key: cord-0298313-6wb1zhqm authors: Mishra, Shalabh; Raj, Athira S; Kumar, Akhilesh; Rajeevan, Ashwathi; Kumari, Puja; Kumar, Himanshu title: Innate Immune sensing of Influenza A viral RNA through IFI16 promotes pyroptotic cell death date: 2021-02-13 journal: bioRxiv DOI: 10.1101/2021.02.13.431067 sha: 23441474ecc3abfff9c6eaf250bf0ef99466563e doc_id: 298313 cord_uid: 6wb1zhqm Programmed cell death pathways are triggered by various stresses or stimuli, including viral infections. The mechanism underlying the regulation of these pathways upon Influenza A virus IAV infection is not well characterized. We report that a cytosolic DNA sensor IFI16 is essential for the activation of programmed cell death pathways in IAV infected cells. We have identified that IFI16 functions as an RNA sensor for influenza A virus by binding to genomic RNA. The activation of IFI16 triggers the production of type I, III interferons, and also other pro-inflammatory cytokines via the STING-TBK1 and Pro-caspase-1 signaling axis, thereby promoting cell death (apoptosis and pyroptosis in IAV infected cells). Whereas, IFI16 knockdown cells showed reduced inflammatory responses and also prevented cell mortality during IAV infection. These results demonstrate the pivotal role of IFI16-mediated IAV sensing and its essential role in activating programmed cell death pathways. The past century has witnessed several pandemics disrupting the socio-economic harmony of infections account for more than 5 million cases annually, severely affecting children and older 47 adults(4, 5). An effective therapeutic strategy against emerging influenza virus strains is 48 perplexing because it mutates very fast and subverts host immunity and cellular machinery. 49 However, novel therapeutic approaches targeting host factors, essential for establishing viral 50 infection, can prove to be more effective. (11) 135 assay was performed as described previously(33). Quantitative real-time reverse transcription-PCR: Total RNA was isolated using TRIzol 137 reagent (Ambion/Invitrogen) and was used to prepare cDNA using iscript cDNA synthesis kit 138 (Bio-Rad) following the manufacturer's protocol. Gene expression was estimated by 139 quantitative real-time PCR using SYBR green chemistry (Bio-Rad) and gene-specific Fluorescence-activated cell sorting Cytometry Analysis: Cells were stained with FITC 159 labeled Annexin V and propidium iodide (Invitrogen) based on the manufacturer's instructions. Stained cells were analysed using a FACS Aria III (Becton Dickinson), and data were analysed 161 by using FlowJo software (FlowJo, Ashland, OR, USA). down viral RNA expression was quantified using qPCR, as described previously (9). Caspase-1 Assay: Caspase 1 activity was determined using FAM-FLICA caspase 1 assay kit 199 (Immunochemistry Technologies) following the manufacturer's protocol. Annexin-PI FACS. We found that IFI16 knockdown cells were more than 80% resistant to 235 IAV-induced cell death ( Figure 1A, 1B, 1C, 1D, Supplementary figure 2B) . Notably, IFI16 236 knockdown cells were resistant to cell death induced by IAV but not by another RNA virus, Newcastle Disease virus (NDV), suggesting an IAV-specific, IFI16-dependent cell death. Taken together, these data suggest a critical role of IFI16 in the regulation of cell death/survival 239 during IAV infection. To gain mechanistic insights into IFI16-mediate cell death, we performed whole transcriptome Figure 2D) . We noticed that a large number of genes associated with apoptosis pathway were 251 dysregulated ( Figure 2E ). The mRNA expression of selected pro-and anti-apoptotic genes 252 was again confirmed by qRT-PCR ( Figure 2F) . These results suggest that IAV induced cell 253 death is mediated through type I interferons, pro-inflammatory cytokines, and caspases and is 254 IFI16-dependent. 256 We noticed that apart from upregulating apoptotic genes, knocking down of IFI16 also 257 downregulates interferon signaling and pro-inflammatory cytokine production pathways Next, we asked if binding of IFI16 to viral RNA also affects IAV replication; overexpression 301 of IFI16 led to a decrease in IAV RNA inside cells as well as in supernatant ( Figure 5B ). Similarly, Knockdown of IFI16 led to an increase in IAV infection ( Figure 5C ) in both A549 303 and in primary small airway epithelial cells (SAEC). Besides, levels of pro-inflammatory 304 cytokines also decreased in IFI16 knocked-down cells infected with IAV. To test the effect of IFI16's subcellular localization on its viral RNA sensing, we mutated 308 nuclear localization sequences (NLS) on the N-terminus of the IFI16 (Figure 6A ). NLS 309 mutation was able to restrict most of the (>70%) IFI16 protein to the cytosol ( Figure 6B) . Cells Insights gained from this study will improve our understanding of the mechanisms of regulation 369 of IAV pathogenesis and lead to identifying more possible targets for therapeutic intervention. The mutation made in the NLS sequence is marked in red. Are RNA Viruses Candidate 373 Agents for the Next Global Pandemic? A Review Why are RNA virus mutation rates so damn high? Molecular mechanisms of variation in influenza 376 viruses Estimates of 378 global seasonal influenza-associated respiratory mortality: a modelling study Mortality 381 associated with influenza and respiratory syncytial virus in the United States Cutting Edge: Influenza 384 A virus activates TLR3-dependent inflammatory and RIG-I-dependent antiviral responses in human 385 lung epithelial cells Influenza A Virus Panhandle Structure Is Directly 387 Involved in RIG-I Activation and Interferon Induction The intracellular 389 sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of 390 caspase-1 Virus Genomic RNA and Activates RIPK3-Dependent Cell Death A novel influenza A virus 394 mitochondrial protein that induces cell death Necrotic 396 Response to Low Pathogenic H9N2 Influenza Virus in Chicken Hepatoma Cells Compromised 399 respiratory function in lethal influenza infection is characterized by the depletion of type I alveolar 400 epithelial cells beyond threshold levels Influenza A Virus Infection Triggers 402 Pyroptosis and Apoptosis of Respiratory Epithelial Cells through the Type I Interferon Signaling 403 Pathway in a Mutually Exclusive Manner Influenza A mutant 405 viruses with altered NS1 protein function provoke caspase-1 activation in primary human 406 macrophages, resulting in fast apoptosis and release of high levels of interleukins 1beta and 18 Influenza A Virus NS1 Protein Inhibits 409 the NLRP3 Inflammasome The RNA-and 411 TRIM25-Binding Domains of Influenza Virus NS1 Protein Are Essential for Suppression of NLRP3 412 Inflammasome-Mediated Interleukin-1beta Secretion Cell death regulation during 414 influenza A virus infection by matrix (M1) protein: a model of viral control over the cellular survival 415 pathway Influenza virus PB1-F2 protein induces 417 cell death through mitochondrial ANT3 and VDAC1 Influenza A virus nucleoprotein 419 induces apoptosis in human airway epithelial cells: implications of a novel interaction between 420 nucleoprotein and host protein Clusterin IFI16 is an innate 422 immune sensor for intracellular DNA Acetylation modulates cellular distribution and DNA sensing 424 ability of interferon-inducible protein IFI16 IFI16 acts as a 426 nuclear pathogen sensor to induce the inflammasome in response to Kaposi Sarcoma-associated 427 herpesvirus infection Kaposi's sarcoma-429 associated herpesvirus latency in endothelial and B cells activates gamma interferon-inducible protein 430 16-mediated inflammasomes IFI16 senses DNA 432 forms of the lentiviral replication cycle and controls HIV-1 replication Human cytomegalovirus tegument protein pUL83 inhibits IFI16-435 mediated DNA sensing for immune evasion Listeria 437 monocytogenes induces IFNbeta expression through an IFI16-, cGAS-and STING-dependent pathway Nuclear IFI16 induction of IRF-3 signaling during herpesviral 440 infection and degradation of IFI16 by the viral ICP0 protein Interferon 443 gamma-inducible protein (IFI) 16 transcriptionally regulates type i interferons and other interferon-444 stimulated genes and controls the interferon response to both DNA and RNA viruses Interferon-447 inducible protein (IFI) 16 regulates Chikungunya and Zika virus infection in human skin fibroblasts Efficient 450 generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments Generation of recombinant influenza virus from plasmid 453 DNA Suppresses H5N1 Virus Replication by Targeting the Viral PB1 and Host CUEDC2 MAVS-mediated 457 apoptosis and its inhibition by viral proteins Trimmomatic: a flexible trimmer for Illumina sequence data Moderated estimation of fold change and dispersion for RNA-461 seq data with DESeq2 MicroRNA-30e-5p has 463 an Integrated Role in the Regulation of the Innate Immune Response during Virus Infection Kaposi's sarcoma-associated herpesvirus ORF45 interacts with 466 kinesin-2 transporting viral capsid-tegument complexes along microtubules ZBP1/DAI 469 ubiquitination and sensing of influenza vRNPs activate programmed cell death A549 cells were transfected with IFI16 Wt and IFI16 NLS* and infected with IAV (MOI 10) 542 for 24 h. The cell viability was determined using the MTT assay E) Relative expression of NP and IFNβ RNAs were measured in total RNA by qRT-PCR RIG-I were overexpressed in HEK293T cells and 545 then infected with IAV (MOI 10) for 24 h. RNA-IP was performed using Anti-flag M2 Affinity 546 gel, and expression of viral RNA in the IP-ied sample was analysed by qRT-PCR using Input 547 RNA for normalization 550 We thank Professor David Knipe for providing us IFI16 plasmid, R. Fouchier for providing the 551 A/PR8/H1N1 reverse genetics system, Professor Yan Yuan for providing the pCMV3Tag1a 552 plasmid. We thank IISER Bhopal for providing the Central Instrumentation Facility.