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.]
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