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）是非常重要的，因为它在免疫功能低下的人群中具有活动性感染。活动性感染导致进行性多灶性脑白质病（PML）。PML会破坏髓磷脂，导致痴呆、昏迷和死亡。JC病毒的细胞内通路尚未确定，这为治疗活动性感染提供了障碍。本研究的目的是确定JC和其他多瘤病毒如何在细胞中运输。这包括确定用于运输的囊泡和驱动运动的运动蛋白。我认为，特定数量囊泡的运输与所载货物无关。这一假设将通过两个步骤进行验证：1。人类、猿猴和小鼠的多瘤病毒将在细胞内运输过程中成像，以确定使用了哪些囊泡和运动蛋白。将使用二色和三色荧光成像来确定病毒使用哪些囊泡进行运输。参与运输的动力蛋白的表征将基于病毒在细胞骨架上的单个步骤。这些实验将首次使用最近开发的一项技术，一纳米精度荧光成像（FIONA），在单个运动蛋白步骤的水平上探测病毒运输。2. 开发的实验技术随后将扩展到非病毒货物，以测试运输动力学和囊泡种群之间的一般关系。为此，多瘤病毒将作为三种主要内吞途径的探针。非病毒货物将被装载到特定的囊泡中，并在细胞内运输过程中成像。观察到的动力学将与多瘤病毒的动力学进行比较。总体而言，本研究有望为我们理解以下三个相互关联的领域做出重大贡献：病毒感染、成像技术和细胞内转运。

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.