Assembly of the Herpes Simplex Virus Capsid

1型单纯疱疹病毒（HSV-1）的衣壳是一个二十面体（T=16）蛋白壳，厚度约为15nm，直径约为125nm。在完整的病毒中，衣壳包含病毒的双链dna，并被脂质膜包围。本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，用解离/变性剂（如胍- hc1或尿素）解离成它们的六组分蛋白。然后纯化单个衣壳蛋白，并在设计的条件下将其添加到一起，以促进它们重新组装成二十面体衣壳。反应混合物将通过电子显微镜、蔗糖密度梯度超离心和sds -聚丙烯酰胺凝胶电泳检查是否存在重新组装的衣壳。此外，还将开展涉及使用特异性单克隆抗体的研究，以确定衣壳蛋白VP19、VP23和VP26的位置。该项目的具体目标是：(1)确定纯化衣壳蛋白VP22a体外自组装的环状结构的形态、总质量和蛋白质化学计量；(2)确定原生VP26可以重新附着在缺乏它的HSV-1衣壳上的条件，并确定VP26在重组衣壳中的位置；(3)确定纯化后的主要衣壳蛋白VP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;(4)制备特异性衣壳蛋白VP19， VP23和VP26的单克隆抗体，并使用它们（结合电子显微镜方法）在HSV-1衣壳中定位其同源蛋白。

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.