LONG DISTANCE AXONAL TRANSPORT OF HSV CAPSID AND DNA

HSV 衣壳和 DNA 的长距离轴突运输

• 批准号: 7724217
• 负责人: JENNIFER Hart LAVAIL
• 金额: $ 0.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724217
• 关键词: Affinity; Antibodies; Axonal Transport; Biochemical; Capsid; Capsid Proteins; Cell model; Computer Retrieval of Information on Scientific Projects Database; Coupled; Cytoplasm; DNA; Funding; Gel; Grant; Herpesvirus 1; Immunohistochemistry; Institution; Label; Mass Spectrum Analysis; Modeling; Motor Neurons; Mus; Neurons; Nucleocapsid; Presynaptic Terminals; Process; Proteins; Research; Research Personnel; Resources; Retinal Ganglion Cells; Sepharose; Silver Staining; Source; United States National Institutes of Health; Viral; Viral Envelope Proteins; Viral Proteins; Virus; Visual system structure; anterograde transport; mutant; neuronal cell body; therapeutic target; tool; transmission process; vesicle-associated membrane protein; viral DNA

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 单纯疱疹病毒 1 型病毒 (HSV) 从一个感染的神经元传播到下一个的关键步骤是病毒 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，以便随后释放。 病毒如何运输以及需要哪些病毒蛋白是长期存在的问题。 使用缺乏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蛋白对于病毒衣壳和DNA的长距离顺行轴突运输是必需的。 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。 使用与琼脂糖珠偶联的 Us9 抗体的免疫亲和基质，我们共同浓缩了 Us9 和主要衣壳蛋白 VP5。 这一生化证据表明衣壳运输依赖于与 Us9 蛋白的关联的机制。 这种关联通过 EM 免疫组织化学进一步得到证实，其中 Us9 抗体标记了野生型病毒感染的视网膜神经节细胞细胞质中的无包膜衣壳。 我们得出的结论是，HSV DNA 和衣壳的有效轴突运输取决于 Us9 蛋白的表达，并且不需要与许多宿主细胞马达相关的传统膜囊泡蛋白。 我们将在亲和矩阵上亲和纯化与 Us9 结合的蛋白质，然后在 PAGE 上将它们分离。 蛋白质将被银染并从凝胶中切下，以使用质谱进行鉴定。 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的这一功能过程提供了一种新的生化工具。 此外，Us9 蛋白是对抗 HSV 在视觉系统中传播的潜在治疗靶点。