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; 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以及DEXD/H-box解旋酶，RIG-I和MDA5 Sense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 带有SARS-COV-2的树突状细胞（DC）或MF导致流产感染而没有病毒复制。相比之下， 与SARS-COV-2的正常人支气管上皮细胞（NHBE）感染可导致稳健的病毒复制。 SARS-COV-2感染两种细胞谱系都会产生与测量的类似的稳健宿主抗病毒反应 通过诱导1型干扰素（IFN1），干扰素刺激的基因（ISGS），TNF-A，IL-1，IL-6，IL-8，其他 细胞因子，趋化因子和其他促炎性介质。 Alu元素占人类基因组的10％。 Alu RNA在人类细胞中很丰富，因为 其重复性的性质可以形成双链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，alu rnas迅速 A-to-i由针对DSRNA的腺苷脱氨酶编辑，Adar。我们的初步研究表明严重 COVID-19疾病（COV-S）与两者中内源性Alu RNA的A至I丧失显着丧失有关 血液和肺部，而轻度的Covid-19疾病（COV-M）与A到I编辑的部分损失有关。感染 DC以及NHBE的内源性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传感器并刺激转录响应导致 ISGS，IL-6和IL-8的诱导。相反，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，这些结果表明我们建议解决以下假设。首先，未经编辑的Alu RNA是连续合成的，并且在细胞中存在高水平。如果未经编辑，则会形成DSRNA 刺激潜在的致病性抗病毒反应。但是，alu rnas经过了一对我的编辑，所以他们 无法形成DSRNA。响应病毒感染，这种连续周期因损失A到I而迅速破坏 ADAR编辑允许积累未经编辑的Alu DSRNA和刺激下游抗病毒宿主 回答。很容易推测这个唯一连续循环的主机的值是快速 刺激Alu DSRNA刺激抗病毒和促炎的宿主反应，以防止病毒感染 致病病毒颗粒的积累和扩散。 为了探讨这一假设，我们建议用SARS-COV-2感染MF，DC和NHBE，并遵循 使用RNA-seq和我们的计算丢失了内源性ALU RNA和宿主响应的A对I编辑 管道。我们还将确定刺激宿主反应的RNA是病毒起源还是ALU DSRNA。 在AIM II中，我们将调查未经编辑和编辑的Alu RNA刺激抗病毒反应并采用的能力 siRNA介导的Alu RNA敲低，证明了Alu RNA在宿主抗病毒反应中的直接作用。