Nucleocapsid envelopment Herpes simplex virus-1

描述(申请人提供)：这些研究的长期目标是了解疱疹病毒核衣壳被膜的分子机制。所有疱疹病毒最初都是从受感染细胞的内核膜中萌发的，这表明从这些研究中获得的信息将适用于这个具有重要医学意义的病毒家族的所有成员。该应用侧重于单纯疱疹病毒1型UL31和UL34在包膜过程中的作用。对基于大量初步数据的假设进行了检验。第一种假说认为，UL31编码的蛋白质(PUL3t)与UL34基因产物(PUL34)相互作用，介导这两种蛋白质正确地靶向内核膜。为了评估体内相互作用的重要性，将测试携带缺少pUL34相互作用域的UL31基因的病毒突变体和缺少pUL31相互作用区的UL34突变体，以确定它们正确靶向感染细胞中的两种蛋白质和介导核衣壳被膜的能力。要检验的第二个假说试图了解pUL31核层关联的作用。核层是排列在核缘内表面的核细胞骨架或核基质的一部分。一个可能的作用是pUL31的核板结合介导了pUL31/pUL34复合体在核缘的锚定。因此，在瞬时表达分析中，将测试缺少片层结合结构域而含有取代这些结构域的已知片层结合结构域的UL31突变体是否能够介导pUL31和pUL34正确靶向核边缘。表达这些蛋白质的突变病毒将通过对感染这些病毒的细胞进行电子显微镜检查来评估它们产生被包裹的病毒粒子的能力。第二个可能的作用是pUL31导致部分板层解聚，使核衣壳进入内核膜。为了评估这种可能性，感染野生型病毒和缺失UL31的细胞的叶片将用免疫电子显微镜进行鉴定。如果野生型病毒感染的细胞含有多孔板，而感染UL31突变体的细胞没有，将探索pUL31介导局部板层解聚的机制。

DESCRIPTION (provided by applicant): The long-term goal of these studies is to understand the molecular mechanisms of herpesvirus nucleocapsid envelopment. All herpesviruses bud initially from the inner nuclear membranes of infected cells suggesting that information gained from these studies will be applicable to all members of this medically important virus family. The application focuses on the role of herpes simplex virus 1 UL31 and UL34 in the envelopment process. Hypotheses based on extensive preliminary data are tested. The first hypothesis proposes that the protein encoded by UL31 (pUL3 t) interacts with the UL34 gene product (pUL34) to mediate proper targeting of both proteins to the inner nuclear membrane. To assess the importance of the interaction in vivo, viral mutants bearing UL31 genes that lack pUL34 interacting domains, and UL34 mutants that lack pUL31 interacting regions will be tested for their abilities to correctly target both proteins in infected cells and to mediate nucleocapsid envelopment. The second hypothesis to be tested seeks to understand the role of nuclear lamina association of pUL31. The nuclear lamina is part of the nucleocytoskeleton or nuclear matrix that lines the inside surface of the nuclear rim. One possible role is that nuclear lamina association of pUL31 mediates anchoring of the pUL31/pUL34 complex at the nuclear rim. Therefore UL31 mutants lacking lamina association domains and containing known lamina binding domains replacing these domains will be tested for their abilities to mediate correct targeting of pUL31 and pUL34 to the nuclear rim in transient expression assays. Mutant viruses expressing these proteins will then be assessed for their ability to produce enveloped virions by electron microscopic examination of cells infected with these viruses. A second possible role is that pUL31 causes partial lamina depolymerization to allow nucleocapsids access to the inner nuclear membrane. To assess this possibility, the lamina of cells infected with wild type virus and a deletion virus lacking UL31 will be characterized by immunoelectron microsopy. If the wild type virus infected cells contain a porous lamina whereas cells infected with the UL31 mutant do not, mechanisms by which pUL31 could mediate local lamina depolymerization will be explored.