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: 10206579
• 负责人: Susan C. Baker
• 金额: $ 78.99万
• 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 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10206579
• 关键词: 2019-nCoV; Address; Animals; Antibodies; Antiviral Agents; Antiviral Therapy; 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; betacoronavirus; drug development; experience; 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：研究干扰素拮抗剂延迟对 SARS-CoV-2 的先天免疫反应 PI：Susan C. Baker，博士，洛约拉大学芝加哥斯特里奇医学院 该提案的目标是确定病毒干扰素拮抗剂在复制和复制过程中如何发挥作用。 冠状病毒的发病机制，特别是在严重急性呼吸综合征的复制过程中 冠状病毒 2（SARS-CoV-2）。冠状病毒 (CoV) 是一个多样化的正链 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传播 水库进入人类循环系统，导致严重的疾病和流行病，正如我们目前正在经历的那样 与 SARS-CoV-2。针对新出现的冠状病毒（包括 SARS-CoV）制定适当的保护措施 2，将取决于对冠状病毒与宿主相互作用的了解。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成分，可减少 dsRNA 被宿主模式识别受体 MDA5 识别的物种，延迟干扰素的诱导。我们 报道称，表达非活性形式 EndoU 的病毒与野生型病毒在非干扰素中的复制效率相同。 反应细胞。重要的是，EndoU 突变 CoV 在干扰素反应细胞中的复制激活了强大的 免疫反应，消除病毒复制并减少动物的发病机制。最近，我们 确定了 EndoU 活性的目标是含有负义 RNA 的聚尿苷，我们称之为 PUN 核糖核酸。 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MDA5。我们假设 EndoU 活动导致了延迟 对 SARS-CoV-2 复制的先天免疫反应。在此，我们建议研究其机制 EndoU 如何在 SARS-CoV-2 中发挥作用、EndoU 如何与复制酶复合物结合以及 PUN RNA 如何 有助于激活 MDA5。在目标 1 中，我们将评估 EndoU 和其他 IFN 拮抗剂的作用 原代人气道细胞中 I 型和 III 型 IFN 对 SARS-CoV-2 感染反应的调节剂 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 EndoU 失活与其他病毒蛋白 IFN 拮抗剂失活突变相结合的效果。在 目标 2 我们将描述并破坏冠状病毒复制酶复合物内的 EndoU 相互作用。结果 这些研究将指导从 CoV 复制酶复合物中破坏 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，我们将识别多聚尿苷区域 负义 RNA，称为 PUN RNA，是 EndoU 和 MDA5 识别所必需的。这些研究将 提供有关 EndoU 和 MDA5 如何识别 PUN RNA 的新信息。总体而言，这些研究将 定义核糖核酸内切酶如何充当毒力因子的新机制。这个新信息可以 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。