Structural determinants of membrane fusion by HSV-1 gB

描述（由申请人提供）：单纯疱疹病毒终身感染其宿主，引起唇疱疹（HSV-1）、眼睛和生殖器感染（HSV-1和HSV-2）以及脑炎。此外，它们可以对免疫功能低下的个体或新生儿造成严重破坏。HSV-1进入宿主细胞需要四种病毒包膜糖蛋白：gB、gD和gH/gL复合物。已经从晶体学上研究了gD与细胞表面受体的结合，其触发融合事件。相比之下，其他三种蛋白质没有结构信息，并且对它们如何参与细胞进入知之甚少。了解膜融合机器的所有组件的结构是揭示HSV细胞融合机制的必要步骤。我们的长期目标是在原子水平上阐明疱疹病毒进入宿主细胞的机制，并利用这些知识设计有效的病毒进入细胞的抑制剂，用于预防和治疗目的。在初步研究中，我们已经结晶的gB的胞外域，目前正在确定其结构。在本申请中，我们提出了对gB的其他结构域和更大形式的生物化学和结构研究，以建立其活性和相互作用的更完整的图像。三个具体目标是1）确定gB的胞质结构域的结构; 2）产生包含用于结构研究的多个结构域的纯的可溶形式的gB;（3）从晶体学角度研究G-四联体寡核苷酸的抗病毒作用机制。这些结构信息将为阐明gB在细胞进入中的功能提供框架。例如，已知的功能突变将被映射到结构上。此外，战略开发用于生产可溶性多结构域形式的gB的结构研究将使我们能够接近gH/gL，并最终组装多蛋白复合物。

DESCRIPTION (provided by applicant): Herpes simplex viruses infect their hosts for life, causing cold sores (HSV-1), eye and genital infections (HSV-1 and -2), and encephalitis. Furthermore, they can wreak havoc in immuno-compromised individuals or in newborns. Host cell entry by HSV-1 requires four viral envelope glycoproteins: gB, gD, and gH/gL complex. Binding of gD to a cell-surface receptor, which triggers fusion events, has been studied crystallographically. By contrast, no structural information is available on the other three proteins, and little is known about how they participate in cell entry. Knowing the structures of all the components of the membrane fusion machinery is a necessary step in unveiling the mechanism of HSV cell fusion. Our long-term goal is to elucidate the mechanism of Herpes virus entry into host cells at the atomic level and to use this knowledge to design effective inhibitors of viral entry into cells for preventative and therapeutic purposes. In preliminary studies, we have crystallized the ectodomain of gB and are currently determining its structure. In this application we propose biochemical and structural studies of additional domains and larger forms of gB, in order to build a more complete picture of its activities and interactions The three specific aims are 1) to determine the structure of the cytoplasmic domain of gB; 2) to produce pure, soluble forms of gB encompassing multiple domains for structural studies; and 3) to study the mechanism of the antiviral effect of a G-quartet oligonucleotide crystallographically. This structural information will provide the framework for elucidating the function of gB in cell entry. For example, known functional mutations will be mapped onto the structure. Moreover, strategies developed for producing soluble multidomain forms of gB for structural studies will allow us to approach gH/gL and, eventually, to assemble multi-protein complexes.