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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(申请人提供)：单纯疱疹病毒1型和2型(HSV-1和HSV-2)是人类病原体，可建立终身潜伏感染并导致许多疾病。感染是由病毒被膜和宿主细胞膜的合并启动的，这是由病毒FusoGen GB在附加蛋白Gd和Gh/gl以及细胞内的GD受体的帮助下催化的。与其他病毒融合原相似，GB被认为是从融合后的前融合形成了一系列大的构象变化，这些变化将复性与膜融合强烈地结合在一起。虽然后融合形式的结构已知，但尚未表征再融合形式。缺乏gB的融合结构是我们知识中的一大空白，阻碍了破译疱疹病毒细胞进入机制。这项研究的长期目标是阐明疱疹病毒进入和细胞传播过程中膜融合的原子水平机制。这项建议的目的是稳定HSV-1 GB的灌流形式，并获得其结构。到目前为止，对其他病毒融合原起作用的方法在GB中都失败了；因此，需要新的策略来获得GB的预融合结构。这一R21建议是由一个创新的中心假设推动的，该假设认为GB的预融合形式是通过与膜的相互作用来稳定的，并且需要存在膜-近端、跨膜和细胞质结构域。这一假设将在两个特定的目标中得到验证：Aim 1将使用GB伪型VSV G病毒粒子，一种在初步数据中产生的新试剂，以获得病毒被膜中GB预融合形式的低分辨率冷冻重建，以确定其预融GB在其天然状态下的整体结构。在目标2中，将使用膜模拟物-洗涤剂/脂胶束和纳米盘-来提取可溶的、预融合形式的全长gB基因在昆虫细胞中表达。这种形式将被用于结晶试验和低温EM单粒子3D重建，这将被用来生成伪原子模型。除了灌流的GB胞外区的结构外，拟议的工作还将产生膜近端、跨膜区和细胞质结构域的结构。这一提议是创新的，因为它提出了新的假设和旨在稳定融合形式的创新方法。这一建议意义重大，因为它解决了一个阻碍HSV和其他疱疹病毒细胞进入机制解体的重要问题。在任何分辨率下，gB融合形式的结构都将是一项重要发现，对疱疹病毒领域具有重大影响。它将通过提供绘制功能数据的3D框架，为许多其他研究人员未来的工作提供信息。此外，它可能最终解释了为什么GB需要其他蛋白质作为FusoGen发挥功能。这里获得的结果将成为未来R01建议的基础，以确定灌流GB的原子级晶体结构，并利用结构和生物物理方法阐明其融合机制。除了这种结构，找到一种策略来稳定Gb的预融合形式，可能会为成功研制针对HSV的亚单位疫苗铺平道路。