The prefusion form of HSV-1gB

HSV-1gB 的预灌输形式

基本信息

批准号：
8967556
负责人：
Ekaterina Heldwein
金额：
$ 20.63万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8967556
关键词：
Address Architecture Blindness Cell membrane Cells Collaborations Cryoelectron Microscopy Crystallization Cytoplasmic Tail Data Detergents Electron Microscopy Encephalitis Environment Future Glycoproteins Goals Health Herpes Labialis Herpesviridae Herpesvirus 1 Human Human Herpesvirus 2 Imagery Immunocompromised Host Individual Infection Insecta Knowledge Lead Length Life Lipids Maps Mediating Membrane Membrane Fusion Micelles Modeling Molecular Conformation Molecular Machines Nature Newborn Infant Pathway interactions Proteins Reagent Research Research Personnel Resolution Resources Series Simplexvirus Structure Subunit Vaccines Surface Therapeutic Therapeutic Intervention Vaccines Vesicular stomatitis Indiana virus Viral Viral Proteins Virion Virus Virus Diseases Work base biophysical techniques combat design electron tomography genital herpes innovation insight latent infection mimetics nanodisk novel novel strategies particle pathogen receptor reconstitution reconstruction research study tomography

项目摘要

DESCRIPTION (provided by applicant): Herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) are human pathogens that establish lifelong latent infections and cause a number of ailments. Infection is initiated by the merger of the viral envelope and a host cell membrane, which is catalyzed by the viral fusogen gB with the help of additional proteins, gD and gH/gL plus a cellular receptor for gD. By analogy with other viral fusogens, gB is thought to refold from the prefusion to the postfusion form in a series of large conformational changes that energetically couple refolding to membrane fusion. While the structure of the postfusion form is known, the prefusion form has not yet been characterized. The lack of the prefusion structure of gB is a major gap in our knowledge that has hindered the deciphering of the herpesvirus cell entry mechanism. The long-term goal of this research is the elucidation of the atomic-level mechanism of membrane fusion during herpesvirus entry and cell spread. The objective of this proposal is to stabilize the prefusion form of HSV-1 gB and to obtain its structure. Approaches that worked for other viral fusogens have so far failed with gB; thus, new strategies towards obtaining the prefusion structure of gB are needed. This R21 proposal is driven by an innovative central hypothesis that the prefusion form of gB is stabilized by interaction with membrane and requires the presence of membrane-proximal, transmembrane, and cytoplasmic domains. This hypothesis will be examined in two Specific Aims: Aim 1 will use gB-pseudotyped VSV?G virions, a novel reagent generated in preliminary data, to obtain a low-resolution cryoET reconstruction of the prefusion form of gB in the viral envelope to determine its overall architecture of the prefusion gB in its native-like state. In Aim 2, membrane mimetics - detergent/lipid micelles and nanodiscs - will be used to extract the soluble, prefusion form of the full-length gB expressed in insect cells. This form will be used in crystallization trials and in cryoEM single-particle 3D reconstruction, which will be used to generate a pseudoatomic model. In addition to the structure of the prefusion gB ectodomain, the proposed work will also yield the structures of the membrane-proximal, transmembrane, and cytoplasmic domains. This proposal is innovative because of its novel hypothesis and innovative approaches aimed at stabilizing the prefusion form. This proposal is significant because it addresses an important problem that has impeded the unraveling of the cell entry mechanism of HSV and other herpesviruses. The structure of the prefusion form of gB, at any resolution, would be an important discovery with a major impact on the herpesvirus field. It would inform the future work of many other investigators by providing a 3D framework for mapping functional data. Moreover, it may finally explain why gB requires other proteins to function as a fusogen. The results obtained here will form the basis for a future R01 proposal to determine the atomic-level crystal structure of prefusion gB and to elucidate its fusogenic mechanism using structural and biophysical approaches. Beyond the structure, finding a strategy to stabilize gB in its prefusion form may pave the way to a successful subunit vaccine against HSV.

描述（由申请人提供）：单纯疱疹病毒类型1和2（HSV-1和HSV-2）是人类病原体，它们建立了终生潜在感染并引起许多疾病。感染是由病毒包膜和宿主细胞膜的合并引发的，宿主细胞膜是由病毒型GB催化的，借助其他蛋白质，GD和GH/GH/GH/GL以及用于GD的细胞受体。通过与其他病毒式融合剂类似，GB被认为是从在一系列大型构象变化中，对后灌注形式的融合将其逐渐融化为膜融合。虽然已知后灌注形式的结构，但尚未表征灌注形式。在我们所知中，缺乏GB的预选结构是一个主要差距，它阻碍了疱疹病毒细胞进入机制的解密。这项研究的长期目标是阐明疱疹病毒进入和细胞扩散期间膜融合的原子水平机制。该提案的目的是稳定HSV-1 GB的预选形式并获得其结构。迄今为止，GB的方法对其他病毒式融合剂有效。因此，需要新的策略获得GB的预融合结构。该R21提案是由一种创新的中心假设驱动的，即GB的预融合形式通过与膜的相互作用稳定，需要膜 - 透明膜，跨膜和细胞质结构域的存在。该假设将在两个具体的目的中进行检查：AIM 1将使用初步数据中产生的一种新型试剂GB型VSV vsv？g Virions，以获取病毒式信封中GB预融合形式的低分辨率低分辨率重建形式，以确定其在其天然型GB状态的整体结构。在AIM 2中，膜模拟物 - 洗涤剂/脂质胶束和纳米散发 - 将使用全长GB在昆虫细胞中表达。该形式将用于结晶试验和冷冻单粒子3D重建中，该重建将用于生成假子模型。除了预灌注GB外生域的结构外，所提出的工作还将产生膜 - 透明体，跨膜和细胞质结构域的结构。该提议具有创新性，因为它具有新颖的假设和旨在稳定预选形式的创新方法。该提案很重要，因为它解决了一个重要的问题，该问题阻碍了HSV和其他疱疹病毒的细胞进入机制的解散。在任何分辨率下，GB的预融合形式的结构将是一个重要的发现，对疱疹病毒场有重大影响。它将通过提供映射功能数据的3D框架来为许多其他研究人员的未来工作提供信息。此外，它最终可能解释了为什么GB需要其他蛋白质作为融合原作用。此处获得的结果将构成未来R01建议的基础，以确定预融合GB的原子水平晶体结构，并使用结构和生物物理方法阐明其融合机制。除了结构之外，寻找以稳定GB形式稳定GB的策略可能为成功使用HSV的亚基疫苗铺平了道路。