Priming and fusion activation of the SARS coronavirus spike glycoprotein

描述（由申请人提供）：严重急性呼吸系统综合症（SARS）是一种由冠状病毒（SARS- cov）引起的新出现的病毒性疾病。与其他病毒系统的情况相反，关于SARS-CoV进入宿主细胞的过程，特别是关于膜融合事件，仍有许多未解之谜。虽然SARS-CoV自首次暴发以来没有再次出现，但仍存在SARS-CoV或其他动物冠状病毒进一步暴发人畜共患疾病的持续威胁。对病毒进入的详细了解对于我们应对未来疫情的能力至关重要。我们提出了两个具体目标：1)确定蛋白水解裂解位点在SARS-CoV S2结构域控制膜融合中的作用。我们的初步结果表明，在SARS冠状病毒（SARS- cov） S蛋白的S2结构域中，一个新的蛋白水解裂解位点793-KPTKR-797在控制病毒融合中起着重要作用。该区域基本残基的突变导致胰蛋白酶介导的融合缺失，在该位置引入一个furin识别位点允许在缺乏ACE2 （SARS-CoV受体）的情况下融合，这表明在宿主范围内具有重要作用。我们还发现，R797的裂解也会通过肺表达的丝氨酸蛋白酶TMPRSS2和HAT引发膜融合。我们提议对S2切割位点（S2’）进行全面的诱变和生化研究，目的是了解不同蛋白酶对SARS-CoV S蛋白的选择性切割如何调节病毒进入过程中S的启动和融合激活。2)研究中性粒细胞弹性酶在SARS-CoV S2结构域裂解活化中的作用。我们还知道，SARS-CoV的进入可被弹性蛋白酶激活，这是一种感染增强机制，在SARS-CoV感染者肺部出现明显炎症反应的背景下尤为重要。我们的初步结果表明，中性粒细胞弹性酶也在S2'裂解位点附近的残基T795处裂解。我们建议对弹性蛋白酶裂解位点S2'进行全面研究，目的是了解弹性蛋白酶裂解SARS-CoV S蛋白如何影响病毒的进入和融合。这个项目的重点是sars冠状病毒，因为它的高度优先地位；然而，我们的工作可能直接适用于其他病毒系统。总的来说，我们的研究将揭示SARS-CoV融合激活和宿主范围的关键特征。值得注意的是，S2中切割位点的存在在整个冠状病毒科中是非常高度保守的，并且可能是普遍的特征；融合反应由不同的蛋白酶引发取决于不同的病毒和环境。这些研究将为SARS-CoV的发病机制提供一个模型，并了解冠状病毒如何克服物种特异性并进入新宿主。公共卫生相关性：由于2003年春季爆发了严重急性呼吸系统综合症（SARS），冠状病毒作为传染病病原体最近受到了广泛关注。虽然sars冠状病毒自首次暴发以来没有再次出现，但仍存在sars冠状病毒或其他新型冠状病毒进一步暴发的持续威胁。sars冠状病毒识别宿主细胞并与宿主细胞进行膜融合的能力是其宿主范围的主要因素。我们的研究旨在阐明sars -冠状病毒融合和进入的分子细节，这将作为SARS-CoV发病机制和新抗病毒药物开发的模型，并帮助我们了解冠状病毒如何克服物种特异性并进入新宿主。

DESCRIPTION (provided by applicant): Severe Acute Respiratory Syndrome (SARS) is a recently emerged viral disease caused by a coronavirus (SARS-CoV). In contrast to the situation with other viral systems, there remain many unanswered questions regarding the process of SARS-CoV entry into host cells, especially with regard to membrane fusion events. While SARS-CoV has not undergone re-emergence since the initial outbreak, there remains a continued threat of further zoonotic outbreaks of SARS-CoV, or other animal coronaviruses. A detailed understanding of virus entry will be essential in our ability to respond to future outbreaks. We propose two specific aims: 1) To determine the role of a proteolytic cleavage site in the SARS-CoV S2 domain controlling membrane fusion. Our Preliminary Results indicate that a novel proteolytic cleavage site 793-KPTKR-797 within the S2 domain of the SARS coronavirus (SARS-CoV) S protein is instrumental in controlling viral fusion. Mutation of basic residues in this region leads to a loss of trypsin-mediated fusion and the introduction of a furin recognition site in this position allows fusion in the absence of ACE2 (the SARS-CoV receptor) suggesting an important role in host range. We have also shown that cleavage at R797 also primes membrane fusion via the lung-expressed serine proteases TMPRSS2 and HAT. We propose a comprehensive mutagenic and biochemical study of the S2 cleavage site (S2'), with the goal of understanding how selective cleavage of the SARS-CoV S protein by different proteases regulate S priming and fusion activation during virus entry. 2) To characterize the role of neutrophil elastase on SARS-CoV activation via cleavage within the S2 domain. It is also known that SARS-CoV entry can be activated by elastase, a mechanism of infection enhancement that is especially important in the context of the pronounced inflammatory response seen in the lungs of SARS-CoV-infected individuals. Our Preliminary Results indicate that neutrophil elastase also cleaves in the vicinity of the S2' cleavage site, at residue T795. We propose a comprehensive study of the elastase cleavage site at S2', with the goal of understanding how cleavage of the SARS-CoV S protein by elastase impacts virus entry and fusion. This project has a focus on the SARS-CoV because of its high priority status; however our work is likely to be directly applicable to other virus systems. Overall our studies will reveal critical features of SARS-CoV fusion activation and host range. Notably the presence of a cleavage site within S2 are very highly conserved across the Coronaviridae, and are likely to be universal features; with the fusion reaction primed by different proteases depending on the individual virus and circumstances. These studies will provide a model for both pathogenesis of SARS-CoV, as well as an understanding how coronaviruses might overcome species-specificity and emerge into new hosts. PUBLIC HEALTH RELEVANCE: Coronaviruses have recently received much attention as agents of infectious disease, due to the outbreak of severe acute respiratory syndrome (SARS) in the spring of 2003. While the SARS-coronavirus has not undergone re-emergence since the initial outbreak, there remains a continued threat of further outbreaks of SARS-CoV, or of other novel coronaviruses. The ability of the SARS-coronavirus to recognize and undergo membrane fusion with host cells is a major factor in its host range. Our studies are designed to elucidate the molecular details of SARS-coronavirus fusion and entry, which will serve as a model for both pathogenesis of SARS-CoV and the development of new antiviral drugs, as well as to help us understand how coronaviruses might overcome species-specificity and emerge into new hosts.