Nucleocapsid Envelopment Herpes Simplex Virus-1

描述(由申请人提供)：UL31和UL34蛋白都是单纯疱疹病毒1型核衣壳内核膜(INM)包膜所必需的。数据表明，这些蛋白质在包膜反应中发挥着多种作用。目标1中拟议的研究将调查pUL31和pUL34是如何针对INM的。这些信息将与理解包膜反应的两个独立方面有关，这两个方面构成了本申请的其余部分：1)核层的破坏，以提供进入INM萌芽部位的核衣壳；2)在INM构筑萌芽部位。对于目标2，初步数据支持这样的假设，即当pUL31和/或pUL34蛋白单独或一起表达时，能够破坏核层。假设pUL31/pUL34复合体干扰核膜完整性所需的相互作用，从而局部解聚核膜，使病毒粒子进入内核膜的萌发部位。瞬时表达分析中确定的片层结合和假定的解聚活性的作用将被测试它们对病毒粒子核出口的影响。对于目的3，初步数据支持pUL34依赖于病毒糖蛋白D(GD)到INM的募集，并表明pUL34结合未成熟的GD，已知的INM膜蛋白被结合到核周病毒粒子中。由于该蛋白与Gb和Gh相互作用，而Gh和Gh也是核周病毒粒子成分，我们假设pUL34/Gd相互作用有助于协调INM出芽部位和核周病毒粒子的蛋白质组。为了测试这种可能性，我们建议在GD中鉴定pUL34结合部位，并产生携带突变的重组病毒，从而排除pUL34/GD相互作用。这一假说预测，gD不应该有效地定位在感染病毒突变体的细胞的INM中，并且不应该存在于免疫金电镜下的核周病毒粒子中。最后，初步数据表明，pUL31对于浓染OsO4的INM结构是必需的，并且代表优先的核衣壳萌发位置。因此，pUL31可能类似于其他病毒的基质蛋白，通过改变局部的脂组成来帮助协调INM上的萌发部位。为了测试这种可能性，将使用薄层层析法比较核周病毒粒子与核膜和高尔基膜的脂类成分。该假说预测不饱和脂肪酸优先招募到萌芽部位和核周病毒粒子。这种假定的脂类成分的改变如何有助于INM萌芽部位的蛋白质募集也将得到测试。公共卫生相关性：这些研究适用于公共卫生，因为包膜机制和涉及的蛋白质在所有疱疹病毒中都是保守的，对病毒复制至关重要。因此，任何阻止这些蛋白质功能的药物都可能对所有疱疹病毒具有广泛的活性。

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. PUBLIC HEALTH RELEVANCE: These studies are applicable to public health because the mechanisms of envelopment, and the proteins involved are conserved in all herpesviruses and essential for viral replication. Thus any drugs that preclude the function of these proteins could have broad activities against all herpesviruses.