Structural mechanism of membrane remodeling during herpesvirus nuclear egress

描述（由申请人提供）：疱疹病毒是一种无所不在的人类病原体，可引起多种重要疾病。设计更好的策略来对抗这些病毒需要深入了解它们是如何复制的。该建议的重点是核出口，在此期间，核衣壳出芽进入内核膜，这是子代病毒体组装和释放的关键步骤。病毒核出口复合物（NEC）是这一过程中的关键参与者，但其功能的详细机制知识是缺乏的。本研究的长期目标是阐明疱疹病毒核出口的原子水平机制，提高这一过程的基础知识，并确定和表征新的抗病毒靶点。本提案的目的是充分探索NEC介导的膜变形的机制，在核出口通过使用结构，生物物理和细胞为基础的策略的合并。这一建议是由中心假设驱动的，基于大量的初步数据，即NEC单独介导膜出芽，并可能以寡聚化依赖的方式断裂。目的1将集中于通过使用巨单层囊泡（GUV）的体外膜出芽试验结合光学显微镜和冷冻电子显微镜来确定HSV-1 NEC的膜变形机制。膜出芽测定也将用于在体外重建衣壳出芽。目的2的目标是确定HSV-1 NEC的结构，并通过结构导向诱变结合体外测定和重组病毒的体内表征来定位功能重要区域。目的3将集中于揭示保守的和病毒特异性的特征，NEC机制在α和γ-疱疹病毒通过扩展的生物化学和结构研究NEC从伪狂犬病，EB病毒，卡波西肉瘤疱疹病毒。拟议的工作将利用生物化学和结构生物学（Heldwein实验室）的专业知识与病毒诱变和活细胞荧光成像（Smith实验室）的专业知识相结合。这些研究的结果将是NEC的结构和机制，它使膜变形在核出口。获得NEC的晶体结构将在疱疹病毒学领域产生重大影响，产生任何其他方法都无法获得的机制和功能见解。此外，详细的体外研究NEC介导的膜出芽可能会发现一个新的膜出芽机制。重要的是，通过将NEC突变体的体外性质与相应重组病毒的体内表型相关联，NEC结构和来自体外出芽模型的观察结果将与感染细胞中的核排出直接相关。详细了解NEC在核出口过程中膜重塑的结构机制不仅有利于疱疹病毒领域，而且还显着推进了细胞过程的现有知识。

DESCRIPTION (provided by applicant): Herpesviruses are omnipresent human pathogens that cause a number of important diseases. Devising better strategies to combat these viruses requires the in-depth understanding of how they replicate. This proposal focuses on nuclear egress during which nucleocapsids bud into the inner nuclear membrane, a critical step in the assembly and release of progeny virions. The viral nuclear egress complex (NEC) is the key player in this process, but the detailed mechanistic knowledge of its function is lacking. The long-term goal of this research is to elucidate the atomic-level mechanism of herpesvirus nuclear egress, to improve the fundamental knowledge of this process and to identify and characterize novel antiviral targets. The objective of this proposal is to fully explore the mechanism of NEC-mediated membrane deformation during nuclear egress by using an amalgamation of structural, biophysical, and cell-based strategies. This proposal is driven by the central hypothesis, based on substantial preliminary data, that the NEC alone mediates membrane budding and, possibly, scission in oligomerization-dependent manner. Aim 1 will focus on determining the mechanism of membrane deformation by the HSV-1 NEC by using the in vitro membrane budding assay with giant unilamellar vesicles (GUVs) in combination with light microscopy and cryoelectron microscopy. The membrane budding assay will also be used to reconstitute capsid budding in vitro. The goal of Aim 2 is determine the structure of the HSV-1 NEC and to map functionally important regions by structure- guided mutagenesis in combination with in vitro assays and the in vivo characterization of recombinant viruses. Aim 3 will concentrate on revealing the conserved and the virus-specific features of the NEC mechanism in alpha and gamma-herpesviruses by extending the biochemical and the structural studies to NECs from pseudorabies, Epstein-Barr, and Kaposi's Sarcoma herpesviruses. The proposed work will leverage the combined expertise in biochemistry and structural biology (Heldwein lab) with expertise in virus mutagenesis and live-cell fluorescent imaging (Smith lab). The outcome of these studies will be the structure of the NEC and the mechanism by which it enables membrane deformation during nuclear egress. Obtaining the crystal structure of the NEC will have a major impact in the field of herpes virology by yielding mechanistic and functional insights unavailable by any other approaches. Furthermore, detailed in vitro studies of the NEC-mediated membrane budding may uncover a novel membrane budding mechanism. Importantly, by correlating the in vitro properties of the NEC mutants with the in vivo phenotypes of the corresponding recombinant viruses, the NEC structure and the observations from in vitro budding models will be directly related to the nuclear egress in infected cells. A detailed knowledge of the structural mechanism of membrane remodeling by the NEC during nuclear egress will benefit not just the herpesvirus field, but also significantly advance the current knowledge of cellular processes.