Proteomic and molecular analysis of early events in HSV assembly

描述(申请人提供)：所有疱疹病毒都在感染的细胞核中组装DNA包装的衣壳。然后，这些衣壳经历了一个生物学上独一无二的过程：它们发芽进入内核膜，然后掐断进入核周空间，形成一个初级被包裹的粒子。这些颗粒随后将其包膜与外核膜融合，现在的细胞质衣壳在细胞质中经历二次包膜，形成成熟的病毒粒子。在每个包膜步骤中，一个被称为被膜的蛋白质复合体被组装在衣壳和包膜之间，并可能驱动包膜过程。原始的包膜和成熟的单纯疱疹病毒颗粒在形态上非常不同，被认为含有完全不同的被膜和包膜蛋白阵列。然而，核周单纯疱疹病毒(PnHSV)的结构和组成尚不清楚，因为到目前为止，只能通过超微结构研究来检查它。在本申请中，我描述了我们开发的以生物化学有用的数量提纯pnHSV颗粒的方法。我们建议通过对pnHSV进行蛋白质组学分析来扩展我们正在进行的研究，并测试我们已经证明存在的细胞蛋白质的作用。此外，使用被膜蛋白VHS的模型，我们将剖析被膜多肽被靶向包膜病毒颗粒的分子机制。

DESCRIPTION (provided by applicant): All Herpes viruses assemble their DNA-packaged capsids in the infected cell nucleus. These capsids then undergo a process unique in biology: they bud into the inner nuclear membrane and pinch off into the perinuclear space to form a primary enveloped particle. These particles subsequently fuse their envelopes with the outer nuclear membrane and the now cytoplasmic capsids undergo a secondary envelopment in the cytoplasm to form the mature virion. At each envelopment step a complex of proteins termed the tegument is assembled between the capsid and envelope and likely drives the envelopment process. The primary enveloped and mature HSV particles are morphologically quite distinct and are believed to contain quite different arrays of tegument and envelope proteins. However the structure and composition of the perinuclear HSV virus (pnHSV) is poorly defined because to date it has only been possible to examine it by ultrastructural studies. In this application I describe methodology that we have developed to purify pnHSV particles in biochemically useful quantities. We propose to extend our ongoing studies by performing a proteomic analysis of the pnHSV and to test the role of cellular proteins which we have already shown to be present. Furthermore, using the model tegument protein vhs we will dissect the molecular mechanism by which a tegument polypeptide is targeted into the enveloping viral particle.