Polyomavirus transport: Vesicles, motor proteins, and endocytosis.

描述（由申请人提供）：了解人类多瘤病毒（JC 病毒）非常重要，因为它会主动感染免疫功能低下的人。活动性感染会导致进行性多灶性白质脑病 (PML)。 PML 会导致髓磷脂破坏，导致痴呆、昏迷和死亡。 JC病毒的细胞内途径尚未确定，为活动性感染的治疗提供了障碍。这项研究的目的是确定 JC 和其他多瘤病毒如何在细胞中转运。这包括确定用于运输的囊泡和驱动运动的运动蛋白。我建议特定囊泡群体的运输与所装货物无关。这一假设将分两步进行检验： 1. 人类、猿猴和鼠多瘤病毒在细胞内运输过程中进行成像，以确定使用哪些囊泡和运动蛋白。两色和三色荧光成像将用于确定病毒使用哪些囊泡进行运输。参与运输的马达蛋白的表征将基于病毒在细胞骨架上的各个步骤。这些实验将首次利用最近开发的技术，即一纳米精度荧光成像（FIONA），来探测单个运动蛋白步骤水平上的病毒运输。 2. 所开发的实验技术将扩展到非病毒货物，以测试运输动力学与囊泡群体之间的一般关系。为此，多瘤病毒将作为三个主要内吞途径的探针。非病毒货物将被加载到特定的囊泡中并在细胞内运输过程中成像。观察到的动态将与多瘤病毒的动态进行比较。总体而言，这项研究预计将极大地促进我们对三个相互关联领域的理解；病毒感染、成像技术和细胞内运输。

DESCRIPTION (provided by applicant): An understanding of the human polyomavirus, JC virus, is of great importance due to its active infection of immunocompromised persons. Active infection results in progressive multifocal leukoencephalopathy (PML). PML causes the destruction of myelin resulting in dementia, coma, and death. The intracellular pathway of JC virus has not been determined, providing a barrier to the treatment of active infection. The goal of this investigation is to determine how JC and other polyomaviruses are transported in cells. This includes determining the vesicles used for transport and the motor proteins that drive the motion. I propose that the transport of specific populations of vesicles is independent of the cargo contained. This hypothesis will be tested in two steps: 1. The human, simian, and murine polyomaviruses will be imaged during intracellular transport to determine which vesicles and motor proteins are used. Two- and three-color fluorescence imaging will be used to determine which vesicles the virus uses for transport. Characterization of the motor proteins involved in transport will be based on the individual steps of the virus on the cytoskeleton. These experiments will be the first to make use of a recently developed technique, Fluorescence Imaging with One Nanometer Accuracy (FIONA), to probe viral transport on the level of individual motor protein steps. 2. The experimental techniques developed will then be extended to non-viral cargo to test the general relationship between transport dynamics and vesicle population. To this end the polyomaviruses will serve as probes of the three major endocytic pathways. Non-viral cargo will be loaded into specific vesicles and imaged during intracellular transport. The dynamics observed will be compared to those of the polyomaviruses. Overall, this research is expected to contribute significantly to our understanding of three interrelated fields; viral infection, imaging technology, and intracellular transport.