Structural insight into the herpesvirus core nuclear egress complex - investigating its role as initiator of virus nuclear egress and its potential as an antiviral therapy target.

疱疹病毒家族的病毒在宿主细胞核内开始基因组复制，这也是衣壳组装的地方。由于这些核衣壳太大，不能通过核孔离开细胞核，疱疹病毒依赖于另一种退出机制，称为核出口。这是病毒复制的关键步骤，由两种病毒蛋白之间的复合体启动，即在人巨细胞病毒(HCMV)中称为pUL50的完整核膜锚定病毒蛋白和在HCMV中称为pUL53的核质可溶性病毒蛋白之间的复合体。这个建筑群被认为是对多个角色的整理。它(I)形成一个招募额外病毒和宿主细胞蛋白的平台，(Ii)启动膜芽的形成，(Iii)将核衣壳招募到萌芽位置。我们最近发表了HCMV pUL50-pUL53复合体的晶体结构。该复合体的一个具体特征是在pUL53中有一个钩状的N-末端延伸。钩子(氨基酸59-87)包含pUL50，贡献了1510 Ang.**2的总蛋白质界面面积1880 Ang.**2，代表了pUL50-pUL53相互作用的标志。在晶体中，pUL50-pUL53杂二聚体进一步组装成六面体环状结构，形成蜂窝状的层。这些组件在文献中被讨论为促进内核膜的弯曲，从而启动萌发过程。这一研究领域竞争激烈，几种核心NEC结构，即来自HCMV、HSV-1和PRV的结构已经几乎同时发表。在此，我们建议极大地扩展我们关于核心NEC作为病毒核出口启动子的结构生物学知识，同时探索pUL50-pUL53复合体作为抗病毒治疗靶点的潜力。特别是，我们的目标是确定EB病毒(EBV)和水痘带状疱疹病毒(VZV)核心NEC的晶体结构。这两种病毒都具有很高的医学相关性，到目前为止还没有解决伽马疱疹病毒核心NEC(例如EBV)的结构。此外，我们的目的是通过分子动力学计算比较不同疱疹病毒的NEC复合体及其分子性质，解决核心NEC与额外的病毒或宿主细胞蛋白形成的复合体的晶体结构，并从结构上探索核心NEC与核衣壳蛋白之间的相互作用，作为启动萌发过程的关键步骤。最后，我们的目标是表征HCMV pUL50和pUL53之间的钩槽相互作用，作为开发具有潜在治疗价值的抑制剂的蓝图。后者将通过计算设计和结晶学片段筛选来实现。这一方法是非常有希望的，因为我们最近在获得衍射率高于1.5Angs的工程化的HCMV核心NEC复合体晶体方面取得了进展。决议。

Viruses from the herpesviridea family start genomic replication in the host cell nucleus, which is also the place of capsid assembly. Because these nucleocapsids are too large to exit the nucleus via the nuclear pores, herpesviruses rely on an alternative exit mechanism, termed nuclear egress. This is a key step in virus replication and is initiated by the formation of a complex between two viral proteins, namely between an integral nuclear membrane-anchored viral protein, termed pUL50 in human cytomegalovirus (HCMV) and a nucleoplasmic-soluble viral protein, termed pUL53 in HCMV. The complex is thought to collate multiple roles. It (i) forms a platform for the recruitment of additional virus and host cell proteins, (ii) initiates the formation of membrane buds and (iii) recruits the nucleocapsids to the budding sites. We recently published the crystal structure of the HCMV pUL50-pUL53 complex. A specific feature of the complex is a hook-like N-terminal extension in pUL53. The hook (amino acids 59-87) embraces pUL50, contributes 1510 Angs.**2 to the total protein interface area of 1880 Angs.**2 and represents a hallmark of the pUL50-pUL53 interaction. In the crystals the pUL50-pUL53 heterodimers further assemble into hexameric ring-like structures that form a honeycomb-like layer. These assemblies are discussed in the literature as facilitating the bending of the inner nuclear membrane and thereby initiating the budding process. The research field is highly competitive and several core NEC structures, namely from HCMV, HSV-1 and PrV, have been published almost simultaneously.Here, we propose to considerably expand our structural biology knowledge of the core NEC as the virus nuclear egress initiator and at the same time explore the potential of the pUL50-pUL53 complex as an antiviral therapy target. In particular, we aim at determining the crystal structures of the core NEC from Epstein-Barr virus (EBV) and varizella zoster virus (VZV). Both are of high medical relevance, and until now no structure of a gammaherpesvirus core NEC (e.g. EBV) has been solved. In addition, we aim at comparing the NEC complexes from different herpesviruses and their molecular properties using molecular dynamics calculations, solving crystal structures of the core NEC in complex with additional virus or host cell proteins and structurally exploring the interaction between the core NEC and the nuclear capsid proteins as a key step in the initiation of the budding process. Finally, we aim at characterizing the hook-into-groove interaction between HCMV pUL50 and pUL53 as a blueprint for the development of inhibitors of potential therapeutic value. The latter will be achieved by computational design and fragment screening by crystallography. This approach is highly promising due to our recent advances in obtaining crystals of an engineered HCMV core NEC complex diffracting to better than 1.5 Angs. resolution.