Structural mechanism of herpesvirus nuclear egress

描述（由申请人提供）：疱疹病毒是一个人类病原体家族，可建立终身潜伏感染，病毒周期性重新激活，导致许多疾病。再激活不仅是造成重大疾病负担的原因， 新感染率高。在再活化过程中，多个感染性子代病毒体组装并在称为出口的过程中从细胞释放。该过程的第一步是核出口，由此组装的核衣壳离开核进入细胞质。虽然已经确定了关键的病毒核出口参与者，但缺乏关键的机制细节。这项研究的长期目标是阐明两种α疱疹病毒（单纯疱疹病毒（HSV）和伪狂犬病病毒（PRV））在原子水平上的核出口过程。本计划的目的是启动HSV-1核出口复合物UL 31/UL 34（NEC）的生物物理、生物化学和结构表征。这项研究是由一个假设驱动的，即NEC能够通过与负链RNA病毒和细胞ESCRT-III蛋白的基质蛋白相似的机制进行初级抑制，该机制通过以寡聚化依赖的方式使膜变形来促进出芽。目标1将侧重于确定UL 31/UL 34相互作用的亲和力和化学计量，绘制可以形成NEC的每种蛋白质的最小片段，并启动结晶试验，以确定UL 31，UL 34和UL 31/UL 34复合物的结构。目的2将测试纯化的HSV-1 NEC可以在没有其他病毒或宿主蛋白的情况下以寡聚化依赖性方式在体外使膜变形的假设。拟议的研究将提供重要的见解NEC的结构和机制，使膜变形核出口期间。获得UL 31、UL 34和NEC的晶体结构将是一项重大成就，将对疱疹病毒学领域产生重大影响。已知的功能数据可以映射到结构上，以获得对功能表型的结构基础的洞察。在未来，这些知识将允许针对这些界面的药物的结构导向设计。表明纯化的NEC单独可以在体外使膜变形不仅将证明其对于该过程是必要和足够的，而且还将允许开发体外测定，其中UL 31和UL 34突变体的膜变形活性可以与它们的体内出芽表型相关。在未来，在拟议的工作过程中获得的结果将形成一个后续的R 01应用程序，以充分阐明疱疹病毒的机制出口的基础。 公共卫生相关性：疱疹病毒引起终身感染，由此病毒保持在休眠状态，它们周期性地从休眠状态重新激活，从而在免疫功能低下的个体和新生儿中引起许多疾病，从唇疱疹和生殖器疱疹到失明、脑炎、癌症和危及生命的病症。这项提案的主题是疱疹病毒核出口-受感染细胞产生和释放新病毒颗粒过程中的一个关键阶段。在本提案中，我们将研究核出口综合体，核出口的关键球员，及其组成部分。作为拟议工作的结果获得的新知识可能会导致基于阻断新病毒颗粒释放的成功治疗干预。

DESCRIPTION (provided by applicant): Herpes viruses are a family of human pathogens that establish lifelong latent infections from which viruses periodically reactivate, causing a number of ailments. Reactivations are responsible not only for a significant disease burden but also for a high rate of new infections. During reactivation, multiple infectious progeny virions are assembled and released from the cell in a process called egress. The first step in this process is nuclear egress whereby assembled nucleocapsids exit the nucleus into the cytoplasm. Although key viral nuclear egress participants have been identified, critical mechanistic details are lacking. The long-term goal of this research is to elucidate the process of nuclear egress at the atomic level for two alpha herpes viruses, herpes simplex (HSV) and pseudorabies (PRV). The objective of this proposal is to initiate a biophysical, biochemical, and structural characterizatin of the HSV-1 nuclear egress complex UL31/UL34 (NEC). This research is driven by a hypothesis that the NEC enables primary envelopment by a mechanism similar to that of matrix proteins of negative-stranded RNA viruses and cellular ESCRT-III proteins, which promote budding by deforming the membrane in an oligomerization-dependent manner. Aim 1 will focus on the determination of the affinity and stoichiometry of UL31/UL34 interactions, mapping of the minimal fragment of each protein that can form the NEC, and initiation of crystallization trials with the goal of determining the structures of UL31, UL34 and the UL31/UL34 complex. Aim 2 will test the hypothesis that purified HSV-1 NEC can deform membranes in vitro in the absence of other viral or host proteins, in oligomerization-dependent manner. The proposed studies will provide important insights into the structure of the NEC and the mechanism by which it enables membrane deformation during nuclear egress. Obtaining the crystal structures of UL31, UL34, and the NEC would be a significant achievement that would have a major impact in the field of herpes virology. Known functional data can be mapped onto the structures to obtain insight into the structural basis of functional phenotypes. In the future, this knowledge will allow for the structure-guided design of drugs targeting these interfaces. Showing that the purified NEC alone can deform membranes in vitro not only will demonstrate that it is necessary and sufficient for this process but will also allow the development of an in vitro assay in which the membrane deformation activity of UL31 and UL34 mutants can be correlated with their in vivo budding phenotypes. In the future, the results obtained in the course of the proposed work will form the foundation for a subsequent R01 application to elucidate fully the mechanism of herpes virus egress. PUBLIC HEALTH RELEVANCE: Herpes viruses cause lifelong infections whereby viruses remain in a dormant state from which they periodically reactivate causing a number of ailments ranging from cold sores and genital herpes to blindness, encephalitis, cancers, and life-threatening conditions in the immunocompromised individuals and in newborns. The subject of this proposal is herpes virus nuclear egress - a critical stage in the process by which infected cells produce and release new viral particles. In this proposal we will study the nuclear egress complex, the key player in nuclear egress, and its components. New knowledge obtained as the result of the proposed work may lead to successful therapeutic interventions based on blocking the release of new viral particles.