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TLR 3和DExD/H-box解旋酶、RIG-I和MDA 5正义病毒RNA 并激活IRF和NF-κ B转录因子，最终产生宿主抗病毒应答。感染 树突状细胞（DC）或mf与SARS-CoV-2的结果是流产感染没有病毒复制。与此相反的是， 用SARS-CoV-2感染正常人支气管上皮细胞（NHBE）导致病毒的稳健复制。 用SARS-CoV-2感染两种细胞系产生了类似的强有力的宿主抗病毒应答， 通过诱导1型干扰素（IFN 1）、干扰素刺激基因（ISG）、TNF-α、IL-1、IL-6、IL-8、其他 细胞因子、趋化因子和其它促炎介质。 Alu元件占人类基因组的约10%。Alu RNA在人类细胞中很丰富， 它们的重复性质，可以形成双链RNA（dsRNA）并刺激上述模式识别 受体和强的抗病毒反应，在没有病毒感染。为了防止这种情况，Alu RNA迅速 由对dsRNA特异的腺苷脱氨酶，阿达尔编辑的A至I。我们的初步研究表明， COVID-19疾病（COV-S）与内源性Alu RNA的A-to-I编辑的显著丢失相关， 血液和肺，而轻度COVID-19疾病（COV-M）与A-to-I编辑的部分丧失有关。感染 DC以及NHBE的释放导致内源性Alu RNA的A至I编辑的显著损失。我们的初步研究 显示未编辑Alu RNA激活宿主dsRNA传感器并刺激转录应答，导致 诱导ISG、IL-6和IL-8。相比之下，相同的Alu RNA，如果被编辑，如在健康对照或 模拟感染的细胞，不能激活这些基因表达程序。 综上所述，这些结果表明了我们提出的以下假设。首先，未编辑的Alu RNA持续合成，并在细胞中以高水平存在。如果未经编辑，Alu RNA形成dsRNA， 刺激潜在致病性抗病毒反应。然而，Alu RNA是连续的A到I编辑，因此它们 不能形成dsRNA。在病毒感染时，这种连续的循环被A到I的丢失迅速破坏。 通过阿达尔编辑，允许未编辑的Alu dsRNA的积累和下游抗病毒宿主的刺激 应答人们很容易推测，这种独特的连续循环对宿主的价值是迅速地 通过Alu dsRNA响应病毒感染刺激抗病毒和促炎宿主应答， 病原性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，并跟踪其动力学。 使用RNA-seq和我们的计算方法， 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。 在目的II中，我们将研究未编辑和编辑的Alu RNA刺激抗病毒应答的能力，并采用 siRNA介导的Alu RNA敲低，以证明Alu RNA在宿主抗病毒应答中的直接作用。