Early Events in Herpes Simplex Virus Entry

描述(申请人提供)：单纯疱疹病毒(HSV)可导致多种人类疾病，从轻微的冻疮到更严重的疾病，如脑炎。这种大型DNA病毒的包膜含有11种糖蛋白，其中4种是病毒进入敏感细胞所必需的，分别是gB、gD、Gh和gl。病毒侵入和HSV糖蛋白诱导的细胞-细胞融合涉及gD与特定细胞受体的相互作用，随后囊膜与细胞膜融合，这需要gB和Gh/gl异源二聚体。虽然gD只存在于甲型疱疹病毒中，但gB和Gh/gl同源物在所有疱疹病毒中都存在。这方面的应用主要集中在HSV进入和HSV糖蛋白诱导的细胞-细胞融合过程中。由于GD胞外结构域的C末端向N端折叠，因此受体结合部位被隐藏。因此，GD与受体的结合需要对GD的构象改变，使GD胞外区的C末端远离GD的核心。这种运动和暴露出来的Gd残留物以某种方式触发了Gb和Gh/g1进行的病毒-细胞和细胞-细胞融合的下一步。这些步骤在很大程度上是未知的。结构数据显示，Gb是一种病毒FusoGen，但它只与Gh/gl协同作用。此外，虽然目前已知GB的结构域结构，但这种复杂蛋白的各个部分的功能仍有待充分阐明。我的实验室已经构建了Gd、Gb和Gh的突变体，这些突变体不能在病毒与细胞或细胞与细胞的融合中发挥作用，但它们表型无效的原因尚不清楚。我们将结合生化、物理、化学、突变和可视化的方法来了解HSV四种糖蛋白协同进行病毒-细胞和细胞-细胞融合的机制。最先进的技术，如脂质体浮选、基于FRET的和基于荧光的脂类混合(半融合)和内容混合(完全融合)分析将被用来剖析各种糖蛋白突变体的表型。我们将使用光学生物传感器(Biacore)石英晶体微天平(Q-SENSE)技术来检测在不同环境条件下接触可溶性受体和单抗的固定完整病毒粒子的变化。我们将使用一种新开发的涉及黄色荧光蛋白双分子互补的方法来研究细胞-细胞融合前和细胞-细胞融合期间发生的四个糖蛋白的异源相互作用。这一新方法将与活细胞共聚焦显微镜相结合，实时显示细胞-细胞融合过程中发生的相互作用。提出了两个具体的目标：1)加深我们对gB结构和功能的了解；2)研究HSV糖蛋白如何协同完成病毒与细胞和细胞与细胞的融合。这些研究将增强我们对HSV进入和病毒细胞融合的理解，并可能为新的治疗方法提供靶点。与公共卫生相关：单纯疱疹病毒(HSV)可导致许多人类疾病，但最常见的是唇疱疮和脑炎。病毒的外表面含有进入宿主细胞并导致疾病所需的蛋白质，我使用分子生物学方法来了解这些蛋白质如何协同工作，使病毒能够进入宿主细胞。我的研究可能会提出新的治疗方法来阻止病毒进入它最喜欢的目标细胞。

DESCRIPTION (provided by applicant): Herpes simplex viruses (HSVs) cause a variety of human diseases, from mild cold sores to more serious ones such as encephalitis. This large DNA virus contains 11 glycoproteins in its envelope, of which 4 are essential for virus entry into susceptible cells, called gB, gD, gH and gL. Virus entry and HSV glycoprotein induced cell-cell fusion involve an interaction of gD with a specific cell receptor, followed by fusion of the envelope with a cellular membrane that requires gB and the gH/gL heterodimer. Whereas gD is present only in alphaherpesviruses, gB and gH/gL homologues are found in all herpesviruses. This application focuses on the processes of HSV entry and HSV glycoprotein-induced cell-cell fusion. Because the C-terminus of the gD ectodomain folds back towards the N-terminus of the native protein, receptor binding sites are hidden. Binding of gD to a receptor therefore requires a conformational change to gD that moves the C-terminus of the gD ectodomain away from the core of gD. This movement and the residues of gD that become exposed somehow trigger next steps of virus-cell and cell-cell fusion carried out by gB and gH/gL. These steps are largely unknown. Structural data show that gB is a viral fusogen, but it only functions in concert with gH/gL. Moreover, although the domain structure of gB is now known, the functions of the individual parts of this complex protein remain to be fully elucidated. My laboratory has constructed mutants of gD, gB and gH that are unable to perform their roles in virus-cell or cell-cell fusion, but the reasons for their null phenotypes are not yet understood. We will use a combination of biochemical, physical chemical, mutational and visual approaches to understand the mechanism by which the four HSV glycoproteins cooperate to carry out virus-cell and cell-cell fusion. State of the art techniques such as liposome flotation, FRET based and fluorescent based assays of lipid mixing (hemifusion) and content mixing (full fusion) will be employed to dissect the phenotypes of the various glycoprotein mutants. We will use optical biosensor (BIAcore) Quartz Crystal Microbalance (Q-sense) technology to examine changes to immobilized intact virions exposed to soluble receptors and monoclonal antibodies under various environmental conditions. We will study heterologous interactions of the quartet of glycoproteins that occur before and during cell-cell fusion using a newly developed assay involving bimolecular complementation of yellow fluorescent protein. This novel approach will be coupled with live cell confocal microscopy to visualize interactions that occur during cell-cell fusion in real time. Two specific aims are proposed: 1) to enhance our understanding of gB structure and function; and 2) to study how the HSV glycoproteins cooperate to accomplish virus-cell and cell-cell fusion. These studies will enhance our understanding of HSV entry, of virus cell fusion in general, and may suggest targets for novel therapeutics. PUBLIC HEALTH RELEVANCE: Herpes simplex virus (HSV) causes many human diseases but the most common are cold sores and encephalitis. The outer surface of the virus contains the proteins that are needed to gain entry into host cells and cause disease and I use molecular biological approaches to understand how these proteins work together to allow the virus to enter host cells. My research may suggest new treatments to stop the virus from gaining entry into its favorite target cells.