PMID- 37564646 OWN - NLM STAT- MEDLINE DCOM- 20230814 LR - 20240411 IS - 1664-3224 (Electronic) IS - 1664-3224 (Linking) VI - 14 DP - 2023 TI - RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response. PG - 1197356 LID - 10.3389/fimmu.2023.1197356 [doi] LID - 1197356 AB - INTRODUCTION: The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1alpha (IRE1alpha)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood. METHODS: To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells +/- an IRE1alpha RNAse inhibitor. RESULTS: We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1alpha and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer. DISCUSSION: We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1alpha-XBP1s pathway potentiates airway mucosal anti-viral responses. CI - Copyright (c) 2023 Qiao, Xu, Zhang, Yang and Brasier. FAU - Qiao, Dianhua AU - Qiao D AD - Department of Medicine, University of Wisconsin-Madison School of Medicine and Public Health (SMPH), Madison, WI, United States. FAU - Xu, Xiaofang AU - Xu X AD - Department of Medicine, University of Wisconsin-Madison School of Medicine and Public Health (SMPH), Madison, WI, United States. FAU - Zhang, Yueqing AU - Zhang Y AD - Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, United States. FAU - Yang, Jun AU - Yang J AD - Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, United States. FAU - Brasier, Allan R AU - Brasier AR AD - Department of Medicine, University of Wisconsin-Madison School of Medicine and Public Health (SMPH), Madison, WI, United States. AD - Institute for Clinical and Translational Research (ICTR), University of Wisconsin-Madison, Madison, WI, United States. LA - eng GR - UL1 TR002373/TR/NCATS NIH HHS/United States GR - R21 AI159702/AI/NIAID NIH HHS/United States GR - U01 AI136994/AI/NIAID NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20230726 PL - Switzerland TA - Front Immunol JT - Frontiers in immunology JID - 101560960 RN - EC 3.1.- (Endoribonucleases) RN - 0 (X-Box Binding Protein 1) RN - EC 2.7.11.1 (Protein Serine-Threonine Kinases) RN - 9008-11-1 (Interferons) RN - 0 (IRF1 protein, human) RN - 0 (Interferon Regulatory Factor-1) RN - EC 3.1.3.16 (DUSP10 protein, human) RN - EC 3.1.3.48 (Dual-Specificity Phosphatases) RN - EC 3.1.3.16 (Mitogen-Activated Protein Kinase Phosphatases) RN - 0 (XBP1 protein, human) SB - IM MH - Humans MH - *Endoribonucleases/genetics/metabolism MH - X-Box Binding Protein 1/genetics/metabolism MH - Protein Serine-Threonine Kinases/genetics/metabolism MH - *Respiratory Syncytial Virus Infections MH - Interferons/metabolism MH - Inflammation MH - Interferon Regulatory Factor-1/genetics/metabolism MH - Dual-Specificity Phosphatases/metabolism MH - Mitogen-Activated Protein Kinase Phosphatases/metabolism PMC - PMC10411192 OTO - NOTNLM OT - Cleavage Under Targets and Release Using Nuclease (CUT&RUN) OT - X-box binding protein 1 (XBP1) OT - innate immunity OT - inositol requiring enzyme (IRE1) OT - interferon regulatory factor 1 COIS- The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. EDAT- 2023/08/11 06:43 MHDA- 2023/08/14 06:42 PMCR- 2023/01/01 CRDT- 2023/08/11 04:03 PHST- 2023/03/30 00:00 [received] PHST- 2023/07/06 00:00 [accepted] PHST- 2023/08/14 06:42 [medline] PHST- 2023/08/11 06:43 [pubmed] PHST- 2023/08/11 04:03 [entrez] PHST- 2023/01/01 00:00 [pmc-release] AID - 10.3389/fimmu.2023.1197356 [doi] PST - epublish SO - Front Immunol. 2023 Jul 26;14:1197356. doi: 10.3389/fimmu.2023.1197356. eCollection 2023.