Assembly of the Herpes Simplex Virus Capsid

单纯疱疹病毒1型(HSV-1)衣壳为二十面体(T=16)蛋白质壳，厚约15 nm，直径约125 nm。在完整的病毒中，衣壳含有病毒dsDNA，并被一层脂膜包围。该项目的目标是从纯化的衣壳蛋白开始，在体外组装HSV-1衣壳。衣壳将从HSV-1感染细胞的细胞核中分离出来，并在解离/变性试剂如盐酸胍或尿素的作用下解离成六组分的蛋白质。然后，单个衣壳蛋白将被提纯，并在旨在促进它们重新组装成二十面体衣壳的条件下添加到一起。反应混合物将通过电子显微镜、蔗糖密度梯度超速离心法和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法检查重组衣壳的存在。此外，还将进行涉及使用特定单抗的研究，以确定衣壳蛋白VP19、VP23和VP26的位置。该项目的具体目标是：(1)确定从纯化的衣壳蛋白VP22a在体外自组装的环状结构的形态、总质量和蛋白质化学计量学；(2)确定天然VP26可以重新附着到缺乏HSV-1衣壳的条件下，并确定VP26在重组衣壳中的位置；(3)确定纯化的主要衣壳蛋白VP在体外组装成T=16二十面体的条件。组装可能需要其他衣壳蛋白的存在，如支架蛋白VP22a：和(4)制备针对衣壳蛋白VP19、VP23和VP26的单抗，并使用它们(结合电子显微镜方法)将它们的同源蛋白定位在HSV-1衣壳中。

The Capsid of herpes simplex virus type 1 (HSV-1) is an icosahedral (T=16) protein shell approximately 15 nm thick and 125nm in diameter. In the intact virus, the capsid contains the viral dsDNA and it is surrounded by a lipid membrane. The goal of this project is to assemble the HSV-1 capsid in vitro beginning with purified capsid proteins. Capsids will be isolated from the nuclei of HSV- 1- infected cells and dissociated into their six component protein with dissociating/denaturing agents such as guanidine-HC1 or urea. Individual capsid proteins will then be purified and added together under conditions designed to promote their re-assembly into icosahedral capsids. Reaction mixtures will be examined for the presence of re-assembled capsids by electron microscopy, sucrose density gradient ultracentrifugation and SDS-polyacrylamide gel electrophoresis. Also, studies involving the use of specific monoclonal antibodies will be carried out to define the locations of capsid proteins VP19, VP23, and VP26. Specific goals of the project are to: (1) define the morphology, total mass and protein stoichiometry of torus-shaped structures self-assembled in vitro from purified capsid protein VP22a; (2) identify conditions in which native VP26 can re-attach to HSV-1 capsids that lack it and define the location of VP26 in reconstituted capsids; (3) define conditions in which purified VP, the major capsid protein, can assembly in vitro into T=16 icosahedra. Assembly may require the presence of other capsid proteins such as the scaffolding protein, VP22a: and (4) prepare monoclonal antibodies specific for capsid proteins VP19, VP23, and VP26, and use them (in conjunction with electron microscopic methods) to localize their cognate proteins in the HSV-1 capsid.