Nucleocapsid Envelopment Herpes Simplex Virus-1

项目摘要/摘要 UL31和UL34蛋白都是单纯疱疹病毒1型包膜所必需的 内核膜(INM)有核衣壳。数据表明，这些蛋白质在 包膜反应。目标1中拟议的研究将调查pUL31和pUL34是如何 目标是国际货币基金组织。这些信息将与理解 包膜反应，包括应用的其余部分：1)破坏核层以 提供进入INM萌芽地点的核衣壳，以及2)在INM建设萌芽地点。 对于目标2，初步数据支持这样的假设，即pUL31和/或pUL34蛋白能够破坏 当核膜单独或一起表达时。假设pUL31/pUL34复合体 干扰核层完整性所需的相互作用，从而局部解聚核层以 允许病毒粒子进入内核膜的萌发部位。椎板联结的作用和 在瞬时表达分析中确定的假定解聚活性将被测试它们对 病毒粒子的核外泄。对于AIM 3，初步数据支持依赖pUL34的病毒招募 糖蛋白D(GD)与INM结合，表明pUL34与未成熟的gD结合，这是一种INM膜蛋白 已知被合并到核周病毒粒子中。因为这种蛋白质与Gb和Gh相互作用 这些也是核周病毒粒子组分，我们假设pUL34/Gd相互作用有助于 编排INM萌芽部位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，并产生携带突变的重组病毒 PUL34/Gd互作。该假说预测，GD不应该有效地定位在细胞的INM中 感染了病毒突变体，免疫金法评估不应出现在核周病毒粒子中 电子显微镜。最后，初步数据表明，染色的INM结构需要pUL31 与OsO4密不可分，代表优先的核衣壳萌发部位。因此，PUL31的作用类似于 其他病毒的基质蛋白通过改变局部脂质来帮助协调INM上的萌发部位 组成。为了测试这种可能性，将使用薄层层析法来比较脂质 核周病毒粒子与核膜和高尔基膜的组成。这一假说预言了 不饱和脂肪酸优先补充到萌芽部位和核周病毒粒子。这是怎么回事 推测脂类成分的改变有助于蛋白质重新聚集到INM萌芽部位 测试过。

Project Summary / Abstract 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.