Structural mechanism of herpesvirus nuclear egress

描述(申请人提供)：疱疹病毒是一种人类病原体家族，它建立终身潜伏感染，病毒周期性地重新激活，导致许多疾病。重新激活不仅会造成重大的疾病负担，还会导致 新感染率高。在重新激活过程中，多个传染性子代病毒粒子被组装在一起，并在一个称为出口的过程中从细胞中释放出来。这一过程的第一步是核出口，即组装好的核衣壳离开细胞核进入细胞质。尽管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。本研究的长期目标是在原子水平上阐明单纯疱疹病毒(HSV)和伪狂犬病(PRV)这两种阿尔法疱疹病毒的核外移过程。这项建议的目的是启动HSV-1核出口复合体UL31/UL34(NEC)的生物物理、生化和结构特征。这项研究是由一个假设推动的，即NEC通过类似于负链RNA病毒基质蛋白和细胞ESCRT-III蛋白的机制实现初级包膜，后者通过以寡聚依赖的方式变形膜来促进萌发。目的1将侧重于确定UL31/UL34相互作用的亲和力和化学计量学，绘制形成NEC的每个蛋白质的最小片段图，并开始结晶试验，目的是确定UL31、UL34和UL31/UL34复合体的结构。目的2验证纯化的HSV-1 NEC可以在没有其他病毒或宿主蛋白的情况下以寡聚依赖的方式在体外使膜变形的假说。拟议的研究将为了解NEC的结构以及它在核外流过程中使膜变形的机制提供重要的见解。获得UL31、UL34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，这一知识将允许针对这些界面的药物的结构指导设计。仅证明纯化的NEC在体外可以使膜变形，这不仅将证明这一过程是必要的和充分的，而且还将使UL31和UL34突变体的膜变形活性与其体内萌发表型相关的体外试验得以发展。在未来，在拟议的工作过程中获得的结果将为随后的R01应用奠定基础，以全面阐明疱疹病毒外泄的机制。 公共卫生相关性：疱疹病毒导致终生感染，病毒保持休眠状态，然后周期性地重新激活，导致许多疾病，从唇疱疹、生殖器疱疹到失明、脑炎、癌症，以及免疫受损个人和新生儿的危及生命的情况。这项提议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。在这项提案中，我们将研究核出口复合体，核出口的关键参与者，及其组件。作为拟议工作的结果而获得的新知识可能会导致基于阻止新病毒颗粒释放的成功的治疗干预。

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.