key: cord-0798466-ei41p2a7 authors: Comar, Courtney E.; Otter, Clayton J.; Pfannenstiel, Jessica; Doerger, Ethan; Renner, David M.; Tan, Li Hui; Perlman, Stanley; Cohen, Noam A.; Fehr, Anthony R.; Weiss, Susan R. title: MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells date: 2021-12-21 journal: bioRxiv DOI: 10.1101/2021.12.20.473564 sha: 5fb2f5a6c06d0cda233d660e39d6a3abac3c4342 doc_id: 798466 cord_uid: ei41p2a7 Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS-CoV is adept at antagonizing early innate immune pathways – interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L) – generated in response to viral double-stranded (ds)RNA generated during genome replication. This is in contrast to SARS-CoV-2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication. Importance Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes highly lethal respiratory disease. MERS-CoV encodes several innate immune antagonists, accessory proteins NS4a and NS4b unique to the merbeco lineage and the nsp15 protein endoribonuclease (EndoU), conserved among all coronaviruses. While mutation of each antagonist protein alone has little effect on innate immunity, infections with recombinant MERS-CoVs with mutations of EndoU in combination with either NS4a or NS4b, activate innate signaling pathways and are attenuated for replication. Our data indicate that EndoU and accessory proteins NS4a and NS4b together suppress innate immunity during MERS-CoV infection, to optimize viral replication. This is in contrast to SARS-CoV-2 which activates these pathways and consistent with greater mortality observed during MERS-CoV infection compared to SARS-CoV-2. Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, 28 causing highly lethal respiratory disease. The severity of disease may be in part because 29 MERS-CoV is adept at antagonizing early innate immune pathways -interferon (IFN) 30 production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L 31 (OAS/RNase L) -generated in response to viral double-stranded (ds)RNA generated during 32 genome replication. This is in contrast to SARS-CoV-2, which we recently reported activates 33 PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV processing, proteolytic processing of replicase proteins, and host innate immune antagonism. 95 The 3' one-third of the genome encodes the structural genes and the unique, lineage-specific 96 accessory genes, which are not necessary for viral replication but play important roles in 97 immune evasion and viral pathogenesis. and then phosphorylates translation initiation factor eIF2α leading to translation arrest (10). 113 Oligoadenylate synthetases (OAS1-3), upon detecting dsRNA, produce 2',5' -oligoadenylates 114 (2-5A) that activate the host enzyme ribonuclease L (RNase L) which cleaves viral and host 115 single-stranded (ss) RNA (11, 12) . Activation of each these pathways leads to restriction of virus 116 replication and spread. RNase L and PKR activation can promote IFN production, cellular 117 stress, inflammation, and/or apoptotic death (13) (14) (15) (16) (MHV) EndoU has been shown to limit dsRNA accumulation and consequently reduces IFN 150 production and signaling as well as activation of OAS/RNase L and PKR pathways during 151 infection of murine bone marrow-derived macrophages (33, 34) . While the exact mechanism of 152 action of EndoU is not completely understood, two different mechanisms have been reported. 153 One report concludes that EndoU cleaves genomic RNA to limit the production of dsRNA ( 168 and A549 DPP4 cells 169 In order to study the effects of EndoU activity on the dsRNA-induced antiviral innate immune 170 pathways during MERS-CoV infection, several recombinant mutants were constructed using the 171 Lambda Red recombination system and a Bacterial Artificial Chromosome (BAC) that encodes 172 the full-length MERS-CoV genome (37). Recombinant viruses (summarized in Fig 1A) were 173 MERS-CoV-nsp15 H231A with an amino acid substitution inactivating EndoU, MERS-CoV-DNS4a 174 with interruption of NS4a expression by insertion of two termination codons at positions 11 and 175 12, MERS-CoV-NS4b H182R with an amino acid substitution inactivating the PDE, and double 176 mutants MERS-CoV-nsp15 H231A /DNS4a and MERS-CoV-nsp15 H231A / NS4b H182R . We confirmed 177 that MERS-DNS4a and -nsp15 H231A /DNS4a infection produced undetectable expression of NS4a 178 protein by Western blot (Fig 1B) . As we observed previously, an NS4b H182R mutant expressed 179 less NS4b than WT MERS-CoV ( Fig 1B) CoV recombinant mutant viruses and collected total intracellular RNA at 24 and 48 hpi (Fig 3) . 229 We used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to quantify MERS-CoV, as well as ciliated epithelial cells). Thus, it presents an optimal system in which to 385 study viral immune evasion. We found that MERS-nsp15 H231A /∆NS4a and -nsp15 H231A /NS4b H182R 386 are attenuated for replication and induce the IFN pathway significantly as compared to WT 387 MERS-CoV in this culture system (Fig 6) . Thus, MERS-CoV nsp15 EndoU, NS4a, and NS4b 388 play pivotal roles in evading host innate immunity in the upper airways well as in lung-derived 389 cell lines. infection of both lung derived A549 cells and primary nasal cells (Fig 3, 6) . using the same Lenti-CRISPR system as previously described (51) TAAGCCCAGTGTACCAAGAG CCAGGTGGAAAGGTAAGAGG TTGGCATTAAGAGGTACACC TAATTTTTCTGTCCTGTCTC CAGTTGCTTAATTCCATTCC TCCAGTGTAGTAGAAGTACC TTAACAGCCCGGAATGGGAG ATGGACCCAAACGATGCCAT TCCCAAAAGTTGAAGGAGCA GAATCATTGTTAGGGTTCCG GGGCGCGAATTGTGTAACAA CCCTCAATGTGGAAGTTTTT TGATTGACTATTGCCTCCAG TTAAGCTAGAGGCTCTTGAA CTAGATCTGGAAGAGTTTCT CAGATGGACCTGGAGAAGTG AAGTAGATCACCTCCTACTG GCTTGTAGTCTGTTCAGAAG CTTTACTTTGCCAGACTCAA TGATTACTTTTGGCTGCGAT AACTTTTGGTGGAAGTGCGC AAGACCAAAAGCTTGCACCA GAAGATCACCAAAGTTTCCC TCAGTGCCGAGTTTATTCAA TCAGCAATTTGGGGCCAACG AAAAGCACTGGCTGTAGGAG GTTTAAATTGCGACATACCC TCATCATTGTTCTGATGGGT ACCGAAGGAAGTACACAGGG TCAAGTTTAATGGCTCCACT AGGTTCAGACATTTGGTCTG TGAGTGATGCTACCTTGCAC AACACTTGGACGGGTGCGAG ACATCAATCATTGGACCAGG SSC. Coverslips were mounted onto slides as stated above. DsRNA was detected using 585 commercial monoclonal antibody J2 (Scions) at 1:500 and nsp8 using anti-nsp8 rabbit serum (Applied Biosystems). cDNA was amplified using specific qRT-PCR primers (see Table 3 .3), 620 iQÔ SYBR Ò Green Supermix (Bio-Rad), and the QuantStudioÔ 3 PCR system (Thermo Fisher). Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia MERS-CoV in Upper Respiratory Tract and Lungs of Dromedary Camels Geographic Distribution of 658 MERS Coronavirus among Dromedary Camels A phylogenetically distinct Middle East respiratory syndrome coronavirus 663 detected in a dromedary calf from a closed dairy herd in Dubai with rising seroprevalence with 664 age Rooting the Phylogenetic Tree of Middle East Respiratory Syndrome 667 Coronavirus by Characterization of a Conspecific Virus from an African Bat A metagenomic viral discovery 671 approach identifies potential zoonotic and novel mammalian viruses in Neoromicia bats within 672 Further Evidence for Bats as the Evolutionary Source of 677 Middle East Respiratory Syndrome Coronavirus Murine Coronavirus Mouse Hepatitis Virus Is Recognized by MDA5 and Induces Type I Interferon in Brain Macrophages/Microglia. 680 RIG-I-like receptors: their regulation and roles in RNA 682 sensing Double-Stranded RNA Sensors and Modulators in Innate Immunity Ribonuclease L mediates the cell-lethal 687 phenotype of double-stranded RNA editing enzyme ADAR1 deficiency in a human cell line Viral encounters with 2',5'-oligoadenylate synthetase and RNase L during 690 the interferon antiviral response RNase L activates the NLRP3 inflammasome during viral infections PKR-dependent inflammatory signals Small self-RNA generated by 696 RNase L amplifies antiviral innate immunity Interferon action and apoptosis are 699 defective in mice devoid of 2',5'-oligoadenylate-dependent RNase L Activation of RNase L by Murine Coronavirus in Myeloid Cells Is Dependent 702 on Basal Oas Gene Expression and Independent of Virus-Induced Interferon SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in 708 respiratory epithelial-derived cells and cardiomyocytes Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of 712 MAVS Signaling Interaction of SARS and MERS Coronaviruses with 714 the Antiviral Interferon Response Antagonism 716 of dsRNA-Induced Innate Immune Pathways by NS4a and NS4b Accessory Proteins during 717 MERS Coronavirus Infection Middle East Respiratory Syndrome Coronavirus 720 NS4b Protein Inhibits Host RNase L Activation Inhibition of stress granule 722 formation by Middle East respiratory syndrome coronavirus 4a accessory protein facilitates viral 723 translation, leading to efficient virus replication Middle East respiratory syndrome coronavirus 726 accessory protein 4a is a type I interferon antagonist Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress 730 Responses Middle East Respiratory Syndrome Coronavirus 4a Protein Is a 733 Double-Stranded RNA-Binding Protein That Suppresses PACT-Induced Activation of RIG-I and 734 MDA5 in the Innate Antiviral Response Middle East respiratory 736 syndrome coronavirus ORF4b protein inhibits type I interferon production through both 737 cytoplasmic and nuclear targets MERS coronaviruses from camels in Africa exhibit 743 region-dependent genetic diversity MERS-CoV 4b protein 747 interferes with the NF-κB-dependent innate immune response during infection The 750 structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory 751 syndrome coronavirus (MERS-CoV) are potent interferon antagonists ORF4b-encoded accessory proteins of Middle East respiratory syndrome coronavirus and two 754 related bat coronaviruses localize to the nucleus and inhibit innate immune signalling Viral phosphodiesterases that antagonize RNA signaling to RNase L by degrading 2-5A Early endonuclease-mediated evasion 762 of RNA sensing ensures efficient coronavirus replication Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and 765 limits apoptosis in macrophages Physiologic RNA targets and refined sequence specificity 768 of coronavirus EndoU Coronavirus endoribonuclease targets viral 770 polyuridine sequences to evade activating host sensors Bacterial Artificial Chromosome-Based Lambda Red Recombination with the I-773 SceI Homing Endonuclease for Genetic Alteration of MERS-CoV nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation 777 and primer extension Defective Viral Genomes Alter How Sendai Virus Interacts 779 with Cellular Trafficking Machinery, Leading to Heterogeneity in the Production of Viral Particles 780 among Infected Cells Solitary chemosensory cells are a primary epithelial source of IL-25 in patients with chronic 784 rhinosinusitis with nasal polyps Bitter and sweet taste receptors regulate human upper respiratory innate immunity T2R38 taste receptor 792 polymorphisms underlie susceptibility to upper respiratory infection IFN-I response 795 timing relative to virus replication determines MERS coronavirus infection outcomes Coronavirus Endoribonuclease Activity in Porcine Epidemic Diarrhea 638 We thank the members of the Weiss