Biophysical and structural analysis of the herpesviral nuclear budding machinery

项目摘要/摘要 疱疹病毒是双链dna包裹的病毒，是感染最复杂的病毒之一。 动物。这项提案的重点是核出口，这是组装和释放核燃料的关键、保守的步骤 子代病毒粒子，在此期间，核衣壳从细胞核转移到细胞质中，在那里它们 成熟为具有感染性的病毒粒子。病毒核出口复合体(NEC)是这一过程中的关键角色。vbl.使用 在体外模型系统中，我们先前发现NEC是一个完整的、病毒编码的膜 在核膜上运行的萌芽机器。然而，理解核能的一个主要障碍是 出口是缺乏关于NEC如何产生导致发芽的膜曲率的知识。这个 这项研究的长期目标是阐明疱疹病毒核外泄的详细机制，两者都 对这一不寻常的过程有一个基本的了解，并确定和描述新的目标 抗病毒治疗设计。这一提议是由中心假设驱动的，基于实质性的 初步数据表明，NEC/膜相互作用和NEC齐聚成膜是主要的 使负膜曲率形成和发芽的驱动力。这项提议的目的是 是系统地剖析单纯疱疹病毒(HSV)中NEC的萌发机制 必需的蛋白质/蛋白质和蛋白质/膜相互作用和萌发中间体 多学科方法，包括低温电子显微镜和 电子自旋共振。拟议工作的科学前提是全面剖析 NEC介导的负膜曲率的形成是解开异常的关键 疱疹病毒核外泄的机制及其阻断策略的发展。除了病毒，这项研究还将 扩展我们对膜变形机制的有限的机械理解。这个 提案之所以具有创新性，是因为它研究了一种不同寻常的机制，并以原始假设为指导， 采用新颖的方法。这项提议意义重大，因为它旨在推动我们的机械化 了解病毒复制周期中的一个重要步骤，目的是确定新的目标 治疗干预，因为它提供了开发负曲率模型的机会 编队，目前是一个黑匣子。

PROJECT SUMMARY/ABSTRACT Herpesviruses are double-stranded-DNA enveloped viruses that are among the most complex viruses infecting animals. This proposal focuses on nuclear egress, a critical, conserved step in the assembly and release of progeny virions during which nucleocapsids are translocated from the nucleus into the cytoplasm where they mature into infectious virions. The viral nuclear egress complex (NEC) is the key player in this process. Using in vitro model systems, we previously discovered that the NEC is a complete, virally encoded membrane budding machine that operates at the nuclear envelope. However, a major barrier to understanding nuclear egress is the lack of knowledge of how the NEC generates membrane curvature that results in budding. The long-term goal of this research is to elucidate the detailed mechanism of herpesvirus nuclear egress, both to gain a fundamental knowledge of this unusual process and to identify and characterize novel targets for antiviral therapeutic design. This proposal is driven by the central hypothesis, based on substantial preliminary data, that both NEC/membrane interactions and NEC oligomerization into a coat are the major driving forces that enable negative membrane curvature formation and budding. The objective of this proposal is to systematically dissect the NEC budding mechanism in Herpes Simples virus (HSV) by characterizing essential protein/protein and protein/membrane interactions and budding intermediates by employing a multidisciplinary approach, which includes the cutting-edge approaches of cryoelectron microscopy and electron spin resonance. The scientific premise of the proposed work is that a comprehensive dissection of the NEC-mediated formation of negative membrane curvature is essential for unraveling the unusual mechanism of herpesviral nuclear egress and developing strategies to block it. Beyond viruses, this study will expand our limited mechanistic understanding of the mechanisms of membrane deformation in general. The proposal is innovative because it investigates an unusual mechanism, is guided by an original hypothesis, and employs novel approaches. The proposal is significant because it aims to advance our mechanistic understanding of an essential step in viral replication cycle with the goal of identifying new targets for therapeutic interventions and because it provides an opportunity to develop models of negative curvature formation, currently a black box.