Nucleocapsid Envelopment Herpes Simplex Virus-1

描述（由申请人提供）：UL31和UL34蛋白都是在核膜（INM）包裹单纯疱疹病毒1型核衣壳所必需的。数据表明，这些蛋白质在包膜反应中起着多种作用。Aim 1中提出的研究将研究pUL31和pUL34如何靶向INM。这些信息将有助于理解包膜反应的两个不同方面，这两个方面构成了应用的其余部分：1)破坏核层，使核衣壳能够进入INM中的出芽位点；2)INM中的出芽位点的构建。对于Aim 2，初步数据支持pUL31和/或pUL34蛋白在单独或一起表达时能够破坏核层的假设。假设是pUL31/pUL34复合体干扰核层完整性所需的相互作用，从而使核层局部解聚，使病毒粒子能够进入核膜内的出芽位点。在瞬时表达试验中鉴定的层关联和假定的解聚合活性的作用将测试它们对病毒粒子核出口的影响。对于Aim 3，初步数据支持pUL34依赖的病毒糖蛋白D （gD）募集到INM，并表明pUL34结合未成熟的gD，一种已知被并入核周病毒粒子的INM膜蛋白。由于这种蛋白与同样是核周围病毒粒子成分的gB和gH相互作用，我们假设pUL34/gD相互作用有助于协调INM出芽位点和核周围病毒粒子的蛋白质组。为了验证这一可能性，我们提出在gD中鉴定pUL34结合位点，并产生携带阻止pUL34/gD相互作用的突变的重组病毒。该假设预测gD不应该有效地定位于感染病毒突变体的细胞的INM中，并且不应该存在于核周病毒粒子中，通过免疫金电子显微镜评估。最后，初步数据表明，被OsO4密集染色的INM结构需要pUL31，并代表优先的核衣壳出芽位点。因此，pUL31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上的出芽位点。为了验证这种可能性，将使用薄层色谱法来比较核周病毒粒子与核膜和高尔基膜的脂质组成。该假说预测不饱和脂肪酸优先招募出芽部位和核周病毒粒子。脂质组成的这种假定的改变如何有助于INM出芽部位的蛋白质募集也将被测试。公共卫生相关性：这些研究适用于公共卫生，因为包膜机制和所涉及的蛋白质在所有疱疹病毒中都是保守的，并且对病毒复制至关重要。因此，任何阻断这些蛋白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.