GENETIC ANALYSIS OF HERPES VIRUS NEUROTROPISM AND ENCEPHALITIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在单纯疱疹病毒1型病毒(HSV)从一个感染神经元到下一个感染神经元的传播过程中，一个关键步骤是病毒DNA从宿主感染的神经细胞体向轴突末端的两极化顺行运输，以便随后释放。病毒是如何传播的，需要哪些病毒蛋白是长期存在的问题。利用缺乏US9蛋白表达的HSV突变病毒和感染的小鼠视网膜神经节细胞模型，我们发现HSV US9蛋白是病毒衣壳和DNA长距离顺行轴突运输所必需的。病毒被膜蛋白的顺行运输或HSV的逆行轴突运输是不必要的。利用偶联到琼脂糖珠上的US9抗体的免疫亲和基质，我们共浓缩了US9和主要衣壳蛋白VP5。这一生化证据表明，衣壳的运输依赖于与US9蛋白的联系。这种联系进一步被EM免疫组织化学证实，在野生型病毒感染的视网膜神经节细胞胞浆中，US9抗体标记的无包膜衣壳。我们的结论是，HSV DNA和衣壳的有效轴突运输依赖于US9蛋白的表达，而不需要传统的与许多宿主细胞马达相关的膜泡蛋白。我们将在亲和矩阵上亲和纯化与US9相关的蛋白质，然后在PAGE上将它们分离。蛋白质将被银染，并从凝胶中切割出来进行鉴定，使用质谱学进行鉴定。作为非囊泡转运的模型，单纯疱疹病毒核衣壳的顺行轴突转运为理解正常神经元的这一功能过程提供了新的生化工具。此外，US9蛋白是一个潜在的治疗靶点，可防止HSV在视觉系统中的传播。

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. A critical step in the transmission of Herpes simplex virus type 1 virus (HSV) from one infected neuron to the next is the polarized anterograde axonal transport of viral DNA from the host infected nerve cell body to the axon terminal for subsequent release. How the virus is transported and what viral proteins are necessary are long-standing questions. Using an HSV mutant virus that lacks expression of the Us9 protein and the infected murine retinal ganglion cell model, we found that HSV Us9 protein is necessary specifically for long distance anterograde axonal transport of viral capsid and DNA. It is unnecessary for anterograde transport of viral envelope proteins or for retrograde axonal transport of HSV. Using an immunoaffinity matrix of Us9 antibody coupled to Sepharose beads, we co-concentrated Us9 and VP5, the major capsid protein. This biochemical evidence suggests a mechanism by which the capsid transport depends on an association with Us9 protein. This association was further confirmed with EM immunohistochemistry in which Us9 antibody labeled unenveloped capsids in wild-type virus infected retinal ganglion cell cytoplasm. We conclude that efficient axonal transport of HSV DNA and capsid depends on expression of Us9 protein and does not require the traditional membrane vesicle proteins that are associated with many host cell motors. We shall affinity purify proteins that associate with Us9 on the affinity matrix and then separate them on PAGE. The proteins will be silver stained and cut out of the gels for identification using mass spectroscopy for identification. As a model of non-vesicular transport, the anterograde axonal transport of HSV nucleocapsid offers a new biochemical tool for understanding this functional process in normal neurons. Furthermore, Us9 protein is a potential therapeutic target against the spread of HSV in the visual system.