Structural determinants of membrane fusion by HSV-1 gB

描述(申请人提供)：单纯疱疹病毒终生感染宿主，导致唇疱疹(HSV-1)、眼睛和生殖器感染(HSV-1和HSV-2)以及脑炎。此外，它们还可能对免疫受损的个人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复合体。已经用结晶学的方法研究了GD与细胞表面受体的结合，该受体触发了融合事件。相比之下，没有关于其他三种蛋白质的结构信息，对它们如何参与细胞进入也知之甚少。了解膜融合机械的所有部件的结构是揭示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，并利用这一知识设计有效的病毒进入细胞的抑制剂，以达到预防和治疗的目的。在初步研究中，我们已经结晶了GB的胞外结构域，目前正在确定其结构。在这一应用中，我们建议对更多结构域和更大形式的GB进行生化和结构研究，以建立其活性和相互作用的更完整的图景。三个具体目标是1)确定GB的细胞质结构域的结构；2)生产包含多个结构域的纯的、可溶的GB形式用于结构研究；以及3)从结晶学角度研究G-四重组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，为生产可溶的多结构域形式的GB用于结构研究的策略将使我们能够接近Gh/Gl，并最终组装多蛋白质复合体。

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.