Structural mechanism of membrane remodeling during herpesvirus nuclear egress

描述(申请人提供)：疱疹病毒是无处不在的人类病原体，可导致许多重要疾病。制定更好的战略来对抗这些病毒，需要深入了解它们是如何复制的。这项提议的重点是核出口，在此期间，核衣壳发芽进入内核膜，这是组装和释放子代病毒粒子的关键步骤。病毒核出口复合体(NEC)是这一过程中的关键角色，但对其功能的详细机制缺乏了解。这项研究的长期目标是阐明疱疹病毒核外泄的原子水平机制，提高对这一过程的基础知识，并识别和表征新的抗病毒靶点。这项建议的目的是通过融合结构、生物物理和基于细胞的策略来充分探索NEC介导的核外泄过程中膜变形的机制。这一建议是由一个中心假设推动的，基于大量的初步数据，即NEC单独介导膜出芽，并可能以寡聚依赖的方式发生断裂。目的1采用单层巨泡体外发芽实验，结合光镜和冷冻电子显微镜技术，探讨HSV-1细胞膜变形的机制。膜萌发试验也将用于在体外重建衣壳萌发。目标2的目标是确定HSV-1 NEC的结构，并通过结构导向突变结合体外试验和重组病毒的体内特征来定位功能重要的区域。目的3通过将生化和结构研究扩展到伪狂犬病、Epstein-Barr和Kaposi肉瘤疱疹病毒的NEC，重点揭示NEC机制在α和伽马疱疹病毒中的保守和病毒特异性特征。拟议的工作将结合生物化学和结构生物学的专业知识(Heldwein实验室)和病毒诱变和活细胞荧光成像的专业知识(Smith实验室)。这些研究的结果将是NEC的结构以及它在核外泄过程中使膜变形的机制。获得NEC的晶体结构将对疱疹病毒学领域产生重大影响，因为它提供了任何其他方法无法获得的机制和功能见解。此外，对NEC介导的膜出芽的详细体外研究可能会揭示一种新的膜出芽机制。重要的是，通过将NEC突变体的体外特性与相应重组病毒的体内表型相关联，NEC的结构和体外萌发模型的观察将直接与感染细胞的核外泄有关。详细了解NEC在核外移过程中膜重塑的结构机制不仅有利于疱疹病毒领域，还将极大地促进当前对细胞过程的了解。

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.