Despite the cytopathic nature of influenza A virus (IAV) replication, we recently reported that a subset of lung epithelial club cells is able to intrinsically clear the virus and survive infection. However, the mechanisms that drive cell survival during a normally lytic infection remained unclear. Using a loss-of-function screening approach, we discovered that the DNA mismatch repair (MMR) pathway is essential for club cell survival of IAV infection. Repair of virally induced oxidative damage by the DNA MMR pathway not only allowed cell survival of infection, but also facilitated host gene transcription, including the expression of antiviral and stress response genes. Enhanced viral suppression of the DNA MMR pathway prevented club cell survival and increased the severity of viral disease in vivo. Altogether, these results identify previously unappreciated roles for DNA MMR as a central modulator of cellular fate and a contributor to the innate antiviral response, which together control influenza viral disease severity.
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The raw RNA-seq data files from Fig. 5i–k are available at NCBI GEO (series GSE130189). The raw data for Figs. 2b–d, 3d and 5i–k are available in Supplementary Tables 1–5. Raw data from all other figures and unique materials, including viruses and plasmids, are available from the corresponding authors upon request.
Downey, J., Pernet, E., Coulombe, F. & Divangahi, M. Dissecting host cell death programs in the pathogenesis of influenza. Microbes Infect. 20, 560–569 (2018).
Turpin, E. et al. Influenza virus infection increases p53 activity: role of p53 in cell death and viral replication. J. Virol. 79, 8802–8811 (2005).
Orzalli, M. H. & Kagan, J. C. Apoptosis and necroptosis as host defense strategies to prevent viral infection. Trends Cell Biol. 27, 800–809 (2017).
Ehrhardt, C. et al. Influenza A virus NS1 protein activates the PI3K/Akt pathway to mediate antiapoptotic signaling responses. J. Virol. 81, 3058–3067 (2007).
Zhirnov, O. P. & Klenk, H. D. Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling. Apoptosis 12, 1419–1432 (2007).
van de Sandt, C. E., Kreijtz, J. H. & Rimmelzwaan, G. F. Evasion of influenza A viruses from innate and adaptive immune responses. Viruses 4, 1438–1476 (2012).
Heaton, N. S. et al. Long-term survival of influenza virus infected club cells drives immunopathology. J. Exp. Med. 211, 1707–1714 (2014).
Reuther, P. et al. Generation of a variety of stable influenza A reporter viruses by genetic engineering of the NS gene segment. Sci. Rep. 5, 11346 (2015).
Burdeinick-Kerr, R. & Griffin, D. E. Gamma interferon-dependent, noncytolytic clearance of sindbis virus infection from neurons in vitro. J. Virol. 79, 5374–5385 (2005).
Guidotti, L. G. et al. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity 4, 25–36 (1996).
Guidotti, L. G. et al. Noncytopathic Clearance of Lymphocytic Choriomeningitis Virus from the Hepatocyte. J. Exp. Med. 189, 1555–1564 (1999).
Chisari, F. V. Viruses, immunity, and cancer: lessons from hepatitis B. Am. J. Pathol. 156, 1117–1132 (2000).
Griffin, D. E. Recovery from viral encephalomyelitis: immune-mediated noncytolytic virus clearance from neurons. Immunol. Res. 47, 123–133 (2010).
Kudchodkar, S. B. & Levine, B. Viruses and autophagy. Rev. Med Virol. 19, 359–378 (2009).
Hamilton, J. R. et al. Club cells surviving influenza A virus infection induce temporary nonspecific antiviral immunity. Proc. Natl Acad. Sci. USA 113, 3861–3866 (2016).
Bridge, G., Rashid, S. & Martin, S. A. DNA mismatch repair and oxidative DNA damage: implications for cancer biology and treatment. Cancers (Basel) 6, 1597–1614 (2014).
Lei, X., Zhu, Y., Tomkinson, A. & Sun, L. Measurement of DNA mismatch repair activity in live cells. Nucleic Acids Res. 32, e100 (2004).
Macpherson, P. et al. 8-Oxoguanine incorporation into DNA repeats in vitro and mismatch recognition by MutSalpha. Nucleic Acids Res. 33, 5094–5105 (2005).
Nencioni, L. et al. Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2. FASEB J. 17, 758–760 (2003).
Sgarbanti, R. et al. Redox regulation of the influenza hemagglutinin maturation process: a new cell-mediated strategy for anti-influenza therapy. Antioxid. Redox Signal. 15, 593–606 (2011).
Amatore, D. et al. Influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by NOX4-derived ROS. Cell Microbiol. 17, 131–145 (2015).
Li, N. et al. Influenza infection induces host DNA damage and dynamic DNA damage responses during tissue regeneration. Cell Mol. Life Sci. 72, 2973–2988 (2015).
Lin, X. et al. The influenza virus H5N1 infection can induce ROS production for viral replication and host cell death in A549 cells modulated by human Cu/Zn superoxide dismutase (sod1) overexpression. Viruses 8, E13 (2016).
Russell, A. B., Trapnell, C. & Bloom, J. D. Extreme heterogeneity of influenza virus infection in single cells. eLife 7, e32303 (2018).
Shin, N., Pyo, C. W., Jung, K. I. & Choi, S. Y. Influenza A virus PB1-F2 is involved in regulation of cellular redox state in alveolar epithelial cells. Biochem. Biophys. Res. Commun. 459, 699–705 (2015).
Wang, Q. W. et al. Anti-influenza A virus activity of rhein through regulating oxidative stress, TLR4, Akt, MAPK, and NF-kappaB signal pathways. PLoS ONE 13, e0191793 (2018).
Valavanidis, A., Vlachogianni, T. & Fiotakis, C. 8-Hydroxy-2’-deoxyguanosine (8-OHdG): a critical biomarker of oxidative stress and carcinogenesis. J. Environ. Sci. Health C 27, 120–139 (2009).
Singh, N. P., McCoy, M. T., Tice, R. R. & Schneider, E. L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191 (1988).
Collins, A. R., Dusinska, M. & Horska, A. Detection of alkylation damage in human lymphocyte DNA with the comet assay. Acta Biochim. Pol. 48, 611–614 (2001).
Hamad, I., Arda, N., Pekmez, M., Karaer, S. & Temizkan, G. Intracellular scavenging activity of Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) in the fission yeast, Schizosaccharomyces pombe. J. Nat. Sci. Biol. Med. 1, 16–21 (2010).
Kuraoka, I. et al. Effects of endogenous DNA base lesions on transcription elongation by mammalian RNA polymerase II. Implications for transcription-coupled DNA repair and transcriptional mutagenesis. J. Biol. Chem. 278, 7294–7299 (2003).
Charlet-Berguerand, N. et al. RNA polymerase II bypass of oxidative DNA damage is regulated by transcription elongation factors. EMBO J. 25, 5481–5491 (2006).
Mellon, I. & Champe, G. N. Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli. Proc. Natl Acad. Sci. USA 93, 1292–1297 (1996).
Mellon, I., Rajpal, D. K., Koi, M., Boland, C. R. & Champe, G. N. Transcription-coupled repair deficiency and mutations in human mismatch repair genes. Science 272, 557–560 (1996).
Ni, T. T., Marsischky, G. T. & Kolodner, R. D. MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxo-guanine in S. cerevisiae. Mol. Cell 4, 439–444 (1999).
Bercovich-Kinori, A. et al. A systematic view on influenza induced host shutoff. eLife 5, e18311 (2016).
Domingues, P. et al. Global reprogramming of host SUMOylation during influenza virus Infection. Cell Rep. 13, 1467–1480 (2015).
Noh, H., Shoemaker, J. E. & Gunawan, R. Network perturbation analysis of gene transcriptional profiles reveals protein targets and mechanism of action of drugs and influenza A viral infection. Nucleic Acids Res. 46, e34 (2018).
Varble, A. et al. An in vivo RNAi screening approach to identify host determinants of virus replication. Cell Host Microbe 14, 346–356 (2013).
Khanna, M. et al. Detection of influenza virus induced ultrastructural changes and DNA damage. Indian J. Virol. 21, 50–55 (2010).
Vijaya Lakshmi, A. N., Ramana, M. V., Vijayashree, B., Ahuja, Y. R. & Sharma, G. Detection of influenza virus induced DNA damage by Comet assay. Mutat. Res. 442, 53–58 (1999).
Ryan, E. L., Hollingworth, R. & Grand, R. J. Activation of the DNA damage response by RNA viruses. Biomolecules 6, 2 (2016).
Machida, K. et al. Hepatitis C virus infection activates the immunologic (type II) isoform of nitric oxide synthase and thereby enhances DNA damage and mutations of cellular genes. J. Virol. 78, 8835–8843 (2004).
Machida, K. et al. Hepatitis C virus inhibits DNA damage repair through reactive oxygen and nitrogen species and by interfering with the ATM-NBS1/Mre11/Rad50 DNA repair pathway in monocytes and hepatocytes. J. Immunol. 185, 6985–6998 (2010).
Clavarino, G. et al. Induction of GADD34 is necessary for dsRNA-dependent interferon-beta production and participates in the control of Chikungunya virus infection. PLoS Pathog. 8, e1002708 (2012).
Nargi-Aizenman, J. L., Simbulan-Rosenthal, C. M., Kelly, T. A., Smulson, M. E. & Griffin, D. E. Rapid activation of poly(ADP-ribose) polymerase contributes to Sindbis virus and staurosporine-induced apoptotic cell death. Virology 293, 164–171 (2002).
Datta, A. & Jinks-Robertson, S. Association of increased spontaneous mutation rates with high levels of transcription in yeast. Science 268, 1616–1619 (1995).
Morey, N. J., Greene, C. N. & Jinks-Robertson, S. Genetic analysis of transcription-associated mutation in Saccharomyces cerevisiae. Genetics 154, 109–120 (2000).
Naganuma, A., Dansako, H., Nakamura, T., Nozaki, A. & Kato, N. Promotion of microsatellite instability by hepatitis C virus core protein in human non-neoplastic hepatocyte cells. Cancer Res. 64, 1307–1314 (2004).
Olejnik, J. et al. Ebolaviruses associated with differential pathogenicity induce distinct host responses in human macrophages. J. Virol. 91, e00179–17 (2017).
Xue, J. et al. Dynamic interactions between Bombyx mori nucleopolyhedrovirus and its host cells revealed by transcriptome analysis. J. Virol. 86, 7345–7359 (2012).
Beard, P. M. et al. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference. PLoS ONE 9, e98431 (2014).
Maddocks, O. D., Scanlon, K. M. & Donnenberg, M. S. An Escherichia coli effector protein promotes host mutation via depletion of DNA mismatch repair proteins. mBio 4, e00152–00113 (2013).
Sauvonnet, N., Pradet-Balade, B., Garcia-Sanz, J. A. & Cornelis, G. R. Regulation of mRNA expression in macrophages after Yersinia enterocolitica infection. Role of different Yop effectors. J. Biol. Chem. 277, 25133–25142 (2002).
Kim, J. J. et al. Helicobacter pylori impairs DNA mismatch repair in gastric epithelial cells. Gastroenterology 123, 542–553 (2002).
Iyer, R. R., Pluciennik, A., Burdett, V. & Modrich, P. L. DNA mismatch repair: functions and mechanisms. Chem. Rev. 106, 302–323 (2006).
Marteijn, J. A., Lans, H., Vermeulen, W. & Hoeijmakers, J. H. Understanding nucleotide excision repair and its roles in cancer and ageing. Nat. Rev. Mol. Cell Biol. 15, 465–481 (2014).
Robertson, A. B., Klungland, A., Rognes, T. & Leiros, I. DNA repair in mammalian cells: base excision repair: the long and short of it. Cell Mol. Life Sci. 66, 981–993 (2009).
Jagger, B. W. et al. An overlapping protein-coding region in influenza A virus segment 3 modulates the host response. Science 337, 199–204 (2012).
Nemeroff, M. E., Barabino, S. M., Li, Y., 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, 991–1000 (1998).
Vreede, F. T., Chan, A. Y., Sharps, J. & Fodor, E. Mechanisms and functional implications of the degradation of host RNA polymerase II in influenza virus infected cells. Virology 396, 125–134 (2010).
Quinlivan, M. et al. Attenuation of equine influenza viruses through truncations of the NS1 protein. J. Virol. 79, 8431–8439 (2005).
Heaton, N. S. et al. In vivo bioluminescent imaging of influenza A virus infection and characterization of novel cross-protective monoclonal antibodies. J. Virol. 87, 8272–8281 (2013).
Heaton, B. E. et al. A CRISPR activation screen identifies a pan-avian influenza virus inhibitory host factor. Cell Rep. 20, 1503–1512 (2017).
We would like to thank H. Bogerd and B. Cullen (Duke University) for their help with the amiRNA northern blots. We would like to thank P. Palese (Mt. Sinai) for support and reagents during preliminary optimization experiments. We would also like to thank B. tenOever (Mt. Sinai) for his help in designing the amiRNA-expressing viruses. We are also grateful for contributions made by H. Froggatt (Duke University) in researching the literature on other pathogens that downregulate DNA MMR. The RNA-seq mapping pipeline was developed by David Sachs. N.S.H. is partially supported by NIH K22-AI116509-01, R21-AI133444-01, R01-HL142985, R01-AI137031 and the Duke School of Medicine Whitehead Scholarship. B.S.C. is supported by NIH training grant T32-CA009111. R.E.D. is supported by NIH training grant T32-GM007184-41. S.C. is supported by NIH grants R01AI074951, R01AI140539 and R01AI122749, and is a recipient of the Burroughs Wellcome Investigators in the Pathogenesis of Infectious Disease Award.
Duke University has filed a provisional patent for targeting DNA MMR as a method to enhance the growth of influenza vaccine strains.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–7.
Average Z-scores of all genes tested in both replicates of the primary siRNA screen, related to Fig. 2b,c.
Validation siRNA sequence information and results of statistical analysis of secondary siRNA screen, related to Figs. 2d and 2g.
Relative mRNA levels of DNA MMR genes at 9 h postinfection with WT PR8 in A549 and H441 cells compared to mock controls, related to Fig. 3d.
Raw read counts for all genes detected in RNA-seq of WT PR8-infected H441 cells with control or DNA MMR knockdown, related to Fig. 5i–k and Supplementary Fig. 4.
RNA-seq data and analysis for all genes induced >5-fold in WT PR8-infected H441 cells, related to Fig. 5i–k.
List of primers used for RT–qPCR analyses, related to Figs. 3d, 5c and 5l–o.
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Chambers, B.S., Heaton, B.E., Rausch, K. et al. DNA mismatch repair is required for the host innate response and controls cellular fate after influenza virus infection. Nat Microbiol 4, 1964–1977 (2019). https://doi.org/10.1038/s41564-019-0509-3