Uncovering the Regulatory Role of gH/gL in HSV Fusion

描述(申请人提供)：单纯疱疹病毒可引起人类疾病，从冻疮到更严重的感染，其中一些可能危及生命。我们的长期目标是阐明控制HSV进入和HSV诱导的细胞融合的机制。与大多数病毒不同，单纯疱疹病毒在一个多步骤的融合和进入过程中利用多个蛋白质。这些事件的特征很可能确定病毒介导的融合的新范例。四种HSV糖蛋白Gd、Gb、Gh和Gl是进入HSV所必需的。GD是单纯疱疹病毒的受体结合蛋白，而gB和Gh/gl复合体形成了所有疱疹病毒的核心融合机制。与其受体结合的gB、gD和gD的晶体结构为这些蛋白质如何在病毒入侵和细胞融合中发挥作用提供了关键的见解。GB与VSV融合蛋白G的结构同源性表明gB在所有疱疹病毒中都是一种融合蛋白。然而，与G不同的是，GB不是单独发挥作用的，而是需要Gh/Gl。同样，先前的研究表明，Gh/g1本身就是一个糟糕的FusoGen。流行的模型主要基于序列基序、合成肽和突变，即这两个糟糕的融合子的组合通过在同一膜上(如在病毒粒子中)共同作用来促进融合。最近，我们与E.Heldwein博士合作，解决了Gh/Gl的晶体结构。令人惊讶的是，Gh/gl有一个不同于任何已知病毒融合蛋白的新颖结构，强烈反对它作为FusoGen的角色。这一结构意味着gB是HSV固有地能够引起融合的唯一进入蛋白。因此，我们建议Gh/gl扮演一个非常不同的角色。我们推测Gh/Gl通过与Gb结合并激活它进入融合状态而起到融合调节的作用。为了支持这一假说，我们发现Gh/Gl胞外结构域本身(非膜结合)可以触发携带gD和gB的受体细胞的融合。在目标1中，我们将剖析Gh/Gl与Gb与突变体和单抗形成的复合体的性质。生物传感器分析、量热、低温电子显微镜断层扫描和X射线结晶学等技术将用于获得有关络合物的化学计量和构象的信息。双分子荧光互补(BIMC)和融合分析将被用来分析Gb和Gh/Gl在同一细胞(顺式)和不同细胞(反式)中的络合物形成。我们将确定Gh/gl突变形式触发GB进入融合状态的能力。在目标2中，我们将通过改变WT的速率和浓度以及可溶性Gh/gl的突变形式来测试Gh/gl作为融合调节因子的作用。我们将使用BIMC来跟踪和比较HSV进入时通过质膜直接融合或通过内吞形成Gb-Gh/gl复合体。我们在HSV进入和融合方面的工作具有很强的临床意义，因为每一步都是针对HSV介导的疾病的治疗和疫苗的潜在靶点。为了验证我们的主要假设，我们提出了两个具体的目标：1)进一步表征Gh/gl并剖析其与Gb形成的复合体的性质；2)确定Gh/gl如何激活Gh/gl使Gb进入融合状态。

DESCRIPTION (provided by applicant): Herpes simplex virus causes human diseases, ranging from cold sores to more serious infections, some of which can be life threatening. Our long term goal has been to delineate the mechanisms that govern HSV entry and HSV induced cell fusion. In contrast to most viruses, HSV utilizes multiple proteins in a multi-step process for fusion and entry. Characterization of these events will likely identify novel paradigms for virus-mediated fusion in general. Four HSV glycoproteins, gD, gB, gH and gL are essential for entry. gD is the receptor binding protein for HSV, while gB and the gH/gL complex form the core fusion machinery for all herpesviruses. Crystal structures of gB, gD and gD bound to its receptors have provided critical insights about how these proteins function in virus entry and cell-fusion. The structural homology of gB with G, the fusion protein of VSV, suggests that gB is a fusion protein in all herpesviruses. Unlike G however, gB does not function on its own but requires gH/gL. Likewise, prior studies showed that gH/gL is a poor fusogen on its own. The prevailing model, based largely on sequence motifs, synthetic peptides and mutations, is that the combination of these two poor fusogens accomplish fusion by acting together in the same membrane (as in virions) to promote fusion. In collaboration with Dr. E. Heldwein, we recently solved the crystal structure of gH/gL. Surprisingly, gH/gL has a novel architecture distinct from any known viral fusion protein, strongly arguing against its role as a fusogen. This structure implies that gB is the only entry protein of HSV that is intrinsically capable of causing fusion. We therefore suggest that gH/gL has a very different role. We postulate that gH/gL functions as a regulator of fusion by binding to gB and activating it into a fusogenic state. In support of this hypothesis, we discovered that the gH/gL ectodomain itself (not membrane bound) can trigger fusion of receptor-bearing cells transfected with gD and gB. In Aim 1, we will dissect the nature of the complex formed by gH/gL with gB with mutants and monoclonal antibodies. Techniques such as biosensor analysis, calorimetry, cryo-EM tomography and X-ray crystallography will be used to gain information about the stoichiometry and conformation of the complex. Bimolecular fluorescence complementation (BiMC) and fusion assays will be used to analyze complex formation when gB and gH/gL are in the same cell (cis) and in different cells (trans). We will determine the ability of mutant forms of gH/gL to trigger gB into a fusogenic state. In Aim 2, we will test gH/gL as a regulator of fusion by varying such parameters as rate and concentration of the WT and mutant forms of soluble gH/gL. We will use BiMC to follow and compare gB-gH/gL complex formation during HSV entry when it occurs by direct fusion at the plasma membrane or by endocytosis. Our work on HSV entry and fusion has strong clinical significance, as each step is a potential target for therapeutics and vaccines against HSV-mediated disease. To test our main hypothesis, we propose two specific aims: 1) to further characterize gH/gL and dissect the nature of the complex it forms with gB; 2) To determine how gH/gL activates gB into a fusogenic state.