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: 10615086
• 负责人: Thomas M. Aune
• 金额: $ 21.63万
• 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 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615086
• 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; 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 感染SARS-CoV-2的树突状细胞(DC)或MF可导致流产感染，而不会复制病毒。相比之下， SARS-CoV-2感染正常人支气管上皮细胞(NHBE)可导致病毒复制。 两种细胞系感染SARS-CoV-2后产生相似的强健宿主抗病毒反应 通过诱导1型干扰素(IFN1)、干扰素刺激基因(ISGs)、肿瘤坏死因子-α、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 由于它们的重复性，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，受体和强大的抗病毒反应。为了防止这种情况，Alu RNA迅速 A-to-I由dsRNA、ADAR特异的腺苷脱氨酶编辑。我们的初步研究表明，严重 新冠肺炎病(冠状病毒-S)与内源性Alu RNA A-to-I编辑显著丢失有关 血液和肺，而轻度新冠肺炎病(COV-M)与A-to-I编辑的部分丧失有关。感染 DC和NHBE的缺失导致内源Alu RNA的A-to-I编辑显著丧失。我们的初步研究 显示未经编辑Alu RNA激活宿主dsRNA传感器并刺激转录反应导致 诱导胰岛素样生长因子、白介素6和白介素8。相反，如果编辑，相同的Alu RNA，就像在健康对照或 模拟感染的细胞，无法激活这些基因表达程序。 综上所述，这些结果提出了我们建议解决的以下假设。首先，未编辑的Alu RNA是连续合成的，并以高水平存在于细胞中。如果未编辑，Alu RNA会形成dsRNA， 激发潜在的致病抗病毒反应。然而，Alu RNA是连续进行A到I编辑的，因此它们 无法形成dsRNA。作为对病毒感染的反应，这种连续的循环会被A与I的损失迅速打乱 由ADAR编辑，允许积累未编辑的Alu dsRNA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迅速 Alu dsRNAs在病毒感染时刺激抗病毒和促炎宿主反应，以防止 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，并遵循以下动力学 使用RNA-SEQ和我们的计算方法丢失内源Alu RNA和宿主反应的A-to-I编辑 管道。我们还将确定刺激宿主反应的RNA是病毒来源还是Alu dsRNAs。 在AIM 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，并使用 SiRNA介导的Alu RNA的敲除，以证明Alu RNAs在宿主抗病毒反应中的直接作用。