Antiviral innate immune responses to pathogenic coronaviruses in the nasal epithelium

项目总结/摘要 MERS-CoV（MERS）和SARS-CoV-2（SARS-2）是高致病性冠状病毒（CoV）， 在过去的20年里出现并引发了突发公共卫生事件。这两种致病性冠状病毒都是 β冠状病毒，虽然来自不同的谱系（分别为merbeco和sarbeco）。像其他 呼吸道病毒CoV进入呼吸道并在上呼吸道上皮中建立感染， 在那里它们遇到宿主的先天免疫防御。所有CoV都产生双链RNA（dsRNA）作为一种 它们的复制的副产物，并且这种中间体可以在宿主细胞中诱导三种先天免疫途径： 干扰素（IFN）的产生和信号传导、蛋白激酶R（PKR）途径和OAS/RNase L系统。 在下呼吸道细胞系中对MERS的研究表明，这种病毒特别善于逃避这些病毒。 dsRNA诱导的先天免疫途径。这与SARS-2形成对比，SARS-2激活IFN、PKR和RNase L路径。相对而言，很少有人研究这些途径在限制 上呼吸道MERS和SARS-2感染。此外，粘膜先天免疫防御，如 在鼻上皮中高度表达的抗菌肽（AMP）和一氧化氮（NO）， 最近被认为是抗病毒的，它们在CoV感染过程中的作用尚未被表征。同样地， 粘液产生和纤毛功能是上呼吸道上皮中的主要先天性免疫防御， 它们在限制SARS-2和MERS感染中的具体作用尚不清楚。有趣的是，MERS和SARS-2 鼻上皮细胞嗜性不同，MERS主要感染粘液分泌杯 细胞和SARS-2感染纤毛细胞，表明对这些病毒的先天免疫反应可能不同。我 我建议使用原代鼻上皮培养系统来表征这些先天免疫效应子功能 在MERS和SARS-2感染期间的上呼吸道。我假设以前被低估的 上皮先天防御，如AMP产生，NO合成和粘膜纤毛机制 与dsRNA一起限制MERS和SARS-2在鼻上皮中的复制和传播， 诱导先天免疫途径。我的第一个目标是利用一组SARS-2和MERS重组病毒 表达重要的病毒先天免疫拮抗剂的无活性形式，以表征激活和逃避 dsRNA诱导的先天性免疫和这些途径（细胞因子）激活的下游效应 生产、细胞死亡）。我的第二个目标是阐明纤毛和粘液功能在SARS-2和SARS-3中的作用。 MERS感染通过干扰这些先天过程，并将调查激活 以及上皮AMP和NO反应在MERS和SARS-2感染中的潜在抑制作用。的 提出的实验将开始表征上呼吸道中对致病性CoV的免疫应答 上皮，呼吸道病毒感染的主要部位，有可能确定新的目标， 抗病毒治疗可能对这些和未来的人畜共患冠状病毒有效。

Project Summary / Abstract MERS-CoV (MERS) and SARS-CoV-2 (SARS-2) are highly pathogenic coronaviruses (CoVs) that have emerged and caused public health emergencies in the past 20 years. Both of these pathogenic CoVs are betacoronaviruses, although from different lineages (merbeco and sarbeco, respectively). Like other respiratory viruses, CoVs enter the respiratory tract and establish an infection in the upper airway epithelium, where they encounter host innate immune defenses. All CoVs produce double-stranded RNA (dsRNA) as a byproduct of their replication, and this intermediate can induce three innate immune pathways in host cells: interferon (IFN) production and signaling, the protein kinase R (PKR) pathway, and the OAS/RNase L system. Studies of MERS in lower airway cell lines has shown that this virus is particularly adept at evading these dsRNA-induced innate immune pathways. This contrasts with SARS-2, which activates IFN, PKR, and RNase L pathways. Relatively little has been done to characterize the role that these pathways may play in limiting MERS and SARS-2 infection in the upper airway. Additionally, mucosal innate immune defenses such as antimicrobial peptides (AMPs) and nitric oxide (NO) that are highly expressed in the nasal epithelium have only recently been recognized as antiviral, and their role during CoV infection has yet to be characterized. Similarly, mucus production and ciliary function are primary innate immune defenses in the upper airway epithelium, and their specific roles in limiting SARS-2 and MERS infection is unclear. Interestingly, MERS and SARS-2 have different cellular tropisms in the nasal epithelium, with MERS predominantly infecting mucus-producing goblet cells and SARS-2 infecting ciliated cells, suggesting innate immune responses to these viruses may differ. I propose to use a primary nasal epithelial culture system to characterize these innate immune effector functions in the upper airway during MERS and SARS-2 infection. I hypothesize that previously underappreciated epithelial innate defenses such as AMP production, NO synthesis, and mucociliary mechanisms function to limit MERS and SARS-2 replication and spread in the nasal epithelium alongside dsRNA- induced innate immune pathways. My first aim will utilize a panel of SARS-2 and MERS recombinant viruses expressing inactive forms of important viral innate immune antagonists to characterize activation and evasion of dsRNA-induced innate immunity and downstream effects of activation of these pathways (cytokine production, cell death). My second aim will elucidate the role of ciliary and mucus function during SARS-2 and MERS infection by pharmacologically perturbing these innate processes, and will investigate the activation of and the potential inhibitory role of epithelial AMP and NO responses during MERS and SARS-2 infection. The experiments proposed will begin to characterize immune responses to pathogenic CoVs in the upper airway epithelium, the primary site of infection by respiratory viruses, with the potential to identify novel targets for antiviral therapeutics that could be effective against these and future zoonotic CoVs.