Virus-Host interactions that drive and control respiratory epithelial injury in coronavirus infections

驱动和控制冠状病毒感染中呼吸道上皮损伤的病毒与宿主相互作用

• 批准号: 319880762
• 负责人: Professor Dr. Michael Kracht
• 金额: --
• 依托单位: Rudolf-Buchheim-Institut für Pharmakologie
• 依托单位国家: 德国
• 项目类别: Clinical Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/319880762?language=en
• 关键词: Virus; Host; interactions; drive; control

Coronaviruses (CoVs) are a major cause of respiratory tract infections in humans. Lung pathology varies greatly between infections caused by common human CoVs (e.g., HCoV-229E) and zoonotic CoVs (e.g., MERS-CoV). The underlying mechanisms for strain-specific differences in human CoV-induced pathology remain poorly understood. The project aims to define CoV-regulated pathways and molecules and their interactions with CoV-encoded effectors in the lung to provide a framework for developing novel therapeutic strategies to treat coronaviral infections more effectively. Using advanced methods, including human lung cell culture systems, CoV reverse genetics, transcriptomics and single cell mRNA and protein-protein interaction analyses we were able to show that HCoV-229E induces a profound and genome-wide host response through NF-κB-dependent and -independent pathways that differ from classical cytokine-driven inflammatory responses. The data obtained in the first funding period suggest that CoVs fine-tune both the NF-κB system and the unfolded protein response (UPR) in a unique (and virus strain-specific) manner, which prompted us to evaluate the effects of inhibitors directed at NF-κB or the UPR in CoV-infected cells. Second, we determined the cellular lipidome of CoV-infected cells and identified (upregulated) specific lysophospholipids as important host cell factors that are required for the formation of intracellular membrane structures necessary for viral RNA synthesis. Third, we investigated CoV replicase gene-encoded enzymatic functions and established their involvement in (i) evading antiviral host reponses in human cells and/or (ii) modulating the cellular transcriptome. Fourth, we found that an alphacoronavirus-wide conserved accessory protein (called ap4 in HCoV-229E) modulates specific cellular pathways. In the next funding period, we will further define the functional interactions of specific CoV proteins with cellular pathways using HCoV-229E and MERS-CoV mutants lacking non-essential activities (Aim 1). To expand our analysis of human CoV strain-specific differences, we will systematically compare specific human lung cell types infected with HCoV-229E and MERS-CoV, focusing on differences in (i) viral replication, (ii) viral and cellular genome expression, and (iii) cell tropism (Aim 2). To reveal CoV strain-specific differences at the level of signaling and to explore the therapeutic potential of small molecule inhibitors, we will investigate the activation and interactions of PERK and NF-κB pathway components at the protein level (Aim 3). Collectively, these approaches will allow us to evaluate comprehensively our central hypothesis that human CoVs evolved a set of proteins that shape and thereby dampen (parts of) the virus-induced host cell response. Accordingly, failure to appropriately control and/or balance these mechanisms is expected to cause more severe lung injury in infections caused by (specific strains of) HCoVs.

冠状病毒(CoV)是人类呼吸道感染的主要原因。常见的人类冠状病毒(如HCoV-229E)和人畜共患冠状病毒(如MERS-CoV)引起的感染之间的肺部病理差异很大。在人类冠状病毒诱导的病理中，毒株特异性差异的潜在机制仍然知之甚少。该项目旨在确定冠状病毒调控的途径和分子及其与肺中冠状病毒编码的效应器的相互作用，为开发更有效地治疗冠状病毒感染的新的治疗策略提供框架。利用先进的方法，包括人类肺细胞培养系统、冠状病毒反向遗传学、转录学和单细胞基因以及蛋白质-蛋白质相互作用分析，我们能够证明，人类冠状病毒-229E通过不同于经典的细胞因子驱动的炎症反应的不同于经典的细胞因子驱动的炎症反应的依赖和非依赖的途径来诱导深刻的和全基因组范围的宿主反应。在第一个资助期获得的数据表明，冠状病毒以一种独特的(和病毒株特异性的)方式微调NF-κB系统和未折叠蛋白反应(UPR)，这促使我们评估针对NF-κB或UPR的抑制剂在冠状病毒感染细胞中的效果。其次，我们确定了冠状病毒感染细胞的细胞脂质体，并确定(上调)特定的溶血磷脂是形成病毒RNA合成所需的细胞膜结构所必需的重要宿主细胞因子。第三，我们研究了冠状病毒复制酶基因编码的酶功能，并确定它们参与(I)逃避人类细胞的抗病毒宿主反应和/或(Ii)调节细胞转录组。第四，我们发现一种甲型冠状病毒范围内保守的辅助蛋白(在HCoV-229E中称为AP4)调节特定的细胞通路。在下一个资助阶段，我们将使用缺乏非必要活性的HCoV-229E和MERS-CoV突变体进一步定义特定CoV蛋白与细胞通路的功能相互作用(目标1)。为了扩大我们对人类CoV毒株特异性差异的分析，我们将系统地比较感染HCoV-229E和MERS-CoV的特定人类肺细胞类型，重点是(I)病毒复制，(Ii)病毒和细胞基因组表达，以及(Iii)细胞嗜性(目标2)的差异。为了揭示冠状病毒不同毒株在信号水平上的差异，并探索小分子抑制剂的治疗潜力，我们将在蛋白质水平上研究PERK和NF-κB通路组件的激活和相互作用(目标3)。总而言之，这些方法将使我们能够全面评估我们的核心假设，即人类冠状病毒进化出一组蛋白质，这些蛋白质塑造并因此抑制病毒诱导的宿主细胞反应的(部分)。因此，未能适当地控制和/或平衡这些机制预计会在由(特定毒株)HCoV引起的感染中导致更严重的肺损伤。