CORONAVIRUS ASSEMBLY

冠状病毒是一个特征明确的RNA病毒家族， 人类和广泛的家畜物种。人类病毒是 常见的呼吸道病原体，导致约25%的上呼吸道疾病， 呼吸道感染冠状病毒可以靶向中枢神经 中枢神经系统（CNS）疾病，可能与人类CNS疾病有关。 冠状病毒独特地组装在内质网之间的膜上， 网壳和高尔基体。长期目标是了解， 分子水平，冠状病毒组装的机制。拟议 将使用体外和体内测定系统研究 病毒蛋白和RNA在装配中的作用和要求。的 核心假设是冠状病毒颗粒的组装依赖于 （1）病毒RNA的选择性折叠，和（2）一组特定的 核衣壳（Nucleocapsid）和病毒之间的相互作用 膜蛋白M的胞质结构域，定位于 在萌芽室中，功能就像一个“受体”， 与核衣壳一起启动病毒体在这些膜上的组装。 该提案的具体目标是：（1）绘制和描述 牛冠状病毒（BCV）RNA上的腺苷酸化位点。一个最近 建立了体外RNA结合测定和RNA的体内表达 包装的复制子将用于鉴定和表征 这是对BCV病毒RNA进行糖苷化的要求。(2)映射 在BCV核衣壳蛋白（N）上与 核糖核酸在特定目的中使用的相同体外RNA结合测定将是 用于确定N与 病毒RNA在被BCV感染的细胞中，N的两种磷酸化形式将被 研究以深入了解磷酸化在组装中的作用。 (3)为了绘制完整膜蛋白（M）上的结构域， 与核衣壳相互作用。假设M细胞质 结构域功能如“受体”将通过测量结合 野生型M和突变形式的蛋白质的核衣壳，在体内 嵌合蛋白的表达和肽阻断 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.