TRANSPORT OF HERPES SIMPLEX VIRUS IN OCULAR TISSUES

描述：(改编自申请人的摘要)单纯疱疹病毒 1型(HSV-1)是导致儿童传染性失明的最常见原因。 美国每年报告的病例约为50万例。人眼 HSV-1感染导致病毒从感觉神经传播 三叉神经节和中枢神经系统的末梢。HSV获得访问 神经元胞浆和盗用宿主细胞器，胞浆 用于逆行轴突运输的马达和细胞骨架元件 原子核。了解参与轴突运输的蛋白质 HSV是开发这种疗法的重要因素 严重的临床问题。要研究这个问题一直很困难 体内HSV逆行转运中的细胞事件，由于 不可能获得同步感染的神经元和 超微结构研究中固有的采样问题。因此，需要 存在一个可量化的体外模型，在这个模型中研究 逆行或向心的(从细胞外围到细胞中心) HSV在整个细胞内的运输。申请人 提出在极化条件下研究单纯疱疹病毒的向心输运 上皮细胞，人类区域色素上皮(RPE)。RPE是 选择有几个原因：第一，了解优惠 高度极化的根尖或基底外侧表面的感染， 神经外胚层来源的细胞可能提供对选择性途径的洞察 临床上和某些情况下可能发生的单纯疱疹病毒感染的传播 动物模型。第二，单纯疱疹病毒感染体外培养的人RPE细胞 顶面和底侧面，以及向心输运可以 通过免疫细胞化学、生物化学和电子技术进行监测 显微免疫化学和激光共聚焦显微镜。第三，很多人 RPE细胞的运输功能依赖于不同的顶端 而神经元同样是极化的细胞。 因此，该项目旨在测试HSV是 从RPE的心尖和基底外侧极向心运输 使用神经元共有的机制和细胞骨架元件的细胞 轴浆运输。长期目标是将由此产生的 了解HSV在神经元中运输的信息，作为 了解眼部疱疹感染的发病机制。这个 该项目的具体目标是：1)测试病毒颗粒 穿透人视网膜色素上皮细胞的顶膜和基底膜 融合而不是通过内吞作用，并向心地与 作为核衣壳的细胞质。2)测试完整的微管是否 是单纯疱疹病毒向心运输所必需的。3)扩大我们的 了解RPE细胞中微管的极性。4)测试 神经细胞质运动蛋白参与向心性运动 人视网膜色素上皮细胞对单纯疱疹病毒的转运。5)测试是否参与了 向心力中的神经元逆行胞浆运动--动力蛋白 人视网膜色素上皮细胞对单纯疱疹病毒的转运。 [另一个具体目标，上一份文件中的第二项目标是 在修订后的提交书中被删除，但无意中留在了 第26页的目标清单。]

DESCRIPTION: (Adapted from the applicant's abstract) Herpes simplex virus type 1 (HSV-1) is the most common cause of infectious blindness in the U.S., with approximately 500,000 cases reported annually. Human ocular HSV-1 infection results in the spread of virus from sensory nerve endings to the trigeminal ganglion and CNS. HSV gains access to neuronal cytoplasm and pirates the host cell machinery, cytoplasmic motors and cytoskeletal elements for retrograde axonal transport to the nucleus. Understanding the proteins involved in the axonal transport of HSV is an important element in developing therapy for this significant clinical problem. It has been difficult to study the cellular events in the retrograde transport of HSV in vivo, due to the impossibility of obtaining synchronously infected neurons and the sampling problems inherent in ultrastructural studies.Thus, a need exists for a quantifiable, in vitro model in which to study the retrograde or centripetal (from the cell periphery to the cell center) transport of HSV within the context of the whole cell. The applicant proposes to examine the centripetal transport of HSV in a polarized epithelial cell, the human reginal pigment epithelium (RPE). RPE were chosen for several reasons: First, understanding the preferential infection of apical or basolateral surfaces of this highly polarized, neuroectoderm-derived cell may provide insight into the selective route of spread of HSV infection which may occur clinically and in certain animal models. Second, HSV infects human RPE cells in vitro from both the apical and basolateral surfaces, and the centripetal transport can be monitored immunocytochemically, biochemically, and with electron microscopic immunochemistry and confocal laser microscopy. Third, many of the transport functions of RPE cells depend on a distinct apical versus basolateral polarity, and neurons are, likewise, polarized cells. The project is, thus, designed to test the hypothesis that HSV is transported centripetally from the apical and basolateral poles of RPE cells using mechanisms and cytoskeletal elements common to neuronal axoplasmic transport. The long-term goals are to apply the resulting information to understanding HSV transport in neurons, as a basis for understanding the pathogenesis of ocular herpes infections. The specific aims of the project are: 1) To test that viral particles penetrate the apical and basolateral membranes of human RPE cells by fusion rather than by endocytosis and travel centripetally with the cytoplasm as nucleocapsids. 2) To test whether intact microtubules are essential for centripetal transport of HSV. 3) To expand our understanding of the polarity of microtubules in RPE cells. 4) To test the involvement of kinesin, a neuronal cytoplasmic motor, in centripetal transport of HSV by human RPE cells. 5) To test the involvement of dynein, a neuronal retrograde cytoplasmic motor, in centripetal transport of HSV by human RPE cells. [An additional specific aim, #2 in the previous submission, was eliminated in the revised submission, but was inadvertently left in the list of Aims on p. 26.]