Viral Resistance and IFN Signaling in STAT2 Knockout Fish Cells Carola E. Dehler, Katherine Lester, Giulia Della Pelle, Luc Jouneau, Armel Houel, Catherine Collins, Tatiana Dovgan, This information is current as Radek Machat, Jun Zou, Pierre Boudinot, Samuel A. M. of September 26, 2021. Martin and Bertrand Collet J Immunol published online 29 May 2019 http://www.jimmunol.org/content/early/2019/05/28/jimmun ol.1801376 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/05/28/jimmunol.180137 Material 6.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 29, 2019, doi:10.4049/jimmunol.1801376 The Journal of Immunology Viral Resistance and IFN Signaling in STAT2 Knockout Fish Cells Carola E. Dehler,* Katherine Lester,† Giulia Della Pelle,‡ Luc Jouneau,‡ Armel Houel,‡ Catherine Collins,† Tatiana Dovgan,*,† Radek Machat,‡,1 Jun Zou,*,2 Pierre Boudinot,‡ Samuel A. M. Martin,* and Bertrand Collet†,‡ IFN belong to a group of cytokines specialized in the immunity to viruses. Upon viral infection, type I IFN is produced and alters the transcriptome of responding cells through induction of a set of IFN stimulated genes (ISGs) with regulatory or antiviral function, resulting in a cellular antiviral state. Fish genomes have both type I IFN and type II IFN (IFN-g), but no type III (l) IFN has been identified. Their receptors are not simple counterparts of the mammalian type I/II IFN receptors, because alternative chains are used in type I IFN receptors. The mechanisms of the downstream signaling remain partly undefined. In mammals, members of the signal transducer and activator of family of transcription factors are responsible for the transmission of the signal from cytokine Downloaded from receptors, and STAT2 is required for type I but not type II IFN signaling. In fish, its role in IFN signaling in fish remains unclear. We isolated a Chinook salmon (Oncorhynchus tshawytscha) cell line, GS2, with a stat2 gene knocked out by CRISPR/Cas9 genome editing. In this cell line, the induction of ISGs by stimulation with a recombinant type I IFN is completely obliterated as evidenced by comparative RNA-seq analysis of the transcriptome of GS2 and its parental counterpart, EC. Despite a complete absence of ISGs induction, the GS2 cell line has a remarkable ability to resist to viral infections. Therefore, other STAT2-independent pathways may be induced by the viral infection, illustrating the robustness and redundancy of the innate antiviral defenses in http://www.jimmunol.org/ fish. The Journal of Immunology, 2019, 203: 000–000. nterferon is a family of cytokines responsible for driving innate immune response. Specialized immune cells such as antiviral immune processes (1). Upon a viral infection, most lymphocytes produce type II (g-) IFN, initializing the adaptive I nucleated cells produce type I IFN directing the early antiviral immune response to viruses. Cells respond to IFN through a signaling process involving specific recognition and activation of *University of Aberdeen, AB24 2TZ Aberdeen, United Kingdom; †Marine Scotland, type I, II, and III IFN by specific receptor complexes expressed Marine Laboratory, AB11 9DB Aberdeen, United Kingdom; and ‡Virologie et Immu- at the cell surface: IFNAR1/2–Jak1–Tyk2, IFNGR1/2–Jak1/2, by guest on September 26, 2021 nologie Mole´culaires, Institut National de la Recherche Agronomique, Universite´ and IFNlR1/IL–10R2–Jak1/2–Tyk2, respectively (2, 3). The Paris-Saclay, 78352 Jouy-en-Josas cedex, France resulting conformational changes of the receptors create docking 1Current address: Department of Immunology, Veterinary Research Institute, Brno, Czech Republic. sites for key downstream signaling molecules, the STAT-1 or 2, 2Current address: The National Pathogen Collection Center of Aquatic Animals, that get phosphorylated at specific sites (Y701, S708, S727 for College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China. STAT1 and Y690 for STAT2). ORCIDs: 0000-0003-1732-9884 (C.E.D.); 0000-0002-8110-1932 (G.D.P.); 0000- The type I IFN receptor complex docks and phosphorylates both 0002-5094-7302 (L.J.); 0000-0002-9130-6261 (T.D.); 0000-0002-7220-6038 STAT1 and STAT2, which combine with IFN regulatory factor (R.M.); 0000-0003-2795-2653 (B.C.). (IRF)–9 to form a heterotrimeric complex termed IFN stimulated Received for publication October 11, 2018. Accepted for publication April 30, 2019. gene (ISG) factor 3 (ISGF3) that translocates into the nucleus and The work was partially funded by Biotechnology and Biological Sciences Re- activates IFN-stimulated response elements (ISRE) within the search Council/Natural Environment Research Council (NE/P010946/1), the Institut National de la Recherche Agronomique, and the Scottish Government promoters of a set of ISGs. The ISG consists of a distinct panel of (Marine Scotland). T.D. benefited from a Marine Alliance for Science and Technol- genes, often with direct antiviral or regulatory functions respon- ogy for Scotland small grant. G.D.P. benefited from an ERASMUS grant. R.M. was sible for the establishment of an antiviral state that blocks viral supported by project LO1218 of Ministry of Education, Youth and Sports of the Czech Republic under the NPUI program. replication and limits spread of the virus (4). The dataset presented in this article has been submitted to the National Center for In contrast, the type II IFN receptor complex exclusively docks Biotechnology Information (https://www.ncbi.nlm.nih.gov/sra/) under accession and phosphorylates STAT1, resulting in the formation of a STAT1/ number PRJNA495492. STAT1 homodimeric complex that translocates into the nucleus and Address correspondence and reprint requests to Dr. Bertrand Collet, Institut National de la activates IFN-g activation site (GAS) elements in the genome Recherche Agronomique, Jouy-en-Josas, France. E-mail address: [email protected] resulting in the induction of a GAS-specific set of genes (5). In The online version of this article contains supplemental material. fish, functional ISRE and GAS have been identified in promoter of Abbreviations used in this article: DEG, differentially expressed gene; dpi, day postin- a discrete number of ISGs (6–8). fection; EHNV, epizootic hematopoietic necrosis virus; elf1a, elongation factor 1 a; EMEM, Eagle’s MEM; FC, fold change; GAS, IFN-g activation site; GO, Gene On- The genome of early teleost fish was duplicated 320–350 million tology; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; m.o.i., multiplicity of years ago (9) whereas salmonid fish went through an additional infection; mEGFP, monomeric enhanced green fluorescence protein; NCBI, National ∼ Center for Biotechnology Information; NMD, nonsense-mediated mRNA decay; ORF, genome duplication event 100 million years ago (10–13). Al- open reading frame; QPCR, quantitative RT-PCR; RNA-seq, RNA sequencing; sgRNA, though many genes are lost after a whole genome duplication single guide RNA; SPDV, salmon pancreatic disease virus; VHSV, viral hemorrhagic event, salmonid IFN and ISG repertoires are complex, with septicemia virus. potentially four paralogous genes for each gene described in Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 mammals (14, 15). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801376 2 CHARACTERIZATION OF A STAT2 KNOCKOUT SALMONID CELL LINE Typical genes coding for type I and type II, but not type III, IFN carried out using the Q5 Taq polymerase (New England Biolabs). Each have been identified in fish genomes. In fact, IFN and many ISG sgRNA template was amplified in five 50-ml reactions each with 25 ml 3 genes have shown high rates of change during evolutionary time in 2 Q5 master mix, 400 nM DR274F forward primer (Table I), 40 nM STAT2R1-2 reverse primer, and 20 nM template STAT2T1-2 (see Table I). fish as in tetrapods, and the number of paralogs can be greatly Cycling was as follows: 98˚C for 30 s then 35 cycles of 98˚C for 5 s, 60˚C expanded or reduced to a single gene. Fish type I IFN genes were for 10 s, 72˚C for 10 s, and a final extension of 72˚C 30 s. The five re- prone to extensive diversification, especially in Salmonids (16), as actions were pooled and purified using QIAquick PCR purification kit is also observed in mammalian genomes (17). Importantly, fish (Qiagen) according to the manufacturer’s instructions and eluted in 50 ml water. The sgRNAs were synthesized using the RiboMAX Express T7 kit homologs of the key signaling factors downstream of IFNR, such (Promega) purified using TRIzol (Thermo Fisher Scientific) according to as STAT1/2 and IRF9, have been found, indicating that the general the manufacturers’ instructions. The