STRUCTURE-FUNCTION ANALYSIS OF ENVELOPED VIRUSES

长曲棍球(LAC)病毒是加州血清群的一员 布尼亚病毒科。Lac是一种包膜病毒，含有两种病毒 脂膜外的糖蛋白(G1和G2)。在 在这个包膜上，有三个单链核糖核酸基因组片段(L，M 和S)与核衣壳蛋白、N和 病毒聚合酶，L.利用冷冻剂的初步实验 电子显微镜产生了LAC病毒粒子的图像，低温- 保存在无定形冰中，包含结构信息不 出现在负色颗粒的图像中。这个冷冻机- 这项技术允许人们准备冷冻的、水合的和不沾染的 不同化学环境下的生物标本 消除常见的潜在伪影 传统的方法。数字化图像的计算机分析 已经得出了拉丁病毒具有 二十面体对称。目前的建议旨在研究 完整LAC病毒粒子结构与功能关系的研究 它的大分子成分由冷电子结合而成 显微镜、计算机图像重建和生化 分析。提案的第一部分旨在产生一个 拉丁美洲和加勒比病毒粒子的三维重建，包括 糖蛋白几何构型的描述 在中性和酸性pH值时出现尖峰。这些结构性数据将 提供有关病毒粒子稳定结构的信息 以及它在不同条件下的潜在不同构象 在低pH值下细胞融合发生的地方。第二部分： 建议确定的是三维排列的 完整的病毒粒子内有核衣壳。此信息可以是 用于了解病毒粒子的结构稳定性，该病毒粒子没有 基质蛋白，并推断病毒的可能机制 集合。建议的第三部分是研究分子 N蛋白与病毒核糖核酸的相互作用这些数据将 加深我们对病毒基因组包装的理解 以及N蛋白在细胞周期调控中的作用 转录和复制。

LaCrosse (LAC) virus is a member of the California serogroup of the Bunyaviridae. LAC is an enveloped virus, containing two viral glycoproteins (G1 and G2) external to a lipid membrane. Within this envelope, three single-stranded RNA genome segments (L, M and S) are complexed with the nucleocapsid protein, N and the viral polymerase, L. Preliminary experiments utilizing cryo- electron microscopy have resulted in images of LAC virions, cryo- preserved in amorphous ice, containing structural information not present in the images of negative stained particles. This cryo- technique allows one to prepare frozen, hydrated and unstained biological specimens in different chemical environments and to eliminate the potential artifacts generally encountered in conventional methods. Computer analysis of the digitized images has led to the tentative conclusion that the LAC virion possesses icosahedral symmetry. The present proposal is aimed at studying the structure-function relationships of the intact LAC virion and its macromolecular components by a combination of cryo-electron microscopy, computerized image reconstruction and biochemical analysis. The first part of the proposal is designed to produce a three-dimensional reconstruction of the LAC virion, including a description to the geometrical arrangement of the glycoprotein spikes at neutral and acidic pH. These structural data will provide information about the stable architecture of the virion as well as its potentially different conformations under conditions where cell fusion occurs at low pH. The second part of the proposal is to determine the 3-dimensional arrangement of the nucleocapsids within the intact virion. This information can be used to understand the structural stability of a virion which has no matrix protein and to deduce possible mechanisms for viral assembly. The third part of the proposal is to study the molecular interactions of N protein with viral RNA species. These data will enhance our understanding of the packaging of the viral genome and the potential role of N protein in the regulation of transcription and replication.