Nucleocapsid Envelopment Herpes Simplex Virus-1

描述（由申请人提供）：UL31 和 UL34 蛋白对于在内核膜 (INM) 处包封单纯疱疹病毒 1 核衣壳至关重要。数据表明这些蛋白质在包封反应中发挥多种作用。目标 1 中拟议的研究将调查 pUL31 和 pUL34 如何靶向 INM。该信息将与理解包络反应的两个不同方面相关，这两个方面构成了应用程序的其余部分：1）破坏核层以提供核衣壳进入 INM 中的出芽位点；2）在 INM 上构建出芽位点。对于目标 2，初步数据支持这样的假设：pUL31 和/或 pUL34 蛋白单独或一起表达时能够破坏核纤层。假设 pUL31/pUL34 复合物干扰核纤层完整性所需的相互作用，从而使核纤层局部解聚，从而允许病毒颗粒进入内核膜中的出芽位点。将测试瞬时表达测定中鉴定的层联结和推定解聚活性的作用，以了解它们对病毒粒子核排出的影响。对于目标 3，初步数据支持 pUL34 依赖性病毒糖蛋白 D (gD) 招募至 INM，并表明 pUL34 结合未成熟的 gD，这是一种已知会掺入核周病毒体的 INM 膜蛋白。由于该蛋白与 gB 和 gH 相互作用，而 gB 和 gH 也是核周病毒粒子的组成部分，因此我们假设 pUL34/gD 相互作用有助于协调 INM 出芽位点和核周病毒粒子的蛋白质组。为了测试这种可能性，我们建议鉴定 gD 中的 pUL34 结合位点，并生成带有阻止 pUL34/gD 相互作用的突变的重组病毒。该假设预测，gD 不应有效定位于感染病毒突变体的细胞的 INM 中，并且根据免疫金电子显微镜评估，不应存在于核周病毒体中。最后，初步数据表明 pUL31 是 INM 结构所必需的，该结构被 OsO4 密集染色并代表优先的核衣壳出芽位点。因此，pUL31 的作用可能与其他病毒的基质蛋白类似，通过改变局部脂质成分来帮助协调 INM 的出芽位点。为了测试这种可能性，将使用薄层色谱来比较核周病毒粒子与核膜和高尔基膜的脂质组成。该假设预测不饱和脂肪酸会优先招募到出芽位点和核周病毒体。脂质成分的这种假定改变如何促进蛋白质招募到 INM 出芽位点也将得到测试。

DESCRIPTION (provided by applicant): Both the UL31 and UL34 proteins are essential for envelopment of herpes simplex virus 1 nucleocapsids at the inner nuclear membrane (INM). Data imply that these proteins play multiple roles in the envelopment reaction. The proposed studies in Aim 1 will investigate how pUL31 and pUL34 are targeted to the INM. This information will be relevant to understanding two separate aspects of the envelopment reaction that comprise the rest of the application: 1) Disruption of the nuclear lamina to provide nucleocapsids access to budding sites in the INM and 2) Construction of budding sites at the INM. For Aim 2, preliminary data support the hypothesis that pUL31 and/or pUL34 proteins are able to disrupt the nuclear lamina when expressed alone or together. The hypothesis is that the pUL31/pUL34 complex interferes with interactions required for nuclear lamina integrity, thereby locally depolymerizing the lamina to allow virions access to budding sites in the inner nuclear membrane. The role of lamina association and putative depolymerizing activities identified in transient expression assays will be tested for their effects on nuclear egress of virions. For Aim 3, preliminary data support a pUL34-dependent recruitment of viral glycoprotein D (gD) to the INM and indicate that pUL34 binds immature gD, an INM membrane protein known to become incorporated into perinuclear virions. Because this protein interacts with gB and gH which are also perinuclear virion components, we hypothesize that the pUL34/gD interaction helps orchestrate the proteome of INM budding sites and perinuclear virions. To test this possibility we propose to identify the pUL34 binding site in gD, and generate recombinant viruses bearing mutations that preclude the pUL34/gD interaction. The hypothesis predicts that gD should not localize efficiently in the INM of cells infected with the viral mutant, and should not be present in perinuclear virions as assessed by immunogold electron microscopy. Finally, preliminary data indicate that pUL31 is required for INM structures that stain densely with OsO4 and represent preferential nucleocapsid budding sites. Thus, pUL31 may act similarly to matrix proteins of other viruses to help orchestrate budding sites at the INM by alteration of localized lipid composition. To test this possibility, thin layer chromatography will be used to compare the lipid composition of perinuclear virions to those of nuclear and Golgi membranes. The hypothesis predicts preferential recruitment of unsaturated fatty acids to budding sites and perinuclear virions. How this putative alteration of lipid composition contributes to protein recruitment to INM budding sites will also be tested.