Investigating Interferon Antagonists in Delaying Innate Immune Responses to SARS-CoV-2

标题：研究干扰素拮抗剂延迟对SARS-CoV-2的天然免疫应答 PI：Susan C.贝克博士，洛约拉大学芝加哥斯特里奇医学院 本提案的目的是确定病毒干扰素拮抗剂如何在复制和 冠状病毒的发病机制，特别是在严重急性呼吸系统综合征复制期间 冠状病毒2（SARS-CoV-2）。冠状病毒（CoV）是一个不同的正义RNA病毒家族， 包括感染人类和动物宿主的致病菌株。冠状病毒多次从动物身上跳下 水库进入人体循环，造成严重的疾病和流行病，因为我们目前正在经历 感染SARS-CoV-2。针对新出现的冠状病毒，包括SARS冠状病毒，制定适当的保护措施， 2，将取决于获得对冠状病毒-宿主相互作用的理解。我们发现 内切核糖核酸酶（EndoU）是CoV复制酶复合物的高度保守组分， 被宿主模式识别受体MDA 5识别的物种，延迟干扰素的诱导。我们 报道，表达失活形式EndoU的病毒在IFN非-γ中与野生型病毒一样有效地复制， 反应细胞重要的是，干扰素应答细胞中EndoU突变体CoV的复制激活了稳定的免疫应答。 免疫反应，从而抑制病毒复制并减少动物的发病率。最近我们 鉴定了EndoU活性的靶点是含有负义RNA的聚尿苷，我们称之为PUN 核糖核酸这种PUN RNA的去除延迟了宿主识别的dsRNA种类的产生 模式识别受体MDA 5。我们假设EndoU活性有助于延迟 对SARS-CoV-2复制的先天免疫反应。在这里，我们建议调查的机制， EndoU如何在SARS-CoV-2中起作用，EndoU如何与复制酶复合物结合，以及PUN RNA如何在SARS-CoV-2中起作用。 有助于激活MDA 5。在目标1中，我们将评估EndoU和其他IFN拮抗剂作为抗肿瘤药物的作用。 原代人气道细胞中对SARS-CoV-2感染的I型和III型IFN反应的调节剂 在肠细胞中。我们将使用反向遗传学来产生具有无活性IFN拮抗剂的病毒，并评估 将EndoU的失活与其它病毒蛋白IFN拮抗剂的失活突变相结合的效果。在 目的2我们将描绘和破坏冠状病毒复制酶复合物内的EndoU相互作用。的结果 这些研究将指导从CoV复制酶复合物中破坏EndoU的策略， 激活对CoV感染的保护性免疫反应。在目标3中，我们将确定多聚尿苷的区域 反义RNA，称为PUN RNA，是EndoU和MDA 5识别所需的。这些研究将 提供了关于PUN RNA如何被EndoU和MDA 5识别的新信息。总的来说，这些研究将 为核糖核酸内切酶作为毒力因子的作用机制提供了新的定义。这些新信息可以 用于开发针对现有和新出现的冠状病毒的抗病毒疗法和疫苗。

TITLE: Investigating Interferon Antagonists in Delaying Innate Immune Responses to SARS-CoV-2 PI: Susan C. Baker, PhD, Loyola University Chicago Stritch School of Medicine The goal of this proposal is to determine how viral interferon antagonists function in the replication and pathogenesis of coronaviruses, particularly during replication of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses (CoVs) are a diverse family of positive-sense RNA viruses that include pathogenic strains infecting human and animal hosts. CoVs have repeatedly jumped from animal reservoirs into human circulation, causing severe disease and pandemics, as we are currently experiencing with SARS-CoV-2. Developing appropriate protective measures against emerging CoVs, including SARS-CoV- 2, will depend upon gaining an understanding of coronavirus-host interactions. We discovered that the endoribonuclease (EndoU), a highly conserved component of the CoV replicase complex, reduces dsRNA species recognized by host pattern recognition receptor MDA5, delaying the induction of interferon. We reported that viruses expressing an inactive form of EndoU replicate as efficiently as wild type virus in IFN non- responsive cells. Importantly, replication of EndoU mutant CoVs in interferon-responsive cells activate robust immune responses, which extinguishes virus replication and reduces pathogenesis in animals. Recently, we identified the target of EndoU activity to be poly-uridine containing negative sense RNA, which we term PUN RNA. This removal of the PUN RNA delays the generation of dsRNA species that are recognized by host pattern recognition receptor MDA5. We hypothesize that EndoU activity contributes to the delay in the innate immune response to SARS-CoV-2 replication. Here, we propose to investigate the mechanism of how EndoU acts in SARS-CoV-2, how EndoU associates with the replicase complex, and how PUN RNA contributes to activating MDA5. In Aim 1, we will evaluate EndoU and other IFN antagonists for their role as modulators of Type I and Type III IFN responses to SARS-CoV-2 infection in primary human airway cells and in enterocytes. We will use reverse genetics to generate viruses with inactive IFN antagonists and evaluate the effects of combining inactivation of EndoU with inactivating mutations of other viral protein IFN antagonists. In Aim 2 we will delineate and disrupt EndoU interactions within the coronavirus replicase complex. The results of these studies will guide strategies for disruption of EndoU from the CoV replicase complex, which would activate protective immune responses to CoV infections. In Aim 3, we will identify regions of poly-uridine negative-sense RNA, termed PUN RNA, required for recognition by EndoU and MDA5. These studies will provide new information on how PUN RNAs are recognized by EndoU and MDA5. Overall, these studies will define a new mechanism for how an endoribonuclease acts as a virulence factor. This new information can be used to develop antiviral therapies and vaccines against existing and emerging coronaviruses.