Single-particle analysis of HSV-1 membrane fusion mechanism

HSV-1膜融合机制的单粒子分析

• 批准号: 10252827
• 负责人: Ekaterina Heldwein
• 金额: $ 19.91万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-03 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10252827
• 关键词: Address; Biological; Biological Assay; Blindness; Cell fusion; Cell membrane; Cells; Cellular Membrane; Characteristics; Data; Dyes; Encephalitis; Event; Future; Glycoproteins; Goals; Herpes Labialis; Herpesviridae; Herpesvirus 1; Human; Human Herpesvirus 2; Image; Immunocompromised Host; Individual; Infection; Investigation; Kinetics; Knowledge; Label; Lead; Life; Lipid Bilayers; Lipids; Liquid substance; Measurement; Measures; Mediating; Membrane; Membrane Fusion; Methodology; Microscopy; Modeling; Molecular Conformation; Mutagenesis; Mutation; Nature; Newborn Infant; Pathway interactions; Process; Protocols documentation; Reagent; Reporter; Research; Role; Series; Signal Transduction; Simplexvirus; Structure; System; Technology; Therapeutic; Time; Vaccines; Vesicle; Vesicular stomatitis Indiana virus; Viral; Virion; Virus; Virus Diseases; Visualization; Work; aqueous; bone; combat; conformational conversion; design; env Gene Products; experimental study; genital herpes; innovation; lipophilicity; mutant; novel; particle; protein function; receptor; receptor binding; success; tool; virus envelope

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. This number far exceeds the number of glycoproteins utilized by most other enveloped viruses complicating mechanistic studies of HSV-1-mediated entry and fusion. We now know that in HSV-1, as in other herpesviruses, the receptor-binding, regulatory, and fusogenic functions are distributed among several glycoproteins. gB is the conserved fusogen that, by analogy with other viral fusogens, is thought to facilitate membrane merger by undergoing large-scale refolding that brings the viral and cellular membranes together. However, gB is unusual in requiring several additional proteins for function. Therefore, understanding how gB works to mediate fusion during cell entry requires direct measurements of its fusogenic activity. Recently, single-particle tracking (SPT) has emerged as a powerful tool for quantitative studies of fusion of individual virions with fluid, supported lipid bilayers using total internal reflection microscopy. The SPT approach enables direct visualization and kinetic measurements of the viral fusion pathway and has been successfully used with both pH-triggered and receptor-triggered fusogens. The goal of this exploratory proposal is to develop single-particle imaging of HSV-1 fusion with supported lipid bilayers to visualize different stages in fusion, measure their kinetic parameters, identify kinetic intermediates, and correlate them with structural rearrangements in gB. Given that the SPT approach has not yet been applied to viruses that utilize >1 glycoprotein and may be challenging to apply to HSV-1 that has 15 envelope proteins, Aim 1 will utilize Vesicular Stomatitis Virus (VSV) virions lacking the native fusogen G and pseudotyped with HSV-1 entry glycoproteins gB, gD, gH, and gL (VSVDG-BHLD), which retain key characteristics of HSV-1 entry. In Aim 2, the SPT approach will be extended to HSV-1, a more complicated yet more biologically relevant system. The scientific premise of the proposed work is that harnessing the power of single-particle imaging of virion fusion provides a unique opportunity to 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。这个数字远远超过了使用的糖蛋白的数量 大多数其他包膜病毒使HSV-1介导的进入和融合的机械研究变得复杂。我们现在 知道在HSV-1中，与其他疱疹病毒一样，受体结合，调节和融合功能是 分布在几种糖蛋白中。 GB是保守的融合原，与其他病毒相比 熔融剂被认为可以通过进行大规模重新折叠来促进膜合并，从而带来病毒和 细胞膜在一起。但是，GB在需要几种额外的蛋白质功能方面是不寻常的。 因此，了解GB在细胞进入过程中的介导融合的工作方式需要直接测量其 融合活性。最近，单粒子跟踪（SPT）已成为定量的强大工具 使用总内反射显微镜将单个病毒与液体，液体，支持的脂质双层融合的研究。 SPT方法可以直接可视化和动力学测量病毒融合途径，并具有 已成功用于pH触发和受体触发的融合原。这个探索的目标 建议是用支持的脂质双层开发HSV-1融合的单粒子成像，以可视化不同 融合中的阶段，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。鉴于SPT方法尚未应用于使用的病毒 > 1个糖蛋白，可能是适用于具有15个信封蛋白的HSV-1的挑战，AIM 1将使用 缺乏天然富菌素G并使用HSV-1进入伪型的囊泡口腔炎病毒（VSV）病毒 糖蛋白GB，GD，GH和GL（VSVDG-BHLD）保留了HSV-1进入的关键特征。在AIM 2中， SPT方法将扩展到HSV-1，这是一个更复杂，更复杂的生物学相关系统。 拟议工作的科学前提是利用Virion Fusion的单粒子成像的力量 提供了一个独特的机会来解决HSV介导的膜融合中的挥之不去的问题 机制，其最终目标是更充分地重建HSV-1介导的融合途径。