Structural mechanism of membrane remodeling during herpesvirus nuclear egress

疱疹病毒是一种普遍存在的人类病原体，可引起许多重要疾病。制定 更好的防治这些病毒的战略需要深入了解它们如何复制。这 一项建议的重点是核出口，在此过程中，核衣壳出芽进入内核膜， 在装配和释放子代病毒体的关键步骤。病毒核出口复合体（NEC）是 在这一过程中的关键球员，但其功能的详细机制知识是缺乏的。长期 本研究的目的是阐明疱疹病毒核出口的原子水平机制， 这一过程的基础知识，并确定和表征新的抗病毒靶点。的 本研究的目的是充分探讨NEC介导的膜变形机制， 核出口通过使用结构，生物物理，和基于细胞的策略的合并。这项建议 基于大量的初步数据，由中心假设驱动，即NEC单独介导 膜出芽，并可能以寡聚化依赖的方式断裂。目标1将侧重于 通过使用体外膜确定HSV-1 NEC引起膜变形的机制 用巨大单层囊泡（GUV）结合光学显微镜和冷冻电子学的出芽测定 显微镜膜出芽测定也将用于在体外重建衣壳出芽。目标 目的2是确定HSV-1 NEC的结构，并通过结构定位功能重要的区域。 结合体外测定和重组体的体内表征的指导诱变 病毒目的3将集中于揭示NEC的保守性和病毒特异性特征 通过扩展生化和结构研究， 来自伪狂犬病、EB病毒和卡波西肉瘤疱疹病毒的NEC。拟议的工作将 利用生物化学和结构生物学（Heldwein实验室）的综合专业知识和病毒 诱变和活细胞荧光成像（Smith实验室）。这些研究的结果将是结构 的NEC和机制，它使膜变形期间核出口。获得 NEC的晶体结构将通过产生机制而在疱疹病毒学领域产生重大影响。 和其他方法无法获得的功能性洞察。此外，详细的体外研究， NEC介导的膜出芽可能揭示一种新的膜出芽机制。重要的是毫 将NEC突变体的体外性质与相应的突变体的体内表型相关联， 重组病毒，NEC结构和体外出芽模型的观察结果将直接 与受感染细胞的核出口有关。详细了解了其结构机理， NEC在核出口期间的膜重塑不仅有益于疱疹病毒领域， 大大推进了细胞过程的现有知识。

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 gammaherpesviruses 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.