CORONAVIRUS ASSEMBLY

冠状病毒是一类特征明确的rna病毒家族，可感染 人类和广泛的家养动物物种。人类病毒是 导致约25%的上呼吸道感染的常见呼吸道病原体 呼吸道感染。冠状病毒可以攻击中枢神经系统 系统(CNS)，并可能与人类中枢神经系统疾病有关。 冠状病毒独特地聚集在内质之间的膜上 网状和高尔基体。长期目标是理解，在 分子水平，冠状病毒组装的机制。在建议的 体外和体内试验系统将被用来研究 病毒蛋白和RNA在组装中的作用和要求。这个 中心假设是冠状病毒颗粒的组装依赖于 关于(1)选择性地包裹病毒RNA，以及(2)一组特定的 囊化RNA(核衣壳)与病毒的相互作用 膜蛋白。M的细胞质结构域，定位于 在萌芽的隔间里，它的功能就像一个相互作用的“受体” 与核衣壳一起启动病毒粒子在这些膜上的组装。 该提案的具体目标是：(1)绘制和描述 牛冠状病毒(BCV)RNA上的包膜位点。A最近 体外RNA结合试验的建立及体内表达 打包的复制图标将用于标识和表征 BCV病毒RNA的包装要求。(2)绘制地图 与BCV核衣壳蛋白(N)相互作用的结构域(S) 核糖核酸。用于特定靶点的体外核糖核酸结合试验将与L相同 用于确定N与N的特定关联要求 病毒核糖核酸。BCV感染细胞中N的两种磷酸化形式将是 调查以深入了解磷酸化在组装中的作用。 (3)将结构域(S)定位在完整的膜蛋白(M)上 与核衣壳相互作用。M细胞质的假说 将通过测量结合来测试结构域功能，如“受体” 野生型M蛋白和突变形式的蛋白在体内对核衣壳的作用 嵌合蛋白的表达及多肽封闭的研究 核衣壳结合。

Coronaviruses are a well characterized family of RNA viruses that infect humans and a broad range of domestic animal species. The human viruses are common respiratory pathogens that cause approximately 25% of the upper respiratory infections. Coronaviruses can target to the central nervous system (CNS) and may be associated with human CNS disease. Coronaviruses uniquely assemble at membranes between the endoplasmic reticulum and Golgi. The long term objective is to understand, at the molecular level, the mechanism of coronavirus assembly. In the proposed studies both in vitro and in vivo assay systems will be used to study the role and requirements of the viral proteins and RNA in assembly. The central hypothesis is that assembly of coronavirus particles is dependent on (1) selective encapsidation of the viral RNA, and (2) a specific set of interactions between the encapsidated RNA (nucleocapsid) and the viral membrane proteins. The cytoplasmic domain of M, the protein that localizes in the budding compartment, functions like a "receptor" that interacts with the nucleocapsid to initiate assembly of virions at these membranes. The specific aims of the proposal are: (1) To map and characterize the encapsidation site on the bovine coronavirus (BCV) RNA. A recently established in vitro RNA binding assay and in vivo expression of an RNA replicon that is packaged will be used to identify and characterize the requirements for encapsidation of the BCV viral RNA. (2) To map the domains (s) on the BCV nucleocapsid protein (N) that interact with the RNA. The same in vitro RNA binding assay used in specific aim l will be used to determine the requirements for specific association of N with the viral RNA. Two phosphorylated forms of N in BCV infected cells will be investigated to gain insight into the role of phosphorylation in assembly. (3) To map the domain(s) on the integral membrane protein (M) that interacts with the nucleocapsid. The hypothesis that the M cytoplasmic domain functions like a "receptor" will be tested by measuring the binding of wild-type M and mutant forms of the protein to nucleocapsids, in vivo expression of a chimeric protein and the use of a peptide to block nucleocapsid binding.