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中开始基因组复制，这也是衣壳组装的地方。由于这些核衣壳太大，无法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形成复合物启动，即在完整的核膜锚定病毒蛋白（在人巨细胞病毒（HCMV）中称为pUL50）和核质溶性病毒蛋白（在HCMV中称为pUL53）之间形成复合物。该复合体被认为可以整理多种角色。它(i)形成募集额外病毒和宿主细胞蛋白的平台，（ii）启动膜芽的形成，（iii）将核衣壳募集到出芽位点。我们最近发表了HCMV pUL50-pUL53复合体的晶体结构。该复合物的一个特殊特征是pUL53中的一个钩状n端延伸。钩子（氨基酸59-87）包含pUL50，贡献1510 Angs。**2为总蛋白界面面积的1880 Angs。**2是pUL50-pUL53相互作用的标志。在晶体中，pUL50-pUL53异二聚体进一步组装成六聚体环状结构，形成蜂窝状层。这些组合在文献中被讨论为促进内核膜的弯曲，从而启动出芽过程。研究领域竞争激烈，几个核心NEC结构，即HCMV， HSV-1和PrV，几乎同时发表。在这里，我们建议大大扩展核心NEC作为病毒核出口启动器的结构生物学知识，同时探索pUL50-pUL53复合物作为抗病毒治疗靶点的潜力。本研究旨在确定eb病毒（EBV）和水痘带状疱疹病毒（VZV）核心NEC的晶体结构。两者都具有很高的医学相关性，直到现在还没有伽玛疱疹病毒核心NEC（例如EBV）的结构得到解决。此外，我们旨在通过分子动力学计算比较来自不同疱疹病毒的NEC复合物及其分子特性，求解核心NEC复合物与附加病毒或宿主细胞蛋白的晶体结构，并从结构上探索核心NEC与核衣壳蛋白之间的相互作用，作为出芽过程启动的关键步骤。最后，我们的目标是表征HCMV pUL50和pUL53之间的钩入槽相互作用，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晶体学片段筛选来实现。由于我们最近在获得工程HCMV核心NEC复合物衍射优于1.5 Angs的晶体方面取得了进展，因此这种方法非常有前途。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.