Project 4

摘要 为了预防致命的冠状病毒感染，我们建议研究冠状病毒的分子基础- 通过识别(I)信号通路，诱导肺水肿及其消退 导致严重的肺部疾病，以告知作为抗病毒候选的抑制剂；以及(Ii)病毒 毒力基因。这些基因的缺失将导致候选疫苗减毒。三个目标 目的：1.确定水肿症的诱发和消退因素 在冠状病毒感染期间。我们已经证明了SARS-CoV的E和3a蛋白以及E蛋白 MERS冠状病毒的5株包括两个与毒力有关的序列结构域，一个含有PDZ 结合基序(PBM)和另一个编码离子通道(IC)活性的基序。的约束性 SARS冠状病毒E蛋白PBM到含有PDZ基序的蛋白引起急性呼吸综合征 受感染动物的疾病综合症。项目管理的重要性很可能与它的 能够与400多种细胞蛋白结合，从而调节许多细胞信号 小路。我们将研究MERS冠状病毒中PBM之间的全蛋白质组相互作用，以及 蜂窝PDZ。这一相互作用组将为鉴定干扰蛋白的多肽奠定基础。 PBM-PDZ结合，使用多肽文库。炎性小体激活机制的研究进展 具有IC活性的MERS-CoV蛋白将被研究。浮肿消退有两个可能 酶活性：上皮钠通道活性(Ena+C)和移动的Na+/K+ATPase Na+离子从肺泡液进入间质，促进水分排出。我们展示了 SARS冠状病毒E蛋白与Na+/K+ATPase结合，并推测这种结合减少 Na+/K+ATPase活性，导致肺水肿；这将在本项目中进行研究。目标2. 我们建议识别与MERS-CoV致病相关的病毒和宿主非编码RNA 和肺部炎症，作为抗病毒和抗水肿策略的潜在靶点。目标3.至 研制MERS-CoV安全减毒活疫苗。MERS-CoVS的构建 传播有缺陷，并产生衰减的、传播能力强的rMERS- 提出了COVS算法。最大化候选疫苗的生物安全性和遗传稳定性 是该项目的主要目标。

SUMMARY To prevent deadly CoV infections we propose to study the molecular basis of coronavirus- induced lung edema and its resolution, through the identification of (i) Signaling pathways resulting in severe lung disease to inform inhibitors as antiviral candidates; and (ii) Virus virulence genes. Deletion of these genes will lead to attenuated vaccine candidates. Three aims are proposed: Aim 1. To determine the factors involved in edema induction and resolution during CoV infection. We have shown that both E and 3a proteins of SARS-CoV, and proteins E and 5 of MERS-CoV include two sequence domains involved in virulence, one containing a PDZ binding motif (PBM), and another one encoding ion channel (IC) activity. The binding of the SARS-CoV E protein PBM to proteins containing the PDZ motif causes Acute Respiratory Disease Syndrome in infected animals. The importance of the PBM is likely associated with its ability to bind to more than 400 cellular proteins and, therefore, to regulate many cell signaling pathways. We will study the whole-proteome interactions between PBMs in MERS-CoV, and cellular PDZs. This interactome will be the basis for the identification of peptides interfering with PBM-PDZ binding, using peptide libraries. The mechanism of inflammasome activation by MERS-CoV proteins with IC activity will be studied. Edema resolution is possible by two enzymatic activities: epithelial sodium channel activity (ENa+C) and Na+/K+ ATPase that move Na+ ions from the alveolar fluid into the interstitium promoting water elimination. We showed that SARS-CoV E protein binds Na+/K+ ATPase and have postulated that this binding reduces Na+/K+ ATPase activity, leading to lung edema; this will be investigated in this project. Aim 2. We propose to identify viral and host non-coding RNAs involved in MERS-CoV pathogenesis and lung inflammation, as potential targets in antiviral and anti-edema strategies. Aim 3. To develop safe live-attenuated vaccines for MERS-CoV. The construction of MERS-CoVs defective in propagation, and the generation of attenuated, dissemination competent rMERS- CoVs are proposed. Maximizing biosafety and genetic stability of the vaccine candidates are main goals of the project.