STRUCTURE-FUNCTION ANALYSIS OF ENVELOPED VIRUSES

长曲棍球（LAC）病毒是加州血清群的成员， 布尼亚病毒科 LAC是一种包膜病毒，含有两种病毒 糖蛋白（G1和G2）外的脂质膜。 内 该包膜，三个单链RNA基因组片段（L，M 和S）与核衣壳蛋白、N和 病毒聚合酶，L. 初步实验利用低温- 电子显微镜已经产生了LAC病毒体的图像， 保存在无定形冰中，包含结构信息， 存在于负染色颗粒的图像中。 这种低温- 这项技术允许人们准备冷冻，水合和未染色的 不同化学环境中的生物标本， 消除了通常会遇到的潜在伪影 常规方法。 数字化图像的计算机分析 得出了一个初步的结论，即LAC病毒粒子拥有 二十面体对称 本建议旨在研究 完整LAC病毒粒子的结构-功能关系， 其大分子成分通过低温电子结合 显微镜、计算机图像重建和生物化学 分析. 该提案的第一部分旨在产生一个 LAC病毒体的三维重建，包括 糖蛋白几何排列的描述 峰值在中性和酸性pH值。这些结构数据将 提供有关病毒体稳定结构的信息， 以及它在一定条件下可能不同的构象 其中细胞融合在低pH下发生。 建议是确定三维排列的 完整病毒体中的核衣壳。 该信息可以 用于了解没有病毒粒子的病毒粒子的结构稳定性 基质蛋白，并推导出病毒感染的可能机制。 组装件. 第三部分建议是研究分子 N蛋白与病毒RNA种类的相互作用。 这些数据将 增强我们对病毒基因组包装的理解 以及N蛋白在调节 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.