Early Events in Herpes Simplex Virus Entry

描述（由申请人提供）：单纯疱疹病毒（HSV）引起多种人类疾病，从轻度唇疱疹到更严重的疾病，如脑炎。这种大的DNA病毒在其包膜中含有11种糖蛋白，其中4种是病毒进入易感细胞所必需的，称为gB，gD，gH和gL。病毒进入和HSV糖蛋白诱导的细胞-细胞融合涉及gD与特异性细胞受体的相互作用，随后包膜与细胞膜融合，这需要gB和gH/gL异二聚体。gD仅存在于α疱疹病毒中，而gB和gH/gL同源物存在于所有疱疹病毒中。本申请集中于HSV进入和HSV糖蛋白诱导的细胞-细胞融合的过程。由于gD胞外域的C-末端向天然蛋白的N-末端折回，因此受体结合位点被隐藏。因此，gD与受体的结合需要使gD胞外域的C末端远离gD的核心移动的gD构象变化。这种移动和暴露的gD残基以某种方式触发了由gB和gH/gL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。结构数据表明，gB是一种病毒融合子，但它仅与gH/gL一起发挥作用。此外，虽然gB的结构域结构现在是已知的，这个复杂的蛋白质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，它们不能在病毒-细胞或细胞-细胞融合中发挥作用，但其无效表型的原因尚不清楚。我们将使用生物化学，物理化学，突变和视觉方法的组合来了解四种HSV糖蛋白合作进行病毒-细胞和细胞-细胞融合的机制。将采用现有技术，如脂质体浮选、基于FRET和基于荧光的脂质混合（半融合）和内容物混合（全融合）测定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）石英晶体微量天平（Q-sense）技术来检查在各种环境条件下暴露于可溶性受体和单克隆抗体的固定化完整病毒粒子的变化。我们将研究异源相互作用的四糖蛋白，细胞融合之前和过程中使用新开发的检测涉及黄色荧光蛋白的双分子互补。这种新的方法将与活细胞共聚焦显微镜结合，以观察真实的时间内细胞-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）提高我们对gB结构和功能的理解; 2）研究HSV糖蛋白如何合作完成病毒-细胞和细胞-细胞融合。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。公共卫生相关性：单纯疱疹病毒（HSV）引起许多人类疾病，但最常见的是唇疱疹和脑炎。病毒的外表面含有进入宿主细胞并引起疾病所需的蛋白质，我使用分子生物学方法来了解这些蛋白质如何共同作用以允许病毒进入宿主细胞。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靶细胞。

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.