GENETIC ANALYSIS OF HERPES VIRUS NEUROTROPISM AND ENCEPHALITIS

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。子项目和 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 中心，不一定是研究者的机构。 1型单纯疱疹病毒（HSV）从一个感染的神经元到下一个感染的神经元的传播中的关键步骤是病毒DNA从宿主感染的神经细胞体到轴突末端的极化顺行轴突运输，用于随后的释放。 病毒是如何运输的，需要什么样的病毒蛋白是长期存在的问题。 使用缺乏Us 9蛋白表达的HSV突变病毒和感染的鼠视网膜神经节细胞模型，我们发现HSV Us 9蛋白对于病毒衣壳和DNA的长距离顺行轴突运输是必需的。 它对于病毒包膜蛋白的顺行运输或HSV的逆行轴突运输是不必要的。 使用Us 9抗体偶联琼脂糖凝胶珠的免疫亲和基质，我们共浓缩Us 9和VP 5，主要衣壳蛋白。 这一生化证据表明，衣壳转运依赖于与Us 9蛋白的关联的机制。 用EM免疫组织化学进一步证实了这种关联，其中Us 9抗体标记了野生型病毒感染的视网膜神经节细胞胞质中的无包膜衣壳。 我们的结论是，有效的轴突运输HSV DNA和衣壳依赖于Us 9蛋白的表达，并不需要传统的膜泡蛋白，与许多宿主细胞马达。 我们将在亲和基质上亲和纯化与Us 9结合的蛋白质，然后在PAGE上分离它们。 蛋白质将进行银染色，并从凝胶中切出，以使用质谱法进行鉴别。 作为非囊泡运输的模型，HSV核衣壳的顺行轴突运输为理解正常神经元中的这一功能过程提供了新的生化工具。 此外，Us 9蛋白是对抗HSV在视觉系统中传播的潜在治疗靶点。

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.