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

研究干扰素拮抗剂延缓SARS-CoV-2先天免疫反应 派：苏珊·贝克，博士，洛约拉大学芝加哥斯特里奇医学院 这项建议的目标是确定病毒干扰素拮抗剂在复制和 冠状病毒的发病机制，特别是在严重急性呼吸综合征复制期间 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。Cov一再从动物身上跳下 水库进入人类循环，造成严重的疾病和流行病，就像我们目前正在经历的那样 与SARS-CoV-2有关。针对新出现的冠状病毒制定适当的保护措施，包括SARS冠状病毒- 2，将取决于对冠状病毒-宿主相互作用的了解。我们发现， 内切核糖核酸酶(Endou)是冠状病毒复制酶复合体的一个高度保守的组成部分，它能还原dsRNA 由宿主模式识别受体MDA5识别的物种，延迟干扰素的诱导。我们 报道称，表达非活性内豆病毒的病毒在非干扰素中的复制效率与野生型病毒一样高。 响应性细胞。重要的是，Endou突变冠状病毒在干扰素反应细胞中的复制激活了强大的 免疫反应，消除病毒复制，减少动物的致病作用。最近，我们 确定Endou活性的靶点是含有负义RNA的多聚尿苷，我们称之为双关 核糖核酸。这种双关RNA的去除延迟了宿主识别的dsRNA物种的产生 模式识别受体MDA5。我们假设，恩杜活动导致了 SARS-CoV-2复制的先天免疫反应。在此，我们建议研究其作用机制。 Endou如何在SARS-CoV-2中发挥作用，Endou如何与复制酶复合体相关联，以及双关语RNA如何 有助于激活MDA5。在目标1中，我们将评估Endou和其他干扰素拮抗剂在 人原代呼吸道细胞和呼吸道细胞对SARS-CoV-2感染的I型和III型干扰素调节因子 在肠道细胞中。我们将使用反向遗传学产生含有非活性干扰素拮抗剂的病毒，并评估 Endou的失活与其他病毒蛋白干扰素拮抗剂的失活突变联合作用的效果。在……里面 目的2我们将描述和干扰冠状病毒复制酶复合体内的Endou相互作用。结果是 这些研究将指导从冠状病毒复制酶复合体中破坏Endou的策略，这将 激活对冠状病毒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 负义RNA，称为双关RNA，需要被Endou和MDA5识别。这些研究将 提供有关Endou和MDA5如何识别双关语RNA的新信息。总体而言，这些研究将 定义内切核糖核酸酶作为毒力因子的新机制。这一新信息可以 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.