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

项目摘要/摘要 单纯疱疹病毒1型(HSV-1)通过病毒被膜与靶细胞融合进入细胞并感染细胞 膜。膜融合是由HSV-1 FusoGen、糖蛋白B(GB)催化的。基于我们的 由于了解了其他病毒融合原，GB被认为是不可逆转地从不稳定的融合形式重新折叠到其低水平 融合后的能量通过大量的构象变化合并病毒和靶膜。 然而，与大多数被包膜的病毒使用单一蛋白质催化融合不同，融合途径在 HSV-1更复杂，也不寻常，需要三种额外的糖蛋白，Gd，g1和Gh。在 假想的融合途径，Gd与靶细胞上的受体结合并激活Gh/Gl复合体，这 激活GB以重新折叠。然而，目前尚不清楚gB如何保持其亚稳定的融合前构象。 在融合触发之前，也不知道Gh如何激活Gb导致融合。我们之前的工作表明，细胞质 GB和Gh区域调节这一过程，GB细胞质结构域(CTD)稳定灌流 Gh细胞尾巴(CT)激活gB折叠。阐明这些区域调控GB的机制 融合活性是理解病毒糖蛋白如何完成膜融合的关键。 在病毒感染期间。本建议的目的是确定GB CTD如何稳定输液。 融合触发前的构象，以及Gh CT如何与GB相互作用导致融合。在《目标1》中，我将 通过突变和去折叠实验确定GB CTD中负责稳定GB的区域 并确定CTD稳定度增加是否会降低融合。在目标2中，我将确定涉及的残留物 在Gh-GB相互作用中，通过突变和蛋白质-蛋白质相互作用分析，以及它们对融合的影响 细胞-细胞融合实验。这些实验将阐明Gb和Gh的细胞质区域如何相互作用和 调节HSV-1融合途径中的gB活性。这项提案意义重大，因为它调查了 Gh激活和gB折叠之间的未知事件目前限制了我们对疱疹病毒的理解 核聚变。这一提议具有创新性，因为它使用了原始的假设来研究一种新的 独特的疱疹病毒融合途径中的蛋白质活性调节。圆满完成拟议中的 研究将通过增加我们对HSV-1融合的不同步骤的理解来促进HSV-1领域的发展 在分子细节上。根据NIAID的使命，这将是朝着更好的治疗迈出的重要一步 预防HSV-1感染。这项奖学金将为我提供出色的研究培训，帮助我 成为一名内科科学家。

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.