Suppression of Host Antiviral Responses by a SARS-CoV-2 Histone Mimetic

SARS-CoV-2 组蛋白模拟物抑制宿主抗病毒反应

• 批准号: 10312686
• 负责人: Irene Po-Ru Chen
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312686
• 关键词: 2019-nCoV; Acetylation; Antiviral Agents; Antiviral Response; BRD2 gene; Binding; Biochemical Genetics; Biological Assay; Bromodomain; COVID-19; COVID-19 patient; CRISPR/Cas technology; Cell Line; Cells; Cessation of life; Chromatin; Coronavirus; Country; Data; Disease; Foundations; Fractionation; Genes; Genetic Transcription; Histone H3; Histones; IL6 gene; ISG15 gene; Immune; Immune Evasion; Immune response; Immunologics; Immunoprecipitation; Impairment; Infection; Inflammatory; Inflammatory Response; Innate Immune Response; Integration Host Factors; Interferon Type I; Interferons; Knock-out; Knowledge; Label; Lysine; Mass Spectrum Analysis; Mediating; Microscopy; Mission; Modeling; Molecular; Pathogenesis; Pathogenicity; Patients; Phenotype; Process; Production; Protein Family; Protein Microchips; Proteins; Proteomics; Reader; Research; Role; SARS-CoV-2 infection; SARS-CoV-2 pathogenesis; Sequence Analysis; Signal Pathway; System; Tertiary Protein Structure; Testing; Transcription Coactivator; Transcriptional Activation; United States National Institutes of Health; Viral; Viral Load result; Viral Pathogenesis; Viral Proteins; Virulence; Virus; Virus Diseases; Virus Replication; combat; cytokine; env Gene Products; experimental study; genetic approach; human coronavirus; influenzavirus; insight; member; mimetics; mutant; new therapeutic target; non-histone protein; novel; pointed protein; reconstitution; response; therapeutic target; therapeutically effective; transcription factor; virology; virus characteristic

PROJECT SUMMARY/ABSTRACT Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS- CoV-2) and has resulted in more than 1.4 million deaths globally. In contrast to other highly pathogenic influenza viruses, SARS-CoV-2 infection is characterized by low levels of type I interferons and over production of pro- inflammatory cytokines in patients. While concerted efforts have been made to understand the pathogenesis of this virus, how SARS-CoV-2 is able to evade the innate immune response is unclear. The central hypothesis of this proposal is that the envelope (E) protein of the virus interacts with BRD4 in a bromodomain-dependent manner to disrupt the induction of host innate inflammatory and antiviral responses. This hypothesis is supported by the recent proteomic study identifying this interaction and the presence of a histone H3-like motif containing two lysine residues in the E protein. This model is also strongly supported by previous studies demonstrating BRD4 as an important transcriptional coactivator of interferon and inflammatory genes during viral infection. The central hypothesis will be tested in two specific aims: 1) To define the role of bromodomains in the interaction between SARS-CoV-2 E protein and BRD4. The working hypothesis is that the lysine residues of the E protein are acetylated and mediate interaction with the bromodomains of BRD4, acting as a histone mimetic. I will test this model with label-free mass spectrometry to identify acetylated residues in E protein and immunoprecipitation assays with domain constructs to characterize the interaction. 2) To determine the functional relevance of the SARS-CoV-2 E protein and BRD4 interaction on the viral replication. The working hypothesis is that the E protein acts as a histone mimetic to sequester BRD4 from chromatin thereby disrupting the transcription of pro- inflammatory and interferon genes. I will use CRISPR/Cas9-mediated BRD4 knockout cell lines and BRD4 reconstitution studies in infected cells to test this model. My analysis will also focus on the transcriptional activation of canonical pro-inflammatory and interferon genes along with the chromatin occupancy of BRD4 in the presence of the E protein through RT-qPCR and ChIP-qPCR studies. I expect my proposed studies to inform our fundamental understanding of coronavirus pathogenesis and provide novel therapeutic targets to combat SARS-CoV-2 infection and immune evasion.

项目总结/摘要 2019年冠状病毒病（COVID-19）是由严重急性呼吸道综合征冠状病毒-2（SARS- CoV-2），并导致全球140多万人死亡。与其他高致病性流感相比， SARS-CoV-2感染的特征是I型干扰素水平低， 患者的炎症细胞因子。虽然已经做出了一致的努力来了解 这种病毒，SARS-CoV-2如何能够逃避先天免疫反应尚不清楚。的中心假设 该建议是病毒的包膜（E）蛋白以溴结构域依赖性的方式与BRD 4相互作用， 以破坏宿主先天性炎症和抗病毒反应的诱导。这一假设得到了支持 通过最近的蛋白质组学研究，确定了这种相互作用和组蛋白H3样基序的存在， 两个赖氨酸残基这一模型也得到了先前研究的有力支持， BRD 4在病毒感染过程中作为干扰素和炎症基因的重要转录共激活因子。的 中心假设将在两个具体目标中进行检验：1）确定溴结构域在相互作用中的作用 SARS-CoV-2 E蛋白与BRD 4蛋白的同源性。工作假设是E蛋白的赖氨酸残基 被乙酰化并介导与BRD 4的溴结构域的相互作用，充当组蛋白模拟物。我将测试 该模型与无标记质谱鉴定E蛋白中的乙酰化残基和免疫沉淀 用结构域构建体进行测定以表征相互作用。2)为了确定 SARS-CoV-2 E蛋白与BRD 4相互作用对病毒复制的影响。目前的假设是E蛋白 作为组蛋白模拟物从染色质中螯合BRD 4，从而破坏前- 炎症和干扰素基因。我将使用CRISPR/Cas9介导的BRD 4敲除细胞系和BRD 4 在感染细胞中进行重建研究以测试该模型。我的分析也将集中在转录 典型的促炎基因和干扰素基因的激活沿着BRD 4的染色质占据， 通过RT-qPCR和ChIP-qPCR研究确定E蛋白的存在。我希望我提出的研究报告 我们对冠状病毒发病机制的基本理解，并提供新的治疗靶点，以打击 SARS-CoV-2感染与免疫逃避