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

项目总结/摘要 单纯疱疹病毒1型（HSV-1）通过病毒包膜与靶细胞融合进入细胞并感染 膜。膜融合由HSV-1融合原糖蛋白B（gB）催化。基于我们 根据对其他病毒融合原的了解，认为gB从其不稳定的融合前形式不可逆地重折叠到其低融合水平。 能量后融合通过大的构象变化，合并病毒和目标膜。 然而，与使用单一蛋白质催化融合的大多数包膜病毒不同， HSV-1更复杂，并且不常见，需要三种额外的糖蛋白，gD，gL和gH。在 假设的融合途径，gD结合靶细胞上的受体并激活gH/gL复合物， 激活gB重折叠然而，尚不清楚gB是如何维持其亚稳态融合前构象的 也不知道gH如何激活gB以引起融合。我们之前的工作表明细胞质 gB和gH的区域调节该过程，其中gB胞质结构域（CTD）稳定融合前gB， gH cytotail（CT）激活gB重折叠。阐明这些区域调节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。本提案的目的是确定gB CTD如何稳定预融合 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。在目标1中，我将 通过诱变和解折叠试验鉴定gB CTD中负责稳定gB的区域 并确定CTD稳定性增加是否会降低融合。在目标2中，我将确定涉及的残基 通过诱变和蛋白质-蛋白质相互作用测定， 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， 调节HSV-1融合途径中的gB活性。该提案意义重大，因为它调查了 gH激活和gB重折叠之间的未知事件，目前限制了我们对疱疹病毒的理解 核聚变该提案是创新的，因为它使用了原始假设来研究一种新的机制， 在独特的疱疹病毒融合途径中的蛋白活性调节。圆满完成拟议的 研究将通过增加我们对HSV-1融合不同步骤的理解来推进HSV-1领域 在分子细节上。根据NIAID的使命，这将是朝着更好地治疗 预防HSV-1感染。这项奖学金将提供出色的研究培训，我的道路上， 成为一名物理学家兼科学家

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.