Assembly and release of the SARS coronavirus

描述：严重急性呼吸系统综合症（SARS）于2002年底在中国南部的广东省出现，并于2003年迅速在全球蔓延，死亡率很高。这种疾病是由一种新型冠状病毒（SARS CoV）引起的，开发疫苗和抗病毒疗法是当务之急。冠状病毒具有在细胞膜内聚集的不寻常特性：它们将包膜蛋白靶向内质网-高尔基中间室的膜，芽入该室的管腔，然后通过胞吐作用退出细胞。几种冠状病毒包膜蛋白的靶向和组装已经得到了很好的研究，但释放步骤尚未得到检验。有效释放颗粒对于经济生产疫苗至关重要。来自模型冠状病毒的病毒样颗粒（vlp）是通过单独表达两种包膜蛋白（M和E）而形成的，当刺突蛋白(S)存在时，将其合并。然而，SARS vlp形成的条件存在争议。VLP疫苗在免疫原性和生产安全性方面具有许多优点。从产生VLPs的细胞中有效释放VLPs可以从上清中纯化，而不是从均质细胞中纯化，后者更简单，纯度更高。我们对一种新型冠状病毒（传染性支气管炎病毒，IBV）的初步数据表明，小包膜蛋白E是有效的颗粒组装和有效释放所必需的。IBV E蛋白的细胞质尾部是与M相互作用和粒子组装所必需的。相比之下，IBV E的跨膜结构域是有效释放感染性病毒所必需的。在本文提出的实验中，我们将系统地确定SARS VLPs的组装和释放要求，并验证SARS E蛋白在这一过程中具有两种独立的功能。我们的具体目标是：(1)验证SARS E蛋白是VLP组装所必需的结构蛋白的假设；(2)验证SARS E蛋白通过促进完整颗粒的释放而发挥非结构性作用的假设。这些研究将增加我们对细胞内组装病毒释放的理解，为提高疫苗生产中病毒释放的效率提供框架，并确定抗病毒治疗的新靶点。

DESCRIPTION: Severe acute respiratory syndrome (SARS) emerged in the Guangdong province of southern China in late 2002, and rapidly spread worldwide in 2003 with significant mortality. The disease is caused by a novel coronavirus (SARS CoV), and developing vaccines and antiviral therapeutics are high priorities. Coronaviruses have the unusual property of assembling at intracellular membranes: they target their envelope proteins to membranes of the endoplasmic reticulum-Golgi intermediate compartment, bud into the lumen of this compartment, and then exit the cell by exocytosis. Targeting and assembly of the envelope proteins from several coronaviruses have been well studied, but the release step has not been examined. Efficient release of particles is essential for economical vaccine production. Virus-like particles (VLPs) from model coronaviruses are formed by co-expression of two of the envelope proteins alone (M and E), and the spike protein (S) is incorporated when present. However, the requirements for formation of SARS VLPs are controversial. VLP vaccines offer many advantages in terms of immunogenicity and safety of production. Efficient release of VLPs from the cells producing them allows purification from the supernatant rather than homogenized cells, which is simpler and yields preparations with higher purity. Our preliminary data for a model coronavirus (infectious bronchitis virus, IBV) suggest that the small envelope protein E is required for efficient particle assembly and for efficient release. The cytoplasmic tail of the IBV E protein is essential for interaction with M and assembly of particles. By contrast, the transmembrane domain of IBV E is required for efficient release of infectious virus. In the experiments proposed here, we will systematically determine the requirements for assembly and release of SARS VLPs, and test the idea that the SARS E protein has two separate functions in this process. Our specific aims are to (1) test the hypothesis that the SARS E protein is a structural protein that is essential for VLP assembly; and (2) test the hypothesis that the SARS E protein plays a nonstructural role by promoting release of intact particles. These studies will increase our understanding of release of viruses that assemble intracellularly, provide a framework for increasing the efficiency of virus release for vaccine production, and identify new targets for antiviral therapeutics.