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

项目摘要/摘要 冠状病毒病2019年(新冠肺炎)是由严重急性呼吸综合征冠状病毒-2(SARS-2)引起的。 冠状病毒(CoV-2)，已导致全球140多万人死亡。与其他高致病性流感不同 病毒，SARS-CoV-2感染的特点是I型干扰素水平低，前病毒产生过多。 患者体内的炎性细胞因子。虽然人们一直在努力了解该病的发病机制 这种病毒，SARS-CoV-2是如何能够逃避先天免疫反应的尚不清楚。的中心假说 这一推测是病毒的包膜(E)蛋白与BRD4以溴域依赖的方式相互作用 以破坏宿主先天炎症和抗病毒反应的诱导方式。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，其中包含 E蛋白中有两个赖氨酸残基。这一模型也得到了之前的研究的有力支持 BRD4是病毒感染过程中干扰素和炎症基因的重要转录共激活因子。这个 中心假设将在两个具体目标中得到检验：1)确定溴域在相互作用中的作用 SARS-CoV-2E蛋白和BRD4之间的关系工作假说是E蛋白的赖氨酸残基 是乙酰化的，并介导与BRD4的溴结构域的相互作用，作为组蛋白模拟物。我要测试一下 用无标记质谱仪和免疫沉淀鉴定E蛋白中的乙酰化残基 用域结构进行分析以表征交互作用。2)确定 SARS-CoV-2E蛋白与BRD4相互作用对病毒复制的影响工作假说是E蛋白 作为组蛋白模拟物，将BRD4从染色质中隔离出来，从而扰乱PRO-2的转录 炎症和干扰素基因。我将使用CRISPR/Cas9介导的BRD4基因敲除细胞系和BRD4 在受感染的细胞中进行重组研究，以测试这一模型。我的分析还将集中在转录 典型的促炎基因和干扰素基因的激活以及BRD4在染色质中的占据 RT-qPCR和ChIP-qPCR检测E蛋白的存在。我希望我提出的研究能为 我们对冠状病毒发病机制的基本认识，并提供了新的治疗靶点 SARS-CoV-2感染与免疫逃避。

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.