The prefusion form of HSV-1gB

描述（由申请方提供）：1型和2型单纯疱疹病毒（HSV-1和HSV-2）是人类病原体，可建立终身潜伏感染并引起多种疾病。感染是由病毒包膜和宿主细胞膜的合并引发的，这是由病毒融合原gB在另外的蛋白质gD和gH/gL加上gD的细胞受体的帮助下催化的。通过与其他病毒融合子的类比，gB被认为是从 融合前到融合后形成一系列大的构象变化，这些变化将重折叠与膜融合能量耦合。虽然融合后形式的结构是已知的，但融合前形式尚未被表征。缺乏gB的融合前结构是我们知识中的一个主要空白，这阻碍了疱疹病毒进入细胞机制的破译。本研究的长期目标是阐明疱疹病毒进入和细胞扩散过程中膜融合的原子水平机制。该提议的目的是稳定HSV-1 gB的融合前形式并获得其结构。对其他病毒融合体有效的方法迄今为止在gB中失败了;因此，需要获得gB的融合前结构的新策略。这个R21的建议是由一个创新的中心假设，即融合前形式的gB是稳定的相互作用与膜，并需要存在膜近端，跨膜，和胞质结构域。这一假设将在两个特定目标中进行检验：目标1将使用gB假型VSV？G病毒粒子（初步数据中生成的一种新型试剂），以获得病毒包膜中融合前形式gB的低分辨率cryoET重建，以确定融合前gB在其天然样状态下的整体结构。在目标2中，膜模拟物-去污剂/脂质胶束和纳米盘-将用于提取可溶的预融合形式的 在昆虫细胞中表达的全长gB。这种形式将用于结晶试验和cryoEM单粒子3D重建，这将用于生成伪原子模型。除了融合前gB胞外域的结构之外，所提出的工作还将产生近膜、跨膜和胞质结构域的结构。这一建议是创新的，因为它的新假设和创新的方法，旨在稳定的预融合形式。这一建议是重要的，因为它解决了一个重要的问题，阻碍了HSV和其他疱疹病毒的细胞进入机制的解开。在任何分辨率下，gB融合前形式的结构都将是对疱疹病毒领域产生重大影响的重要发现。它将通过提供用于映射功能数据的3D框架来告知许多其他调查人员的未来工作。此外，它可能最终解释为什么gB需要其他蛋白质作为融合剂。这里获得的结果将形成未来的R 01建议的基础，以确定融合前gB的原子级晶体结构，并阐明其融合机制，使用结构和生物物理方法。除了结构，找到一种策略，以稳定gB在其融合前的形式可能铺平了道路，成功的亚单位疫苗抗HSV。

DESCRIPTION (provided by applicant): Herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) are human pathogens that establish lifelong latent infections and cause a number of ailments. Infection is initiated by the merger of the viral envelope and a host cell membrane, which is catalyzed by the viral fusogen gB with the help of additional proteins, gD and gH/gL plus a cellular receptor for gD. By analogy with other viral fusogens, gB is thought to refold from the prefusion to the postfusion form in a series of large conformational changes that energetically couple refolding to membrane fusion. While the structure of the postfusion form is known, the prefusion form has not yet been characterized. The lack of the prefusion structure of gB is a major gap in our knowledge that has hindered the deciphering of the herpesvirus cell entry mechanism. The long-term goal of this research is the elucidation of the atomic-level mechanism of membrane fusion during herpesvirus entry and cell spread. The objective of this proposal is to stabilize the prefusion form of HSV-1 gB and to obtain its structure. Approaches that worked for other viral fusogens have so far failed with gB; thus, new strategies towards obtaining the prefusion structure of gB are needed. This R21 proposal is driven by an innovative central hypothesis that the prefusion form of gB is stabilized by interaction with membrane and requires the presence of membrane-proximal, transmembrane, and cytoplasmic domains. This hypothesis will be examined in two Specific Aims: Aim 1 will use gB-pseudotyped VSV?G virions, a novel reagent generated in preliminary data, to obtain a low-resolution cryoET reconstruction of the prefusion form of gB in the viral envelope to determine its overall architecture of the prefusion gB in its native-like state. In Aim 2, membrane mimetics - detergent/lipid micelles and nanodiscs - will be used to extract the soluble, prefusion form of the full-length gB expressed in insect cells. This form will be used in crystallization trials and in cryoEM single-particle 3D reconstruction, which will be used to generate a pseudoatomic model. In addition to the structure of the prefusion gB ectodomain, the proposed work will also yield the structures of the membrane-proximal, transmembrane, and cytoplasmic domains. This proposal is innovative because of its novel hypothesis and innovative approaches aimed at stabilizing the prefusion form. This proposal is significant because it addresses an important problem that has impeded the unraveling of the cell entry mechanism of HSV and other herpesviruses. The structure of the prefusion form of gB, at any resolution, would be an important discovery with a major impact on the herpesvirus field. It would inform the future work of many other investigators by providing a 3D framework for mapping functional data. Moreover, it may finally explain why gB requires other proteins to function as a fusogen. The results obtained here will form the basis for a future R01 proposal to determine the atomic-level crystal structure of prefusion gB and to elucidate its fusogenic mechanism using structural and biophysical approaches. Beyond the structure, finding a strategy to stabilize gB in its prefusion form may pave the way to a successful subunit vaccine against HSV.