Uncovering the Regulatory Role of gH/gL in HSV Fusion

描述（由申请人提供）：单纯疱疹病毒会引起人类疾病，从唇疱疹到更严重的感染，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。与大多数病毒相反，HSV在多步骤过程中利用多种蛋白质进行融合和进入。这些事件的表征将有可能确定一般病毒介导的融合的新范例。四种HSV糖蛋白gD、gB、gH和gL是进入所必需的。gD是HSV的受体结合蛋白，而gB和gH/gL复合物形成所有疱疹病毒的核心融合机制。gB，gD和gD结合到其受体的晶体结构提供了关于这些蛋白质如何在病毒进入和细胞融合中发挥作用的关键见解。gB与VSV的融合蛋白G的结构同源性表明gB在所有疱疹病毒中均为融合蛋白。然而，与G不同，gB本身并不起作用，而是需要gH/gL。同样，先前的研究表明，gH/gL本身是一种不良的融合剂。主要基于序列基序、合成肽和突变的流行模型是，这两种弱融合子的组合通过在同一膜中（如在病毒体中）一起作用以促进融合来实现融合。与Dr. E合作。Heldwein，我们最近解决了gH/gL的晶体结构。令人惊讶的是，gH/gL具有不同于任何已知病毒融合蛋白的新结构，强烈反对其作为融合剂的作用。这种结构意味着gB是HSV的唯一内在能够引起融合的进入蛋白。因此，我们认为gH/gL具有非常不同的作用。我们假设，gH/gL的功能作为一个调节器的融合结合gB和激活它到融合状态。为了支持这一假设，我们发现gH/gL胞外域本身（不与膜结合）可以触发用gD和gB转染的携带受体的细胞的融合。在目的1中，我们将剖析由gH/gL与gB与突变体和单克隆抗体形成的复合物的性质。诸如生物传感器分析、量热法、低温EM断层扫描和X射线晶体学等技术将用于获得有关复合物的化学计量和构象的信息。当gB和gH/gL在同一细胞（顺式）和不同细胞（反式）中时，将使用双分子荧光互补（BiMC）和融合试验分析复合物形成。我们将确定突变形式的gH/gL触发gB进入融合状态的能力。在目的2中，我们将通过改变诸如WT和可溶性gH/gL的突变形式的速率和浓度等参数来测试gH/gL作为融合的调节剂。我们将使用BiMC来跟踪和比较HSV进入期间gB-gH/gL复合物的形成，当它通过在质膜上直接融合或通过内吞作用发生时。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和剖析它与gB形成的复合物的性质; 2）确定gH/gL如何激活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.