Assembly and release of the SARS coronavirus

产品说明：严重急性呼吸综合征（SARS）于2002年底在中国南部的广东省出现，并于2003年在全球范围内迅速蔓延，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内膜组装的不寻常特性：它们将其包膜蛋白靶向内质网-高尔基体中间隔室的膜，出芽进入该隔室的内腔，然后通过胞吐作用离开细胞。来自几种冠状病毒的包膜蛋白的靶向和组装已经得到了很好的研究，但是释放步骤还没有被检查。颗粒的有效释放对于经济的疫苗生产是必不可少的。来自模型冠状病毒的病毒样颗粒（VLP）通过两种单独的包膜蛋白（M和E）的共表达形成，并且当存在时掺入刺突蛋白（S）。然而，SARS VLP的形成要求存在争议。VLP疫苗在免疫原性和生产安全性方面提供了许多优势。VLP从产生它们的细胞的有效释放允许从上清液而不是均质化细胞纯化，这更简单并且产生具有更高纯度的制剂。我们的模型冠状病毒（传染性支气管炎病毒，IBV）的初步数据表明，小包膜蛋白E是需要有效的颗粒组装和有效的释放。IBV E蛋白的胞质尾区对于与M的相互作用和颗粒的组装是必需的。相反，IBVE的跨膜结构域是有效释放感染性病毒所必需的。在这里提出的实验中，我们将系统地确定组装和释放SARS VLP的要求，并测试SARS E蛋白在此过程中具有两个独立功能的想法。我们的具体目标是：（1）检验SARS E蛋白是VLP组装所必需的结构蛋白的假设;（2）检验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的病毒释放的理解，为提高疫苗生产的病毒释放效率提供框架，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.