Hijacking host cellular motors for the nuclear entry of polyomaviruses

项目概要/摘要 多瘤病毒（PyV）是在人类中引起衰弱性疾病的小DNA肿瘤病毒，包括多瘤病毒。 默克尔细胞多瘤病毒（MCPyV）引起的经常致命的默克尔细胞癌。为了引起感染，这些无包膜 病毒必须穿过拥挤的宿主细胞环境到达细胞核， 病毒基因组的复制导致裂解性感染或细胞转化。在进入时，PyV是 胞吞并运输到内质网（ER），在那里它穿透ER膜到达 细胞质一旦进入细胞质，病毒被分解并转运到细胞核， 通过核孔复合体（NPC）进入细胞核。PyV如何到达核膜， 随后输入到核中的物质尚待确定。细胞内转运主要由 通过驱动蛋白和细胞质动力蛋白宿主马达蛋白的作用 分别朝向单元的外围和中心。研究PyV原型，猿猴病毒40（SV40）， 揭示了病毒利用这些马达逃离ER到达细胞质并进行病毒分解 在这个隔间里这个K99/R00的建议试图了解细胞马达在随后的免疫反应中的作用。 PyV的运输和核进入。在该奖项的指导阶段，Spriggs博士将决定如何 驱动蛋白-1和动力蛋白协调SV40向宿主细胞核的转运， 通过NPC进入细胞核。此时，她将接受高分辨率显微镜的训练 技术，包括相关光学和电子显微镜（CLEM），当与两者结合使用时， 传统的和最先进的生物化学方法，将导致在病毒和细胞核的有影响力的发现， 进口机制。虽然我们对人类PyV感染的大部分知识来自对SV40的研究， 已经观察到它与MCPyV之间的关键差异，MCPyV是唯一与人类明确相关的PyV。 癌一个突出的区别在于它提出的核进入机制，这可能需要 有丝分裂核膜破裂。尽管它对公众健康的影响，很少有人知道的基本生物学， MCPyV感染。在独立阶段，通过她的K99培训，斯普里格斯博士将描绘入口 这一独特的人类病原体的途径，最终在其进入核的机械理解。当 结合她在病毒学和细胞生物学方面的强大背景，学习高分辨率显微镜应该 充分装备斯普里格斯博士运行一个成功的独立研究项目，研究病毒与宿主的相互作用。 此外，沿着她的高度积极的咨询委员会，密歇根大学提供了一个理想的 她的培训环境，鉴于其特殊的研究设施和专业发展资源。

Project Summary/Abstract Polyomaviruses (PyVs) are small DNA tumor viruses that cause debilitating disease in humans, including the often-fatal Merkel cell carcinoma by Merkel cell polyomavirus (MCPyV). To cause infection, these non-enveloped viruses must transport through the crowded host cellular environment to reach the nucleus where transcription and replication of the viral genome leads to lytic infection or cellular transformation. During entry, PyV, is endocytosed and trafficked to the endoplasmic reticulum (ER) where it penetrates the ER membrane to reach the cytosol. Once in the cytosol, the virus is disassembled and transported to the nucleus where it is thought to enter the nucleus through the nuclear pore complex (NPC). How PyV reaches the nuclear membrane and is subsequently imported into the nucleus have yet to be determined. Intracellular transport is mediated largely through the actions of the kinesin and cytoplasmic dynein host motor proteins that transport cellular cargo towards the periphery and center of the cell, respectively. Studies of the archetype PyV, simian virus 40 (SV40), revealed that the virus exploits these motors in escaping the ER to reach the cytosol and for virus disassembly in this compartment. This K99/R00 proposal seeks to understand the role of cellular motors in the subsequent transport and nuclear entry of PyVs. During the mentored phase of this award, Dr. Spriggs will determine how kinesin-1 and dynein coordinate the transport of disassembled SV40 to the host nucleus and the mechanism by which it enters the nucleus through the NPC. At this time, she will receive training in high-resolution microscopy techniques, including correlative light and electron microscopy (CLEM), that when used in combination with both traditional and state-the-art biochemical approaches, will lead to impactful discoveries in viral and cellular nuclear import mechanisms. While much of our knowledge of human PyV infection has come from the study of SV40, key differences have been observed between it and MCPyV, the only PyV definitely associated with human cancer. One prominent distinction lies in its proposed mechanism of nuclear entry, which may instead require mitotic nuclear envelope breakdown. Despite its impact on public health, little is known of the basic biology of MCPyV infection. During the independent phase, and with her K99 training, Dr. Spriggs will delineate the entry pathway of this distinct human pathogen, culminating in a mechanistic understanding of its nuclear entry. When combined with her strong background in virology and cell biology, learning high-resolution microscopy should fully equip Dr. Spriggs to run a successful independent research program studying virus-host interactions. Further, along with her highly motivated Advisory Committee, the University of Michigan provides an ideal environment for her training, given its exceptional research facilities and professional development resources.