Hijacking host cellular motors for the nuclear entry of polyomaviruses

项目摘要/摘要 多瘤病毒(PYV)是一种小的DNA肿瘤病毒，会导致人类虚弱的疾病，包括 由默克尔细胞多瘤病毒(MCPyV)引起的通常致命的默克尔细胞癌。为了引起感染，这些非包膜的 病毒必须穿过拥挤的宿主细胞环境才能到达转录的细胞核 病毒基因组的复制会导致裂解感染或细胞转化。在进入期间，PYV，是 内吞并运输到内质网(ER)，在那里它穿过ER膜到达 细胞质。一旦进入胞浆，病毒就被分解并运输到被认为是核子的地方 通过核孔复合体(NPC)进入细胞核。热带病病毒是如何到达核膜的 随后输入到细胞核中的还有待确定。细胞内转运在很大程度上是由 通过运动蛋白和细胞质动力蛋白宿主马达蛋白来运输细胞货物 分别朝向细胞的外围和中心。PYV原型猴40(SV40)的研究 揭示了病毒利用这些马达逃离内质网到达胞浆并进行病毒分解 在这个隔间里。这份K99/R00提案试图了解蜂窝发动机在随后的 PYV的运输和核进入。在这个奖项的指导阶段，斯普里格斯博士将决定如何 Kinesin-1和Dynein通过以下途径协调拆解的SV40到宿主核的运输和机制 它通过全国人大进入细胞核。此时，她将接受高分辨率显微镜的培训 技术，包括相关光学和电子显微镜(Clem)，当两者结合使用时 传统的和最先进的生物化学方法，将导致病毒和细胞核的重大发现 进口机制。虽然我们对人类PYV感染的大部分知识都来自于对SV40的研究， 已经观察到它与MCPyV之间的关键区别，MCPyV是唯一一种明确与人类有关的PYV 癌症。一个显著的区别在于它提议的核进入机制，这可能需要 有丝分裂核膜破裂。尽管它对公众健康有影响，但人们对猪细小病毒的基本生物学知之甚少 MCPyV感染。在独立阶段，通过她的K99训练，斯普里格斯博士将勾勒出条目 这一独特的人类病原体的途径，最终导致了对其核进入的机械理解。什么时候 再加上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会 让斯普里格斯博士完全有能力开展一项成功的独立研究项目，研究病毒与宿主之间的相互作用。 此外，与她积极进取的咨询委员会一起，密歇根大学提供了一个理想的 鉴于其卓越的研究设施和专业发展资源，该公司为她的培训提供了良好的环境。

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.