Antiviral innate immune responses to pathogenic coronaviruses in the nasal epithelium

项目摘要/摘要 MERS冠状病毒(MERS)和SARS-CoV-2(SARS-2)是高致病性冠状病毒(CoV)，具有 在过去的20年里出现并引发了突发公共卫生事件。这两种致病冠状病毒都是 贝塔冠状病毒，尽管来自不同的谱系(分别为Merbeco和Sarbeco)。像其他人一样 呼吸道病毒，冠状病毒进入呼吸道，在上呼吸道上皮细胞中建立感染， 在那里它们会遇到宿主的先天免疫防御。所有冠状病毒都会产生双链RNA(DsRNA)作为 它们复制的副产品，这种中间体可以在宿主细胞中诱导三条先天免疫途径： 干扰素的产生和信号传递，蛋白激酶R途径，以及美洲呼吸综合征/核糖核酸酶L系统。 对下呼吸道细胞系中MERS的研究表明，这种病毒特别擅长逃避这些 DsRNA诱导的先天免疫途径。这与SARS-2不同，SARS-2激活了干扰素、PKR和核糖核酸酶 L的小路。相对较少的工作是表征这些通路在限制 上呼吸道MERS和SARS-2感染。此外，粘膜的先天免疫防御系统，如 在鼻上皮中高表达的抗菌肽(AMPs)和一氧化氮(NO)只有 最近被认为是抗病毒药物，它们在冠状病毒感染过程中的作用尚未确定。同样， 粘液产生和睫毛功能是上呼吸道上皮的主要先天免疫防御，以及 它们在限制SARS-2和MERS感染方面的具体作用尚不清楚。有趣的是，MERS和SARS-2 鼻上皮中不同的细胞趋向性，MERS主要感染产生粘液的高脚杯 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 建议使用原代鼻腔上皮细胞培养系统来表征这些先天免疫效应功能 在MERS和SARS-2感染期间的上呼吸道。我假设之前低估了 上皮的天然防御，如AMP的产生、NO的合成和粘液纤毛机制 限制MERS和SARS-2复制并与dsRNA一起在鼻黏膜上皮扩散的作用 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 表达重要的病毒天然免疫拮抗剂的非活性形式来表征激活和逃避 DsRNA诱导的先天免疫和这些途径(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在SARS-2和SARS-2期间纤毛和粘液功能的作用 MERS感染通过药物扰乱这些先天过程，并将调查激活 以及上皮型AMP和NO在MERS和SARS-2感染过程中的潜在抑制作用。这个 拟议的实验将开始描述上呼吸道对致病冠状病毒的免疫反应。 上皮细胞，呼吸道病毒感染的主要部位，有可能确定新的靶点 可能对这些和未来的人畜共患病冠状病毒有效的抗病毒疗法。

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.