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

研究干扰素拮抗剂延迟对 SARS-CoV-2 的先天免疫反应

• 批准号: 10449132
• 负责人: Susan C. Baker
• 金额: $ 75.05万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-09 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10449132
• 关键词: 2019-nCoV; Address; Animals; Antibodies; Attenuated; Blood Circulation; Cells; Chicago; Complex; Coronavirus; Coronavirus Infections; Cryoelectron Microscopy; Disease; Disease Outbreaks; Doctor of Philosophy; Double-Stranded RNA; Endoribonucleases; Enterocytes; Excision; Family; Generations; Goals; Human; Immune; Immune response; Individual; Infection; Innate Immune Response; Interferometry; Interferons; Kinetics; Knowledge; Measures; Membrane; Mus; Mutation; Pathogenesis; Pathogenicity; Pattern recognition receptor; Pneumonia; Process; Proteins; RNA; RNA Viruses; Reporting; Role; Structure; Universities; Uridine; Vaccines; Viral; Viral Proteins; Virulence Factors; Virus; Virus Replication; antiviral drug development; betacoronavirus; experience; interferon antagonist; macrophage; medical schools; mutant; novel coronavirus; pandemic disease; preference; replicase; response; reverse genetics; viral RNA

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.

标题：调查干扰素拮抗剂在延迟对SARS-COV-2的先天免疫反应时 PI：苏珊·​​贝克（Susan C. Baker），洛约拉大学芝加哥斯特里奇医学院 该提案的目的是确定病毒干扰素拮抗剂在复制中的作用和 冠状病毒的发病机理，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（COVS）是一个多元化的阳性RNA病毒家族， 包括感染人和动物宿主的致病菌株。 COV反复从动物中跳了起来 水库进入人类循环，引起严重的疾病和大流行，因为我们目前正在经历 与SARS-COV-2。采取适当的保护措施，以防止新兴的COV，包括SARS-COV- 2，将取决于对冠状病毒 - 宿主相互作用的理解。我们发现 内切核酸酶（Endou）是COV复制酶配合物的高度保守成分，可降低dsRNA 通过宿主模式识别受体MDA5识别的物种，延迟了干扰素的诱导。我们 报道说，表达不活跃形式的内户的病毒在IFN非非野生型病毒中像野生型病毒一样有效 响应细胞。重要的是，在干扰素响应细胞中复制endou突变体COV激活了鲁棒 免疫反应，可以消除病毒复制并减少动物的发病机理。最近，我们 确定内向活性的目标是含有负性RNA的多尿尿素，我们将其称为双关语 RNA。双关语RNA的这种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Endou活动有助于延迟 对SARS-COV-2复制的先天免疫反应。在这里，我们建议研究 Endou如何在SARS-COV-2中作用，Endou如何与复制酶复合体相关 有助于激活MDA5。在AIM 1中，我们将评估Endou和其他IFN对抗者的角色 I型和III型IFN对SARS-COV-2感染的反应的调节剂， 在肠肠细胞中。我们将使用反向遗传学来生成具有非活性IFN拮抗剂的病毒，并评估 将endou与其他病毒蛋白IFN拮抗剂的灭活突变相结合的影响。在 AIM 2我们将描绘并破坏冠状病毒复制酶复合物中的内户相互作用。结果 这些研究将指导策略破坏COV复制酶络合物中的Endou，这将 激活对COV感染的保护性免疫反应。在AIM 3中，我们将确定多尿尿氨酸的区域 负态RNA，称为双关rNA，被Endou和MDA5识别所需的RNA。这些研究会 提供有关endou and MDA5如何识别双关语RNA的新信息。总体而言，这些研究将 定义一种新的机制，用于核糖核酸酶如何充当毒力因子。这个新信息可以是 用于开发针对现有和新兴冠状病毒的抗病毒疗法和疫苗。