MOLECULAR GENETICS OF HSV 1 CAPSIDS

描述：1型单纯疱疹病毒的遗传和生化研究 (HSV-1)衣壳病毒被提出。拟议实验的主要目标是 确定B衣壳期间衣壳结构的构象变化 成熟，以识别引起变化的分子中的残基， 并确定分子之间的相互作用，通过这些相互作用， 可能会被调停。衣壳由三种必需蛋白质组成； VP5是主要衣壳成分，VP19C-VP23是一种复合体，它 与VP5相互作用并使其稳定。UL26(VP24和21)及更多 丰富的UL26.5(22a)基因产物决定了蛋白酶活性(VP24)， 以及壳体通过其上组装的脚手架(22a和可能21 与VP5的交互。 特定目的1.已知相互作用的UL26和UL26.5基因产物的残基 对于VP5，将更改(1.1)。阻断蛋白酶的裂解 成熟部位，或(1.2)。防止与VP5发生必要的相互作用。 可能在VP5中的补偿性遗传变化将被选择，以允许 需要克服的障碍。 具体目标2.形状的变化(球形到二十面体)可能伴随着B 衣壳成熟。变异病毒可能被阻断在 在感染非允许细胞的过程中将检查成熟过程 通过电子显微镜，使用VP5特异性的细胞免疫荧光 抗体，并对衣壳蛋白进行了SDS-PAGE分析 沉淀物分析。 具体目标3.主要支架的大小、形状和低聚状态 将测定分子(22a)。酵母GCN4和HIV-1 gp160残基 可以使用指定基本亮氨酸拉链图案的 自我互动领域。突变病毒的跨优势将是 已评估。 具体目标4.将确定与VP5相互作用的VP26残基 使用VP26阴性衣壳与野生型和突变型 体外合成VP26。 具体目标5.将继续进行研究，以确定 用X-射线单晶衍射法测定了VP19C-VP23配合物。中的多个插入 VP19C编码序列将有助于鉴定VP19C的残基 与VP23和VP5交互。VP19C和VP19C体外合成产物 VP23将通过免疫共沉淀和衣壳结合试验进行检测， 分别进行了分析。 具体目的6.提出了将病毒DNA包装到衣壳中的实验， 并确定将DNA包装成衣壳的方向。

DESCRIPTION: A genetic and biochemical study of herpes simplex virus type 1 (HSV-1) capsids is proposed. Major goals of the proposed experiments are to identify conformation changes in capsid structure that occur during B capsid maturation, to identify residues in the molecules that cause the changes, and to identify interactions between the molecules through which the changes may be mediated. Capsid shells are composed of three essential proteins; VP5, the major capsid shell component, and VP19C-VP23, a complex which interacts with and stabilizes VP5. The UL26 (VP24 and 21) and the more abundant UL26.5 (22a) gene products specify the protease activity (VP24), and the scaffold (22a and probably 21) on which the shell is assembled via interactions with VP5. Specific Aim 1. Residues of UL26 and UL26.5 gene products known to interact with VP5 will be altered that (1.1). block protease cleavage at the maturation site, or (1.2). prevent essential interactions with VP5. Compensatory genetic changes, presumably in VP5, will be selected that allow the blocks to be overcome. Specific Aim 2. Changes in shape (spherical to icosahedral) may accompany B capsid maturation. Mutant viruses possibly blocked at steps in the maturation process will be examined during infection of non-permissive cells by electron microscopy, cellular immunofluorescence using a VP5 specific antibody, and by SDS-PAGE analysis of capsid proteins following sedimentation analysis. Specific Aim 3. The size, shape and oligomeric status of the major scaffold molecule (22a) will be determined. The yeast GCN4 and HIV-1 gp160 residues that specify basic leucine zipper motifs may be used to replace the self-interactive domain. The transdominance of mutant viruses will be evaluated. Specific Aim 4. Residues of VP26 that interact with VP5 will be identified using VP26-negative capsids incubated with wild-type and mutant forms of VP26 synthesized in vitro. Specific Aim 5. Studies will be pursued to determine the structure of the VP19C-VP23 complex by x-ray crystallography. A number of insertions in VP19C coding sequences will aid the identification of residues of VP19C that interact with VP23 and with VP5. In vitro synthesized products of VP19C and VP23 will be assayed by co-immunoprecipitation and a capsid binding assay, respectively. Specific Aim 6. Experiments are proposed to package viral DNA into capsids, and to determine the direction of packaging of DNA into capsids.