In-vitro analysis of HSV-1 membrane fusion mechanism

HSV-1膜融合机制的体外分析

基本信息

批准号：
10230779
负责人：
Ekaterina Heldwein
金额：
$ 25.48万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-17 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10230779
关键词：
Address Biological Assay Blindness Cell fusion Cell membrane Cells Data Dissection Encephalitis Future Glycoproteins Goals Herpes Labialis Herpesviridae Herpesvirus 1 Human Human Herpesvirus 2 Image Immunocompromised Host In Vitro Individual Infection Investigation Kinetics Knowledge Label Life Lipid Bilayers Liquid substance Measurement Measures Mediating Membrane Membrane Fusion Membrane Lipids Methodology Microscopy Modeling Molecular Conformation Mutagenesis Nature Newborn Infant Participant Pathway interactions Process Proteins Reagent Reporter Research Role Series Side Simplexvirus Structure System Technology Therapeutic Time Vaccines Vesicular stomatitis Indiana virus Viral Virion Virus Virus Diseases Visualization Work bone combat conformational conversion design env Gene Products follow-up genital herpes innovation novel particle protein function receptor receptor binding reconstitution tool virus envelope

项目摘要

PROJECT SUMMARY/ABSTRACT Herpes Simplex virus type 1 (HSV-1) requires four glycoproteins for cell entry and membrane fusion – gB, gD, gH, and gL – in addition to a cellular receptor. A thorough knowledge of membrane fusion mechanisms is essential for understanding how herpesviruses penetrate cells and finding ways to inhibit this process. Due to the complexity of this process, many fundamental questions endure despite decades of research. Moreover, traditional entry assays rely on downstream reporters and do not measure fusion directly. Studies of cell-cell fusion of uninfected receptor-bearing cells expressing the four essential HSV entry glycoproteins have yielded much of the current mechanistic knowledge of membrane fusion, including the prevalent regulatory cascade model. However, the cell-cell fusion system, while informative, fails to capture the proper context of virus-cell fusion. Accurate, systematic dissection of the HSV-mediated membrane fusion mechanism thus requires an experimentally tractable system that enables direct visualization and kinetic measurements of the viral fusion. The goal of this exploratory proposal is to reconstitute HSV-1 fusion in vitro and to characterize it at a single- virion level by imaging fusion of individual virions with fluid, supported lipid bilayers using total internal reflection microscopy. This approach will be used to visualize different stages in fusion, measure their kinetic parameters, identify kinetic intermediates, and correlate them with structural rearrangements in gB. In addition to HSV-1, the proposed studies will employ Vesicular Stomatitis Virus (VSV) virions lacking the native fusogen G and pseudotyped with HSV-1 entry glycoproteins gB, gD, gH, and gL (VSVDG-BHLD). The use of this simplified, “bare-bones” system will benefit this work by excluding the potential effects of the 12 other envelope proteins. In-vitro reconstitution of fusion and its characterization at a single particle level will address the lingering questions in HSV-mediated membrane fusion mechanism, with the ultimate goal of reconstructing the HSV-1-mediated fusion pathway more fully.

项目摘要/摘要单纯疱疹病毒1型（HSV-1）需要四种用于细胞进入和膜融合的糖蛋白 - GB，GD， GH和GL - 除了细胞接收器。对膜融合机制的透彻了解是了解疱疹病毒如何穿透细胞并找到抑制此过程的方法至关重要。由于这一过程的复杂性，许多基本问题都忍受了目的地数十年的研究。而且，传统的入学分析依赖于下游记者，并且不能直接测量融合。细胞细胞研究表达四种必需HSV进入糖蛋白的未感染接收器的细胞的融合已产生膜融合的当前机械知识的许多知识，包括普遍的调节级联模型。但是，虽然有信息，但细胞电池融合系统虽然无法捕获病毒细胞的适当上下文融合。因此，精确的，系统地解剖HSV介导的膜融合机制需要实验可拖动的系统，可以直接可视化和动力学测量病毒融合。该探索性建议的目的是在体外重新建立HSV-1融合，并在单一表征它。通过将单个病毒与流体，支持的脂质双层融合的成像融合，使用全部反射显微镜。这种方法将用于可视化融合中的不同阶段，测量其动力学参数，识别动力学中间体，并将其与GB中的结构重排相关。此外对于HSV-1，拟议的研究将采用缺乏天然富菌素的囊泡口腔炎病毒（VSV）病毒用HSV-1输入糖蛋白GB，GD，GH和GL（VSVDG-BHLD）进行g和伪型。使用此简化，“裸机”系统将通过排除其他12个信封的潜在影响来使这项工作受益蛋白质。融合及其在单个粒子级别的表征的体外重构将解决在HSV介导的膜融合机制中挥之不去的问题，其最终目标是重建 HSV-1介导的融合途径更全面。