STRUCTURAL STUDIES ON GH/GL COMPLEX OF HERPES SIMPLEX VIRUS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 单纯疱疹病毒1型和2型（HSV-1和HSV-2）是高度流行的人类病原体，其引起口腔和生殖器溃疡、脑炎、角膜炎和新生儿的致命感染。为了进入细胞，HSV像任何包膜病毒一样，必须将其脂质膜与细胞的脂质膜融合。HSV进入宿主细胞由四种保守的表面糖蛋白gD、gB和gH/gL复合物介导。gD结合宿主细胞表面上的特异性受体，并向gB或gH/gL发出信号。gB和gH/gL复合物是膜融合所必需的。最近发表的gB的晶体结构表明，gB是一种病毒融合蛋白。然而，单独的gB不能进行融合; gH/gL也是必需的。没有关于gH/gL的结构信息，并且其在融合中的确切作用是未知的。确定gH/gL的结构将有助于了解gH/gL的功能，从而了解疱疹病毒融合过程的机制。gH/gL是两种病毒表面糖蛋白的异二聚体复合物，gH锚定在病毒膜上，gL与病毒表面上的gH结合。我过表达，纯化，并结晶的重组gH/gL复合物的可溶性形式。晶体生长到~100米的最长和~20？m的最短尺寸，并且尽管是各向异性的，但λ =约3.2 A。我从几种不同的冷冻保护剂中冷冻保存的晶体中收集了几个3.5A的天然数据集。空间群为P41（3）212，a=B=88 A，c=330 A，90块我将优化我的冷冻保存方案，以提高分辨率和降低各向异性。由于没有同源蛋白质的结构可用于分子置换方法，我计划通过MIR，MAD或SAD相位获得相位信息，我想在Rapidata 2009课程中进行。目前正在生产硒-甲硫氨酸衍生物和重原子浸泡液。这将是gH/gL复合物结构的首次描述，并将有助于解释gH/gL的功能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) are highly prevalent human pathogens that cause oral and genital sores, encephalitis, keratitis and fatal infections in newborns. To enter cells, HSV, like any enveloped virus, must fuse its lipid membrane with that of the cell. Entry of HSV into host cells is mediated by four conserved surface glycoproteins, gD, gB, and gH/gL complex. gD binds a specific receptor on the host cell surface and signals to either gB or gH/gL. Both gB and gH/gL complex are indispensable for membrane-fusion. Recently published crystal structure of gB suggested that gB is a viral fusion protein. However, gB alone cannot perform fusion; gH/gL is also essential. No structural information is available on gH/gL, and its exact role in fusion is unknown. Determining the structure of gH/gL will help understand the function of gH/gL and thus the mechanism of the fusion process of herpesviruses. It may also provide important insights for therapeutic intervention.gH/gL is a heterodimeric complex of two viral surface glycoproteins, gH that is anchored on the viral membrane and gL which is bound to gH on the viral surface. I overexpressed, purified, and crystallized the recombinant gH/gL complex in a soluble form. Crystals grow to ~100 m in longest and ~20 ?m in the shortest dimension and diffract to ~3.2 A, albeit anisotropically. I collected several native datasets to 3.5 A from crystals cryopreserved in several different cryoprotectants. The space group is P41(3)212 with a=b=88 A, c=330 A, ?=?=?=90¿. I will optimize my cryopreservation protocol to improve the resolution and reduce anisotropy.Since no structures of homologous proteins are available for molecular replacement methods, I plan to obtain the phase information by MIR, MAD, or SAD phasing, which I would like to do at the Rapidata 2009 course. Production of Se-Methionine derivative and heavy atom soaks are being done right now. This will be the first description of gH/gL complex structure and will help in explaining the functions of gH/gL.