Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection
Bo Li, Sara M Clohisey, Bing Shao Chia, Bo Wang, Ang Cui, Thomas Eisenhaure, Lawrence D Schweitzer, Paul Hoover, Nicholas J Parkinson, Aharon Nachshon, Nikki Smith, Tim Regan, David Farr, Michael U Gutmann, Syed Irfan Bukhari, Andrew Law, Maya Sangesland, Irit Gat-Viks, Paul Digard, Shobha Vasudevan, Daniel Lingwood, David H Dockrell, John G Doench, J Kenneth Baillie, Nir Hacohen, Bo Li, Sara M Clohisey, Bing Shao Chia, Bo Wang, Ang Cui, Thomas Eisenhaure, Lawrence D Schweitzer, Paul Hoover, Nicholas J Parkinson, Aharon Nachshon, Nikki Smith, Tim Regan, David Farr, Michael U Gutmann, Syed Irfan Bukhari, Andrew Law, Maya Sangesland, Irit Gat-Viks, Paul Digard, Shobha Vasudevan, Daniel Lingwood, David H Dockrell, John G Doench, J Kenneth Baillie, Nir Hacohen
Abstract
Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.
Conflict of interest statement
The authors declare no competing interests.
Figures
References
- Shrestha S. S., Swerdlow D. L., Borse R. H., Prabhu V. S., Finelli L., Atkins C. Y., Owusu-Edusei K., Bell B., Mead P. S., Biggerstaff M., Brammer L., Davidson H., Jernigan D., Jhung M. A., Kamimoto L. A., Merlin T. L., Nowell M., Redd S. C., Reed C., Schuchat A., Meltzer M. I. Estimating the Burden of 2009 Pandemic Influenza A (H1N1) in the United States (April 2009-April 2010) Clinical Infectious Diseases. 2010;52(Supplement 1):S75–S82. doi: 10.1093/cid/ciq012.
- Simonsen Lone, Spreeuwenberg Peter, Lustig Roger, Taylor Robert J., Fleming Douglas M., Kroneman Madelon, Van Kerkhove Maria D., Mounts Anthony W., Paget W. John. Global Mortality Estimates for the 2009 Influenza Pandemic from the GLaMOR Project: A Modeling Study. PLoS Medicine. 2013;10(11):e1001558. doi: 10.1371/journal.pmed.1001558.
- Gao R, et al. Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus. N. Engl. J. Med. 2013;368:1888–1897. doi: 10.1056/NEJMoa1304459.
- Webster RG, Govorkova EA. H5N1 influenza–continuing evolution and spread. N. Engl. J. Med. 2006;355:2174–2177. doi: 10.1056/NEJMp068205.
- Yen HL, Webster RG. Pandemic influenza as a current threat. Curr. Top. Microbiol. Immunol. 2009;333:3–24.
- Bright RA, Shay DK, Shu B, Cox NJ, Klimov AI. Adamantane resistance among influenza A viruses isolated early during the 2005-2006 influenza season in the United States. J. Am. Med. Assoc. 2006;295:891–894. doi: 10.1001/jama.295.8.joc60020.
- Team NS-OIAHVI, et al. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N. Engl. J. Med. 2009;360:2605–2615. doi: 10.1056/NEJMoa0903810.
- Nicoll, A., Ciancio, B. & Kramarz, P. Observed oseltamivir resistance in seasonal influenza viruses in Europe interpretation and potential implications. Euro. Surveillance13, pii: 8025 (2008).
- Hayden FG, et al. Baloxavir Marboxil for uncomplicated influenza in adults and adolescents. N. Engl. J. Med. 2018;379:913–923. doi: 10.1056/NEJMoa1716197.
- Takashita, E. et al. Susceptibility of influenza viruses to the novel cap-dependent endonuclease inhibitor baloxavir marboxil. Front. Microbiol. 9, 3026 (2018).
- Vasin AV, et al. Molecular mechanisms enhancing the proteome of influenza A viruses: an overview of recently discovered proteins. Virus Res. 2014;185:53–63. doi: 10.1016/j.virusres.2014.03.015.
- Baillie JK. Targeting the host immune response to fight infection. Science. 2014;344:807–808. doi: 10.1126/science.1255074.
- Warfield, K. L. et al. Lack of selective resistance of influenza A virus in presence of host-targeted antiviral, UV-4B. Sci. Rep. 10.1038/s41598-019-43030-y (2019).
- Vercauteren Koen, Brown Richard J P, Mesalam Ahmed Atef, Doerrbecker Juliane, Bhuju Sabin, Geffers Robert, Van Den Eede Naomi, McClure C Patrick, Troise Fulvia, Verhoye Lieven, Baumert Thomas, Farhoudi Ali, Cortese Riccardo, Ball Jonathan K, Leroux-Roels Geert, Pietschmann Thomas, Nicosia Alfredo, Meuleman Philip. Targeting a host-cell entry factor barricades antiviral-resistant HCV variants from on-therapy breakthrough in human-liver mice. Gut. 2015;65(12):2029–2034. doi: 10.1136/gutjnl-2014-309045.
- Brass AL, et al. The IFITM proteins mediate cellular resistance to influenza A H1N1 Virus, West Nile Virus, and Dengue Virus. Cell. 2009;139:1243–1254. doi: 10.1016/j.cell.2009.12.017.
- Hao L, et al. Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature. 2008;454:890–893. doi: 10.1038/nature07151.
- Karlas A, et al. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature. 2010;463:818–822. doi: 10.1038/nature08760.
- König R, et al. Human host factors required for influenza virus replication. Nature. 2010;463:813–817. doi: 10.1038/nature08699.
- Shapira SD, et al. A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell. 2009;139:1255–1267. doi: 10.1016/j.cell.2009.12.018.
- Sui B, et al. The use of random homozygous gene perturbation to identify novel host-oriented targets for influenza. Virology. 2009;387:473–481. doi: 10.1016/j.virol.2009.02.046.
- Tran A T, Rahim M N, Ranadheera C, Kroeker A, Cortens J P, Opanubi K J, Wilkins J A, Coombs K M. Knockdown of specific host factors protects against influenza virus-induced cell death. Cell Death & Disease. 2013;4(8):e769–e769. doi: 10.1038/cddis.2013.296.
- Stertz S, Shaw ML. Uncovering the global host cell requirements for influenza virus replication via RNAi screening. Microbes Infect. 2011;13:516–525. doi: 10.1016/j.micinf.2011.01.012.
- Watanabe T, Watanabe S, Kawaoka Y. Cellular networks involved in the influenza virus life cycle. Cell Host Microbe. 2010;7:427–439. doi: 10.1016/j.chom.2010.05.008.
- Everitt AR, et al. IFITM3 restricts the morbidity and mortality associated with influenza. Nature. 2012;484:519–523. doi: 10.1038/nature10921.
- Chou Y-C, et al. Variations in genome-wide RNAi screens: lessons from influenza research. J. Clin. Bioinforma. 2015;5:2. doi: 10.1186/s13336-015-0017-5.
- König R, et al. Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell. 2008;135:49–60. doi: 10.1016/j.cell.2008.07.032.
- Brass AL, et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science. 2008;319:921–926. doi: 10.1126/science.1152725.
- Zhou H, et al. Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe. 2008;4:495–504. doi: 10.1016/j.chom.2008.10.004.
- Ma Y, et al. CRISPR/Cas9 screens reveal epstein-barr virus-transformed B cell host dependency factors. Cell Host Microbe. 2017;21:580–591.e7. doi: 10.1016/j.chom.2017.04.005.
- Marceau CD, et al. Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens. Nature. 2016;535:159–163. doi: 10.1038/nature18631.
- Savidis G, et al. Identification of zika virus and dengue virus dependency factors using functional genomics. Cell Rep. 2016;16:232–246. doi: 10.1016/j.celrep.2016.06.028.
- Park RJ, et al. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat. Genet. 2017;49:193–203. doi: 10.1038/ng.3741.
- Han J, et al. Genome-wide CRISPR/Cas9 screen identifies host factors essential for influenza virus replication. Cell Rep. 2018;23:596–607. doi: 10.1016/j.celrep.2018.03.045.
- Doench JG, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 2016;34:184–191. doi: 10.1038/nbt.3437.
- Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016;44:D457–D462. doi: 10.1093/nar/gkv1070.
- Tripathi S, et al. Meta- and orthogonal integration of influenza ‘oMICs’ data defines a role for UBR4 in virus budding. Cell Host Microbe. 2015;18:723–735. doi: 10.1016/j.chom.2015.11.002.
- Kolde R, Laur S, Adler P, Vilo J. Robust rank aggregation for gene list integration and meta-analysis. Bioinformatics. 2012;28:573–580. doi: 10.1093/bioinformatics/btr709.
- Rimmelzwaan GF, Baars M, Claas ECJ, Osterhaus ADME. Comparison of RNA hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro. J. Virol. Methods. 1998;74:57–66. doi: 10.1016/S0166-0934(98)00071-8.
- Huang I-C, et al. Influenza A virus neuraminidase limits viral superinfection. J. Virol. 2008;82:4834–4843. doi: 10.1128/JVI.00079-08.
- Luo M. Influenza virus entry. Adv. Exp. Med. Biol. 2012;726:201–221. doi: 10.1007/978-1-4614-0980-9_9.
- Merkulova M, et al. Mapping the H+ (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation. Sci. Rep. 2015;5:1–15. doi: 10.1038/srep14827.
- Miles AL, Burr SP, Grice GL, Nathan JA. The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular Iron levels. Elife. 2017;6:1–28. doi: 10.7554/eLife.22693.
- Yoshimori T, Yamamoto a, Moriyama Y, Futai M, Tashiro Y. Bafilomycin-a1, a specific inhibitor of vacuolar-type H+-Atpase, inhibits acidification and protein-degradation in lysosomes of cultured-cells. J. Biol. Chem. 1991;266:17707–17712.
- Ochiai H, Sakai S, Hirabayashi T, Shimizu Y, Terasawa K. Inhibitory effect of bafilomycin A1, a specific inhibitor of vacuolar-type proton pump, on the growth of influenza A and B viruses in MDCK cells. Antivir. Res. 1995;27:425–430. doi: 10.1016/0166-3542(95)00040-S.
- Stauffer S, et al. Stepwise priming by acidic pH and a high K+ concentration is required for efficient uncoating of influenza A virus cores after penetration. J. Virol. 2014;88:13029–13046. doi: 10.1128/JVI.01430-14.
- McGuire CM, Forgac M. Glucose starvation increases V-ATPase assembly and activity in mammalian cells through AMP kinase and phosphatidylinositide 3-kinase/Akt signaling. J. Biol. Chem. 2018;293:9113–9123. doi: 10.1074/jbc.RA117.001327.
- Lu, S., Sung, T., Lin, N., Abraham, R. T. & Jessen, B. A. Lysosomal adaptation: how cells respond to lysosomotropic compounds. PLoS ONE12, e0173771 (2017).
- Mauthe M, et al. Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy. 2018;14:1435–1455. doi: 10.1080/15548627.2018.1474314.
- Martina JA, Puertollano R. Protein phosphatase 2A stimulates activation of TFEB and TFE3 transcription factors in response to oxidative stress. J. Biol. Chem. 2018;293:12525–12534. doi: 10.1074/jbc.RA118.003471.
- Brozzi, A., Urbanelli, L., Germain, P. L., Magini, A. & Emiliani, C. hLGDB: a database of human lysosomal genes and their regulation. Database2013, (2013).
- Skehel JJ, Wiley DC. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu. Rev. Biochem. 2000;69:531–569. doi: 10.1146/annurev.biochem.69.1.531.
- Marwaha, R. & Sharma, M. DQ-Red BSA trafficking assay in cultured cells to assess cargo delivery to lysosomes. Bio. Protoc.7, e2571 (2017).
- Costello DA, Whittaker GR, Daniel S. Variations in pH sensitivity, acid stability, and fusogenicity of three Influenza Virus H3 subtypes. J. Virol. 2015;89:350–360. doi: 10.1128/JVI.01927-14.
- Fontana J, Cardone G, Heymann JB, Winkler DC, Steven AC. Structural changes in influenza virus at low pH characterized by cryo-electron tomography. J. Virol. 2012;86:2919–2929. doi: 10.1128/JVI.06698-11.
- Stegmann, T., Booy, F. P. & Wilschut, J. Effects of low pH on influenza virus. Activation and inactivation of the membrane fusion capacity of the hemagglutinin. J. Biol. Chem. 25, 17744–17749 (1987).
- Huotari J, Helenius A. Endosome maturation. EMBO J. 2011;30:3481–3500. doi: 10.1038/emboj.2011.286.
- Li S, et al. PH-ontrolled two-step uncoating of influenza virus. Biophys. J. 2014;106:1447–1456. doi: 10.1016/j.bpj.2014.02.018.
- Bélanger F, Stepinski J, Darzynkiewicz E, Pelletier J. Characterization of hMTr1, a human Cap1 2′-O-ribose methyltransferase. J. Biol. Chem. 2010;285:33037–33044. doi: 10.1074/jbc.M110.155283.
- Smietanski, M. et al. Structural analysis of human 2′-O-ribose methyltransferases involved in mRNA cap structure formation. Nat. Commun. 5, 4321 (2014).
- Bouloy M, Plotch SJ, Krug RM. Both the 7-methyl and the 2′-O-methyl groups in the cap of mRNA strongly influence its ability to act as primer for influenza virus RNA transcription. Proc. Natl Acad. Sci. USA. 1980;77:3952–3956. doi: 10.1073/pnas.77.7.3952.
- Wakai C, Iwama M, Mizumoto K, Nagata K. Recognition of cap structure by Influenza B Virus RNA polymerase is less dependent on the methyl residue than recognition by influenza A virus polymerase. J. Virol. 2011;85:7504–7512. doi: 10.1128/JVI.02375-10.
- Lutz A, Dyall J, Olivo PD, Pekosz A. Virus-inducible reporter genes as a tool for detecting and quantifying influenza A virus replication. J. Virol. Methods. 2005;126:13–20. doi: 10.1016/j.jviromet.2005.01.016.
- Bukhari SIA, et al. A specialized mechanism of translation mediated by FXR1a-associated microRNP in cellular quiescence. Mol. Cell. 2016;61:760–773. doi: 10.1016/j.molcel.2016.02.013.
- Züst R, et al. Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nat. Immunol. 2011;12:137–143. doi: 10.1038/ni.1979.
- Schuberth-Wagner C, et al. A conserved histidine in the RNA sensor RIG-I controls immune tolerance to N1-2′O-methylated Self RNA. Immunity. 2015;43:41–52. doi: 10.1016/j.immuni.2015.06.015.
- Loo YM, Gale M. Immune signaling by RIG-I-like receptors. Immunity. 2011;34:680–692. doi: 10.1016/j.immuni.2011.05.003.
- Sabeti PC, et al. Genome-wide detection and characterization of positive selection in human populations. Nature. 2007;449:913–918. doi: 10.1038/nature06250.
- Matsuoka Yukiko, Matsumae Hiromi, Katoh Manami, Eisfeld Amie J, Neumann Gabriele, Hase Takeshi, Ghosh Samik, Shoemaker Jason E, Lopes Tiago JS, Watanabe Tokiko, Watanabe Shinji, Fukuyama Satoshi, Kitano Hiroaki, Kawaoka Yoshihiro. A comprehensive map of the influenza A virus replication cycle. BMC Systems Biology. 2013;7(1):97. doi: 10.1186/1752-0509-7-97.
- Yang CH, et al. Influenza A virus upregulates PRPF8 gene expression to increase virus production. Arch. Virol. 2017;162:1223–1235. doi: 10.1007/s00705-016-3210-3.
- Jansen JC, et al. CCDC115 deficiency causes a disorder of Golgi homeostasis with abnormal protein glycosylation. Am. J. Hum. Genet. 2016;98:310–321. doi: 10.1016/j.ajhg.2015.12.010.
- Jansen JC, et al. TMEM199 deficiency is a disorder of golgi homeostasis characterized by elevated aminotransferases, alkaline phosphatase, and cholesterol and abnormal glycosylation. Am. J. Hum. Genet. 2016;98:322–330. doi: 10.1016/j.ajhg.2015.12.011.
- Ballabio A, et al. TFEB Links Autophagy to Lysosomal Biogenesis. Science. 2011;332:1429–1433. doi: 10.1126/science.1204592.
- Perreira JM, et al. RNASEK Is a V-ATPase-associated factor required for endocytosis and the replication of rhinovirus, influenza A virus, and dengue virus. Cell Rep. 2015;12:850–863. doi: 10.1016/j.celrep.2015.06.076.
- Decressac M., Mattsson B., Weikop P., Lundblad M., Jakobsson J., Bjorklund A. TFEB-mediated autophagy rescues midbrain dopamine neurons from -synuclein toxicity. Proceedings of the National Academy of Sciences. 2013;110(19):E1817–E1826. doi: 10.1073/pnas.1305623110.
- Rega Laura R., Polishchuk Elena, Montefusco Sandro, Napolitano Gennaro, Tozzi Giulia, Zhang Jinzhong, Bellomo Francesco, Taranta Anna, Pastore Anna, Polishchuk Roman, Piemonte Fiorella, Medina Diego L., Catz Sergio D., Ballabio Andrea, Emma Francesco. Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells. Kidney International. 2016;89(4):862–873. doi: 10.1016/j.kint.2015.12.045.
- Pastore Nunzia, Blomenkamp Keith, Annunziata Fabio, Piccolo Pasquale, Mithbaokar Pratibha, Maria Sepe Rosa, Vetrini Francesco, Palmer Donna, Ng Philip, Polishchuk Elena, Iacobacci Simona, Polishchuk Roman, Teckman Jeffrey, Ballabio Andrea, Brunetti‐Pierri Nicola. Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha‐1‐anti‐trypsin deficiency. EMBO Molecular Medicine. 2013;5(3):397–412. doi: 10.1002/emmm.201202046.
- Carr CM, Kim PS. A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell. 1993;73:823–832. doi: 10.1016/0092-8674(93)90260-W.
- Bouloy M, Plotch SJ, Krug RM. Globin mRNAs are primers for the transcription of influenza viral RNA in vitro. Proc. Natl Acad. Sci. USA. 2006;75:4886–4890. doi: 10.1073/pnas.75.10.4886.
- Dias A, et al. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature. 2009;458:914–918. doi: 10.1038/nature07745.
- Daffis S, et al. 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature. 2010;468:452–456. doi: 10.1038/nature09489.
- Habjan Matthias, Hubel Philipp, Lacerda Livia, Benda Christian, Holze Cathleen, Eberl Christian H., Mann Angelika, Kindler Eveline, Gil-Cruz Cristina, Ziebuhr John, Thiel Volker, Pichlmair Andreas. Sequestration by IFIT1 Impairs Translation of 2′O-unmethylated Capped RNA. PLoS Pathogens. 2013;9(10):e1003663. doi: 10.1371/journal.ppat.1003663.
- Zaraket H., Bridges O. A., Russell C. J. The pH of Activation of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Replication and Pathogenesis in Mice. Journal of Virology. 2013;87(9):4826–4834. doi: 10.1128/JVI.03110-12.
- Omoto, S. et al. Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil. Sci. Rep. 8, 9633 (2018).
- Maydeu-Olivares Albert. Thurstonian modeling of ranking data via mean and covariance structure analysis. Psychometrika. 1999;64(3):325–340. doi: 10.1007/BF02294299.
- Truesdell, S. S. et al. MicroRNA-mediated mRNA translation activation in quiescent cells and oocytes involves recruitment of a nuclear microRNP. Sci. Rep. 2, 842 (2012).
Source: PubMed