialdi, Randy Albrecht, Maria Teresa Sánchez-Aparicio, Juan Ayllon, Adolfo García-Sastre & Ivan Marazzi

  2. Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Nan Zhao, Natasha Moshkina, Nacho Mena, Yixuan Ma, Alex Rialdi, Randy Albrecht, Maria Teresa Sánchez-Aparicio, Juan Ayllon, Adolfo García-Sastre & Ivan Marazzi

  3. Department of Obstetrics and Gynecology, Stanford University, Stanford, CA, USA

    Vittorio Sebastiano, Sweta Ravisankar & Bahareh Haddad

  4. Institute for Stem Cell Biology & Regenerative Medicine, Stanford University, Stanford, CA, USA

    Vittorio Sebastiano, Sweta Ravisankar & Bahareh Haddad

  5. Department of Medicine (Infectious Diseases), Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Judd Hultquist

  6. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA

    David Jimenez-Morales & Nevan Krogan

  7. Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Romain Fenouil & Harm van Bakel

  8. Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

    Jessica Sook Yuin Ho, Diana Low & Ernesto Guccione

  9. Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Jian Jin

  10. Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic

    Vyacheslav Yurchenko

  11. Epinova Epigenetics Discovery Performance Unit, Immuno-Inflammation Therapy Area, GlaxoSmithKline, Medicines Research Centre, Stevenage, UK

    Rab K. Prinjha

  12. Laboratory of Immune Cell Epigenetics and Signaling, The Rockefeller University, New York, NY, USA

    Alexander Tarakhovsky

  13. Cancer Cell Biology Programme, Centro Nacional de Investigaciones Oncológicas, CNIO, Madrid, Spain

    Massimo Squatrito

  14. Department of Psychiatry, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Dalila Pinto

  15. Department of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Kimaada Allette, Melissa Laird Smith & Robert Sebra

  16. Department of Medicine, Clinical Immunology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Minji Byun

  17. Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

    Terrence Tumpey

  18. Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Benjamin Greenbaum

  19. Division of Hematology and Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Benjamin Greenbaum

  20. Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Benjamin Greenbaum

  21. Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Adolfo García-Sastre

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  1. Nan Zhao
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  2. Vittorio Sebastiano
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Contributions

N.Z., N. Moshkina, A.R., and V.Y. performed experiments with modeling of PDZ–peptide–SUMO complexes, transfection, infection, WB, SDD-AGE, purification of recombinant proteins, BLI, pulldown assays, analysis of proteins stability, immunofluorescence, RT–PCR, chemical synthesis of B-isox, and B-isox-mediated precipitation; V.S., S.R., and B.H. performed independent validation of SUMOylation of the SUP domain; N. Mena, R.A., M.T.S.-A., J.A., and T.T. performed experiments with rescue of recombinant influenza viruses, single-cycle growth curves, plaque phenotypes, and analysis of infectious viral progeny; J.H., D.J.-M., and Y.M. performed experiments with preparation of peptides for MS, protein identification by LC–MS/MS, and statistical relative quantification of proteins and enrichment analysis; R.F., M.S., D.P., A.K., M.B., M.L.S., R.S., and H.v.B. performed directional RNA-seq, differential gene expression analysis, TR and intron/exon ratio calculation, and iso-seq analysis. S.-Y.H., D.L., and E.G. performed bioinformatics analysis on splicing and termination; B.G. performed conservation analysis; J.J., R.K.P., A.T., and N.K. provided materials and insights in experimental procedures; and A.G.-S. and I.M. supervised the project. I.M. wrote the paper, on which coauthors provided feedback.

Corresponding author

Correspondence to Ivan Marazzi.

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Competing interests

The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 NS1 C-terminal diversity in different strains of influenza virus.

a, Diagram representing the C-terminus of an NS1 protein in different Influenza strains. b, Visual representation of the conservation score among NS1 proteins averaged over 10 amino acidic residues.

Supplementary Figure 2 Control experiments.

Control experiments for Fig. 2. a, IF of ectopically expressed GFP, NS1, NS1-KR and NS1-SUMO in A549 (scale bar indicates 100um). b, Pull-down assay between recombinant NS1, NS1-KR, NS1-SUMO and PSD95. PBS served as a negative control. Coomassie staining SDS-PAGE showing purified recombinant proteins (left) and eluted complexes (right). c-e, Structural modeling of SUMOylated NS1 tail and the indicated PDZ domain containing-proteins GIPC2 (c), DVL1 (d) and PSD95 (e). f, Schematic diagram of plaque selection and analysis. All plaques selected after 24 hpi with NS1-SUMO virus indicate that the virus has reverted back to NS1 virus (WT virotype) by mutagenesis of its C-term SUMO extension. This result indicates that NS1-SUMO virus can infect but it is compromised in its ability to bud and spread from cell-to-cell.

Supplementary Figure 3 Control experiments for 1918 NS1 and NS1-SUMO viruses.

a, A549 cells were mock infected (M) or infected with NS1 and NS1-SUMO virus for 12 hours and sequentially treated with mg132 (50 nM) or cycloheximide (CHX, 50 ug/mL) for 0/4/8/24hours. Lysates were immunoblotted with anti-NS1 antibody. b, Cumulative expression levels of positive- and negative-sense viral RNAs for all segments in A549 cells at 6 or 12 hours post-infection with NS1 virus (blue), or NS1-SUMO virus (red). Normalized log2 counts of viral RNAs per million sequenced reads (CPM) are shown for two biological replicate experiments.

Supplementary Figure 4 Control experiments.

Control experiments for Fig. 3. a, Synthetic route of B-isox. Reagents and conditions: (1) HOSu, EDC·HCl, THF, RT, overnight; (2) 6-amino-1-hexanol, THF, 5 hours; (3) Biotin, DMAP, EDC·HCl, DCM, 3 days. b, LC-HRMS of synthesized B-isox. c, Silver staining gel showing B-isox-mediated precipitation of proteins from cell lysate. d, Statistical Analyses of the B-isox precipitated proteins after infection. Statistical quantitation of the overlap between proteins precipitated by B-isox during infection compared to a set of 1286 B-isox-precipitated proteins from the McKnight’s lab (Cell, 2013, 155(5): 1049-1060), a set of 1786 RNA binding proteins from UniProt, a set of 126 literature-cited RNA granule proteins, and the entire human proteome. Fisher’s exact test was used to determine the significance of the overlapping between datasets. e, qPCR analysis of run through transcription downstream of the GAPDH and CDC25A genes in A549 cells infected with WT or NS1-GFP A/Vietnam/1203/2004/H5N1 influenza virus at MOI of 3 for 6 and 12 hours. Expression levels were calculated by using primer sets amplifying region downstream of termination sites and calculating fold enrichment comparing 6 hours and 12 hours time post infection (as mock infection has no detectable signal indicating absence of RNAPII run-through). Error bars correspond to mean ± s.e.m.

Supplementary Figure 5 Control experiments.

Control experiments related to Fig. 4. a, TR density at termination region plotted as a function of gene induction (red) or repression (green) during infection with NS1-SUMO virus at 6 hpi (left) and 12 hpi (right). b, Normalized RNA-Seq read coverage tracks showing increased 3’-end transcript levels for representative genes: CXCL1, MYC, NFKBIA, and CDC25A.

Supplementary Figure 6 TR region changes at specific gene sets and human variation in SUMO-modifying genes.

a, TR plots at termination region of active (RPKM > 1 in 50% of samples) multi-exonic (upper panels) and mono-exonic (lower panels) genes in uninfected A549 cells and 6 or 12 hpi with NS1 or NS1-SUMO virus. Data is shown for two replicates in each condition. b, Scatter plot showing the relationship between the log fold-change (logFC) in gene expression between NS1-SUMO virus and NS1 virus-infected A549 cells at 6 hpi, and the logFC in transcript levels in 5,000 bp 3’ gene-flanking regions. Genes with a significant increase or decrease in expression in NS1-SUMO vs NS1 infected A549 cells at 6 hpi are highlighted in red and green, respectively. All other genes are highlighted in grey. Solid and dotted lines correspond to the regression line and 95% confidence interval for genes with significant expression differences, with the R2 and significance shown at the top-left. c, Analysis of SUMO-modifying gene mutations using a compendium of protein-coding genetic variation in 60,706 humans from the ENCODE Consortium. The probability of being loss-of-function intolerant (intolerant of both heterozygous and homozygous of variants) is shown for all genes compared to SUMO-modifying genes.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6

Reporting Summary

Supplementary Table 1

Information of PDZ domain containing proteins used in this study

Supplementary Table 2

Cross-comparison of B-isox precipitated proteins

Supplementary Table 3

Quantitative mass-spectrometry analysis of B-isox precipitated proteins

Supplementary Table 4

RNA-seq analysis of NS1 vs NS1-SUMO viral infection

Supplementary Table 5

Human polymorphism and damaging-mutations of SUMO-modifying enzymes

Supplementary Dataset 1

Uncropped WB images for Figures 1C, 1D, 1E, 3A, and 3C

Supplementary Dataset 2

Supporting data for Figures 2A, 2C, 3H, 3I, and 3J

Supplementary Dataset 3

Supporting data for Figure 4F, containing the raw circular consensus sequencing (CCS) reads, the reads after adapter trimming (cutadapt.fa), removal of Poly(A) tails (cutadapt_polA.fa), and removal of short reads <300 nt (cutadapt_polA_gt300.fa)

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Zhao, N., Sebastiano, V., Moshkina, N. et al. Influenza virus infection causes global RNAPII termination defects. Nat Struct Mol Biol 25, 885–893 (2018). https://doi.org/10.1038/s41594-018-0124-7

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