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

冠状病毒（CoV）是人类呼吸道感染的主要原因。肺病理学在由常见人类CoV引起的感染之间变化很大（例如，HCoV-229 E）和人畜共患CoV（例如，MERS-CoV）。人类冠状病毒诱导的病理学中菌株特异性差异的潜在机制仍然知之甚少。该项目旨在定义CoV调节的途径和分子及其与肺部CoV编码效应物的相互作用，为开发新的治疗策略提供框架，以更有效地治疗冠状病毒感染。使用先进的方法，包括人肺细胞培养系统，CoV反向遗传学，转录组学和单细胞mRNA和蛋白质-蛋白质相互作用分析，我们能够表明HCoV-229 E通过NF-κ B依赖性和非依赖性途径诱导广泛的全基因组宿主反应，这与经典的尼古丁驱动的炎症反应不同。在第一个资助期获得的数据表明，CoV以独特的（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CoV感染细胞中的作用。第二，我们确定了冠状病毒感染细胞的细胞脂质组，并确定（上调）特定的溶血磷脂作为重要的宿主细胞因子，所需的细胞内膜结构的形成所必需的病毒RNA合成。第三，我们研究了CoV复制酶基因编码的酶功能，并确定了它们参与（i）逃避人类细胞中的抗病毒宿主反应和/或（ii）调节细胞转录组。第四，我们发现一种全冠状病毒保守的辅助蛋白（在HCoV-229 E中称为ap 4）调节特定的细胞途径。在下一个资助期内，我们将使用HCoV-229 E和缺乏非必需活性的MERS-CoV突变体进一步确定特定CoV蛋白与细胞途径的功能相互作用（Aim 1）。为了扩展我们对人类CoV毒株特异性差异的分析，我们将系统地比较感染HCoV-229 E和MERS-CoV的特定人类肺细胞类型，重点关注（i）病毒复制，（ii）病毒和细胞基因组表达，以及（iii）细胞嗜性（Aim 2）。为了在信号传导水平上揭示CoV菌株特异性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通路组分的活化和相互作用（目的3）。总的来说，这些方法将使我们能够全面评估我们的中心假设，即人类冠状病毒进化出一组蛋白质，这些蛋白质塑造并从而抑制病毒诱导的宿主细胞反应（部分）。因此，未能适当地控制和/或平衡这些机制预期在由HCoV（的特定菌株）引起的感染中引起更严重的肺损伤。

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.