Loss of A-to-I editing stimulates SARS-CoV-2 anti-viral responses

A-to-I 编辑缺失会刺激 SARS-CoV-2 抗病毒反应

• 批准号: 10353022
• 负责人: Thomas M. Aune
• 金额: $ 25.95万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10353022
• 关键词: 2019-nCoV; Address; Alu Elements; Antiviral Response; Blood; COVID-19; Cell Line; Cell Lineage; Cells; Dangerousness; Dendritic Cells; Disease; Double-Stranded RNA; Elements; Epithelial Cells; Equilibrium; Gene Expression; Generations; Genes; Genetic Transcription; Genomics; Human; Human Genome; IL8 gene; Immune response; Infection; Inflammation Mediators; Inflammatory; Inflammatory Response; Interferons; Interleukin-1; Interleukin-6; Interruption; Kinetics; Lead; Lung; Measures; Mediating; NF-kappa B; Nature; Pathogenicity; Patients; Pattern recognition receptor; Play; RNA; RNA Editing; RNA chemical synthesis; Role; SARS-CoV-2 antiviral; SARS-CoV-2 infection; Signal Transduction; Small Interfering RNA; Structure; TLR3 gene; TNF gene; Testing; Viral; Virus Diseases; Virus Replication; bronchial epithelium; chemokine; computational pipelines; cytokine; dsRNA adenosine deaminase; helicase; knock-down; particle; prevent; programs; response; sensor; severe COVID-19; tissue culture; transcriptome sequencing; viral RNA

Host pattern recognition receptors TLR3, and the DExD/H-box helicases, RIG-I and MDA5 sense viral RNA and activate IRF and NF-kB transcription factors culminating in generation of host anti-viral responses. Infection of dendritic cells (DC) or mf with SARS-CoV-2 results in an abortive infection without viral replication. In contrast, infection of normal human bronchial epithelial cells (NHBE) with SARS-CoV-2 results in robust viral replication. Infection of both cell lineages with SARS-CoV-2 generates similar robust host anti-viral responses as measured by induction of type 1 interferons (IFN1), interferon-stimulated genes (ISGs), TNF-a, IL-1, IL-6, IL-8, other cytokines, chemokines and other pro-inflammatory mediators. Alu elements make up ~10% of the human genome. Alu RNAs are abundant in human cells and, because of their repetitive nature, can form double-stranded RNAs (dsRNA) and stimulate above-cited pattern recognition receptors and a strong anti-viral response in the absence of viral infection. To prevent this, Alu RNAs are rapidly A-to-I edited by adenosine deaminase specific for dsRNA, ADAR. Our preliminary studies show that severe COVID-19 disease (COV-S) is associated with marked loss of A-to-I editing of endogenous Alu RNAs in both blood and lung, while mild COVID-19 disease (COV-M) is associated with a partial loss of A-to-I editing. Infection of DC as well as NHBE causes a marked loss of A-to-I editing of endogenous Alu RNAs. Our preliminary studies show that unedited Alu RNAs activate host dsRNA sensors and stimulate transcriptional response leading to induction of ISGs, IL-6, and IL-8. In contrast, the same Alu RNAs, if edited, as is seen in healthy controls or mock-infected cells, fail to activate these gene expression programs. Taken together, these results suggest the following hypothesis we propose to address. First, unedited Alu RNAs are continuously synthesized and exist at high levels in cells. If unedited, Alu RNAs form dsRNAs that stimulate potentially pathogenic anti-viral responses. However, Alu RNAs are continuously A-to-I edited so they cannot form dsRNAs. In response to viral infection, this continuous cycle is rapidly disrupted by loss of A-to-I editing by ADAR allowing accumulation of unedited Alu dsRNAs and stimulation of downstream anti-viral host responses. It is tempting to speculate that the value to the host of this unique continuous cycle is to rapidly stimulate anti-viral and pro-inflammatory host responses by Alu dsRNAs in response to viral infection to prevent accumulation and spread of pathogenic viral particles. To explore this hypothesis, we propose to infect mf, DC, and NHBE with SARS-CoV-2 and follow kinetics of loss of A-to-I editing of endogenous Alu RNAs and host responses using RNA-seq and our computational pipelines. We will also determine if RNAs that stimulate host responses are of viral origin or are Alu dsRNAs. In aim II, we will investigate ability of unedited and edited Alu RNAs to stimulate anti-viral responses and employ siRNA-mediated knockdown of Alu RNAs to demonstrate a direct role of Alu RNAs in the host anti-viral response.

宿主模式识别受体 TLR3、DExD/H-box 解旋酶、RIG-I 和 MDA5 感知病毒 RNA 并激活 IRF 和 NF-kB 转录因子，最终产生宿主抗病毒反应。感染 树突状细胞 (DC) 或 SARS-CoV-2 的 mf 会导致感染流产，且不会出现病毒复制。相比之下， SARS-CoV-2 感染正常人支气管上皮细胞 (NHBE) 会导致病毒大量复制。 经测量，两种细胞系感染 SARS-CoV-2 都会产生相似的强大宿主抗病毒反应 通过诱导 1 型干扰素 (IFN1)、干扰素刺激基因 (ISG)、TNF-a、IL-1、IL-6、IL-8 等 细胞因子、趋化因子和其他促炎介质。 Alu 元素约占人类基因组的 10%。 Alu RNA 在人类细胞中含量丰富，因为 由于其重复性质，可以形成双链 RNA (dsRNA) 并刺激上述模式识别 在没有病毒感染的情况下，受体和强烈的抗病毒反应。为了防止这种情况发生，Alu RNA 被迅速 A-to-I 由 dsRNA、ADAR 特异性腺苷脱氨酶编辑。我们的初步研究表明，严重 COVID-19 疾病 (COV-S) 与两种疾病中内源 Alu RNA 的 A-to-I 编辑显着丧失相关 血液和肺部，而轻度 COVID-19 疾病 (COV-M) 与 A 到 I 编辑的部分丢失有关。感染 DC 和 NHBE 的缺失会导致内源 Alu RNA 的 A 至 I 编辑显着丧失。我们的初步研究 表明未经编辑的 Alu RNA 激活宿主 dsRNA 传感器并刺激转录反应，从而导致 诱导 ISG、IL-6 和 IL-8。相比之下，相同的 Alu RNA，如果进行编辑，如在健康对照或 模拟感染的细胞无法激活这些基因表达程序。 综上所述，这些结果表明我们提出了以下假设。首先，未经编辑的铝 RNA 不断合成并在细胞中以高水平存在。如果未经编辑，Alu RNA 会形成 dsRNA， 刺激潜在致病性抗病毒反应。然而，Alu RNA 不断进行 A 到 I 编辑，因此它们 不能形成 dsRNA。为了应对病毒感染，这种连续循环会因 A-to-I 的丢失而迅速中断 ADAR 编辑允许未经编辑的 Alu dsRNA 积累并刺激下游抗病毒宿主 回应。人们很容易推测，这种独特的连续循环对宿主的价值是快速 通过 Alu dsRNA 刺激宿主的抗病毒和促炎反应，以应对病毒感染，从而预防 致病病毒颗粒的积累和传播。 为了探索这一假设，我们建议用 SARS-CoV-2 感染 mf、DC 和 NHBE，并遵循其动力学 使用 RNA-seq 和我们的计算，内源 Alu RNA 和宿主反应的 A-to-I 编辑丢失 管道。我们还将确定刺激宿主反应的 RNA 是病毒来源的还是 Alu dsRNA。 在目标 II 中，我们将研究未经编辑和编辑的 Alu RNA 刺激抗病毒反应的能力，并利用 siRNA 介导的 Alu RNA 敲低，证明 Alu RNA 在宿主抗病毒反应中的直接作用。