Human Cytomegalovirus Nuclear Egress: Molecular Mechanisms and Drug Targeting

 描述（由申请方提供）：本研究的长期目标是鉴定、表征和开发人类疱疹病毒的药物靶点。这项研究特别与健康有关，因为需要新的药物来治疗疱疹病毒感染，特别是人类巨细胞病毒（HCMV）感染。在本申请中，研究了两种HCMV蛋白UL 50和UL 53参与核衣壳从细胞核转运到细胞质（核出口）的不寻常过程。这些蛋白质相互作用形成核出口复合物（NEC），可以作为新的药物靶点。这些蛋白质在核出口的两个重要步骤中的作用-衣壳向核边缘的运动和通过内核膜出芽-知之甚少，并且是本申请的主要焦点。具体目标1是研究HCMV衣壳如何从核内部移动到核边缘。核肌动蛋白和肌球蛋白Va在这一过程中的作用将使用活细胞成像和感染细胞的单粒子跟踪分析来研究， 这些蛋白质的显性负突变体或已用肌动蛋白抑制剂处理。将对UL 53突变病毒进行类似分析。UL 53是否可以连接衣壳肌球蛋白Va将使用纯化的衣壳和蛋白质进行生化测试。具体目标2是研究NEC如何通过内核膜协调出芽。AAA ATP酶，VCP，及其辅助因子在这个过程中可能发挥的作用，将初步研究这些主机蛋白与NEC使用免疫荧光，免疫电子显微镜，免疫共沉淀评估协会。这些研究之后将测量阻断宿主蛋白和VCP抑制剂表达的siRNA对感染细胞中的核出口以及表达NEC或其亚基但不表达其他病毒蛋白的细胞中的囊泡形成的影响。VCP及其辅助因子是否可以促进体外出芽将与Heldwein实验室合作使用巨大的单层囊泡进行研究。具体目标3是确定NEC及其亚基的结构。将通过X射线晶体学测定保留疱疹病毒中保守的所有序列的截短形式的UL 50和UL 53的复合物以及类似形式的UL 53的结构.在与瓦格纳实验室的合作中，核磁共振（NMR）将用于解决类似版本的UL 50，以及小鼠CMV同系物的UL 53和复合物。一个长期的目标是使用X射线晶体学解决一个接近全长的复杂结构。具体目标4是使用高通量测定，并与瓦格纳实验室一起，基于NMR的片段筛选抑制NEC亚基相互作用的小分子。然后将对“命中”进行特异性、抗HCMV活性和细胞毒性以及抑制机制的测定，并将与药物化学家合作开发为先导药物。

 DESCRIPTION (provided by applicant): The long-term objective of this research is to identify, characterize, and exploit drug targets of human herpesviruses. This research is especially health-related, as new drugs are needed for treatment of herpesvirus infections, particularly those of human cytomegalovirus (HCMV). In this application, two HCMV proteins UL50 and UL53 are investigated for involvement in the unusual process by which nucleocapsids transit from the nucleus to the cytoplasm (nuclear egress). These proteins interact to form a nuclear egress complex (NEC) that can serve as a new drug target. The roles of these proteins in two important steps of nuclear egress - movement of capsids towards the nuclear rim, and budding through the inner nuclear membrane - are poorly understood and are a major focus of this application. Specific aim 1 is to investigate how HCMV capsids move from the nuclear interior to the nuclear rim. The roles of nuclear actin and myosin Va in this process will be investigated using live cell imaging and single particle tracking analyses of infected cells that either express dominant negative mutants of these proteins or have been treated with actin inhibitors. Similar analyses will be performed on UL53 mutant viruses. Whether UL53 can link capsids to myosin Va will be tested biochemically using purified capsids and proteins. Specific aim 2 is to investigate how the NEC orchestrates budding through the inner nuclear membrane. A possible role for the AAA ATPase, VCP, and its co-factors in this process will be examined initially by assessing associations of these host proteins with the NEC using immunofluorescence, immuno-electron microscopy, and co-immunoprecipitation. These studies will be followed by measuring the effects of siRNAs that block expression of the host proteins and VCP inhibitors on nuclear egress in infected cells, and vesiculation in cells expressing the NEC or its subunits, but no other viral proteins. Whether VCP and its co-factors can promote budding in vitro will be studied using giant unilamellar vesicles in collaboration with the Heldwein laboratory. Specific aim 3 is to determine structures of the NEC and its subunits. The structures of a complex of truncated versions of UL50 and UL53 that retain all sequences that are conserved among herpesviruses, and of a similar version of UL53 will be determined by X- ray crystallography. In collaboration with the Wagner laboratory, nuclear magnetic resonance (NMR) will be used to solve similar versions of UL50, and the mouse CMV homologs of UL53 and the complex. A longer term goal is to solve the structure of a near-full length complex using X-ray crystallography. Specific aim 4 is to use a high throughput assay and, with the Wagner laboratory, NMR-based fragment screening for small molecules that inhibit subunit interactions of the NEC. "Hits" will be then assayed for specificity, for anti-HCMV activity and cytotoxicity, and for their mechanism of inhibition, and will be developed into leads in collaboration with medicinal chemists.