Roles of the cytoplasmic domains of the HSV-1 glycoproteins gH and gB in membrane fusion

HSV-1 糖蛋白 gH 和 gB 胞质结构域在膜融合中的作用

• 批准号: 10382816
• 负责人: Zemplen Pataki
• 金额: $ 4.25万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-16 至 2023-12-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382816
• 关键词: Antibodies; Binding; Biological Assay; Biology; Blindness; Cell fusion; Cell membrane; Cells; Chimera organism; Chimeric Proteins; Closure by clamp; Complex; Cytoplasmic Tail; Data; Disease; Drug Targeting; Encephalitis; Engineering; Event; Fellowship; Fetus; Fluorescence; Follow-Up Studies; Future; Genital; Genitalia; Glycoproteins; Goals; Herpes Labialis; Herpesviridae; Herpesvirus 1; Human; Hydrophobic Interactions; Immunocompromised Host; Individual; Infection; Knowledge; Life; Link; Luciferases; Maps; Measures; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Mission; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mutation; National Institute of Allergy and Infectious Disease; Oral; Pain; Pathway interactions; Physicians; Population; Process; Proteins; Regulation; Regulatory Element; Research; Research Training; Role; Scientist; Side; Signal Transduction; Simplexvirus; Sodium Chloride; Structure; Testing; Viral; Virus Diseases; Work; base; combat; experimental study; innovation; insight; mutant; novel; novel therapeutics; pathogen; prevent; protein protein interaction; receptor; vaccine development; virus envelope

Project Summary/Abstract Herpes Simplex Virus 1 (HSV-1) enters and infects cells by fusing its viral envelope with target cell membranes. Membrane fusion is catalyzed by the HSV-1 fusogen, glycoprotein B (gB). Based on our knowledge of other viral fusogens, gB is thought to irreversibly refold from its unstable prefusion form to its low energy postfusion from through large conformational changes that merge the viral and target membranes. However, unlike most enveloped viruses which catalyze fusion using a single protein, the fusion pathway in HSV-1 is more complex and is unusual, requiring three additional glycoproteins, gD, gL, and gH. In the hypothesized fusion pathway, gD binds a receptor on the target cell and activates the gH/gL complex, which activates gB to refold. However, it is not known how gB is maintained in its metastable prefusion conformation prior to fusion triggering, nor how gH activates gB to cause fusion. Our prior work suggests the cytoplasmic regions of gB and gH regulate this process, with the gB cytoplasmic domain (CTD) stabilizing prefusion gB and the gH cytotail (CT) activating gB to refold. Elucidating the mechanisms by which these regions regulate gB fusogenic activity is essential for understanding how the viral glycoproteins accomplish membrane fusion during viral infection. The objective of this proposal is to determine how the gB CTD stabilizes the prefusion conformation prior to fusion triggering, and how the gH CT interacts with gB to cause fusion. In Aim 1, I will identify regions in the gB CTD that are responsible for stabilizing gB by mutagenesis and an unfolding assay and determine whether increased CTD stabilization decreases fusion. In Aim 2, I will identify residues involved in the gH-gB interaction by mutagenesis and a protein-protein interaction assay, and their effect on fusion via a cell-cell fusion assay. These experiments will illuminate how cytoplasmic regions of gB and gH interact and regulate gB activity in the HSV-1 fusion pathway. The proposal is significant because it investigates the unknown events between gH activation and gB refolding that currently limit our understanding of herpesvirus fusion. The proposal is innovative because it uses original hypotheses to investigate a novel mechanism of protein activity regulation in the unique herpesviral fusion pathway. Successful completion of the proposed research would advance the HSV-1 field by increasing our understanding of the different steps in HSV-1 fusion in molecular detail. In line with the mission of NIAID, this would be an important step towards better treating and preventing HSV-1 infection. This fellowship will provide outstanding research training on my path to becoming a physician-scientist.

项目摘要/摘要 单纯疱疹病毒1（HSV-1）通过将其病毒包膜与靶细胞融合而进入和感染细胞 膜。膜融合是由HSV-1融合原，糖蛋白B（GB）催化的。基于我们 对其他病毒式融合剂的了解，GB被认为是从其不稳定的预融合形式不可逆转地重新折叠为低的 能量后通过大构象变化融合了病毒和靶膜。 但是，与大多数使用单个蛋白质催化融合融合的包围病毒不同，融合途径 HSV-1更为复杂，不寻常，需要另外三种糖蛋白GD，GL和GH。在 假设的融合途径，GD结合了靶细胞上的受体并激活GH/GL复合物，该复合物 激活GB进行重新分配。但是，尚不清楚如何保持GB的亚稳态构象 在融合触发之前，也没有GH激活GB引起融合。我们先前的工作表明细胞质 GB和GH的区域调节了该过程，GB细胞质结构域（CTD）稳定诱发GB和 GH细胞尾（CT）激活GB以重新分配。阐明这些区域调节GB的机制 融合活性对于理解病毒糖蛋白如何完成膜融合至关重要 在病毒感染期间。该提案的目的是确定GB CTD如何稳定预选 在融合触发之前的构象，以及GH CT如何与GB相互作用以引起融合。在AIM 1中，我会 识别GB CTD中负责通过诱变稳定GB的区域 并确定增加的CTD稳定是否会降低融合。在AIM 2中，我将确定涉及的残留物 在通过诱变和蛋白质 - 蛋白质相互作用测定的GH-GB相互作用中，以及它们对融合的影响 细胞 - 细胞融合测定。这些实验将阐明GB和GH的细胞质区域如何相互作用，并且 调节HSV-1融合途径中的GB活性。该提议很重要，因为它调查了 GH激活与GB重新折叠之间的未知事件目前限制了我们对疱疹病毒的理解 融合。该提案具有创新性，因为它使用原始假设来研究一种新的机制 蛋白质活性调节在独特的疱疹病毒融合途径中。成功完成拟议的 研究将通过增加我们对HSV-1融合中不同步骤的理解来推动HSV-1领域 分子细节。符合Niaid的使命，这将是迈向更好地治疗的重要一步 并防止HSV-1感染。该奖学金将在我的道路上提供出色的研究培训 成为医师科学家。