Nucleocapsid Envelopment Herpes Simplex Virus-1

描述（由申请人提供）：UL 31和UL 34蛋白对于单纯疱疹病毒1型核衣壳在内核膜（INM）上的粘附是必需的。数据表明，这些蛋白质发挥多种作用，在消除反应。目标1中的拟议研究将研究pUL 31和pUL 34如何靶向INM。该信息将与理解包括本申请的其余部分的两个单独方面有关：1）破坏核纤层以提供核衣壳进入INM中的出芽位点和2）在INM处构建出芽位点。对于目的2，初步数据支持这样的假设，即pUL 31和/或pUL 34蛋白在单独或一起表达时能够破坏核纤层。假设pUL 31/pUL 34复合物干扰核纤层完整性所需的相互作用，从而局部解聚纤层以允许病毒体进入内核膜中的出芽位点。将检测在瞬时表达试验中鉴定的纤层缔合作用和推定解聚活性对病毒体核逃逸的影响。对于目标3，初步数据支持病毒糖蛋白D（gD）向INM的pUL 34依赖性募集，并表明pUL 34结合未成熟的gD，一种已知并入核周病毒体的INM膜蛋白。由于该蛋白质与gB和gH相互作用，这也是核周病毒体组分，我们假设pUL 34/gD相互作用有助于编排INM出芽位点和核周病毒体的蛋白质组。为了测试这种可能性，我们建议鉴定gD中的pUL 34结合位点，并产生携带排除pUL 34/gD相互作用的突变的重组病毒。该假设预测，gD不应该有效地本地化的病毒突变体感染的细胞的INM中，不应该存在于核周病毒体通过免疫金电子显微镜评估。最后，初步的数据表明，PUL 31是所需的INM结构，染色密集与OsO 4和代表优先核衣壳出芽位点。因此，pUL 31可能与其他病毒的基质蛋白类似，通过改变局部脂质组成来帮助协调INM的出芽位点。为了测试这种可能性，薄层色谱法将被用来比较脂质组成的核周病毒的核膜和高尔基体膜。该假说预测优先招聘的不饱和脂肪酸的出芽位点和核周病毒粒子。这种假定的脂质组成的改变如何有助于蛋白质募集到INM出芽位点也将进行测试。

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.