Nucleocapsid envelopment Herpes simplex virus-1

描述（由申请人提供）：这些研究的长期目标是了解疱疹病毒核衣壳包膜的分子机制。所有疱疹病毒最初都是从受感染细胞的核膜萌发的，这表明从这些研究中获得的信息将适用于这一医学上重要的病毒家族的所有成员。应用的重点是单纯疱疹病毒1 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检验。第一种假说认为UL31编码的蛋白（pul3t）与UL34基因产物（pUL34）相互作用，介导这两种蛋白对核膜的适当靶向。为了评估体内相互作用的重要性，将测试携带缺乏pUL34相互作用区域的UL31基因突变体和缺乏pUL31相互作用区域的UL34突变体在感染细胞中正确靶向蛋白质和介导核衣壳包膜的能力。第二个有待检验的假设旨在了解pUL31的核层关联的作用。核膜是排列在核边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的核层结合介导了pUL31/pUL34复合物在核边缘的锚定。因此，在瞬时表达试验中，缺乏层相关结构域和含有取代这些结构域的已知层结合结构域的UL31突变体将被测试其介导pUL31和pUL34正确靶向核边缘的能力。然后通过对感染这些病毒的细胞进行电子显微镜检查，评估表达这些蛋白质的突变病毒产生包膜病毒粒子的能力。第二个可能的作用是pUL31引起部分层解聚，使核衣壳能够进入内层核膜。为了评估这种可能性，将用免疫电子显微镜对感染野生型病毒和缺乏UL31的缺失病毒的细胞层进行表征。如果野生型病毒感染的细胞含有多孔层，而UL31突变体感染的细胞没有，那么pUL31介导局部层解聚的机制将被探索。

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.