Apoptosis is a specific type of cell death involved in embryo-genesis, control of cellular proliferation and regulation of immune responses. Apoptotic cell death is a common feature of such diverse neurological diseases as dementia, stroke, neuro-degenerative diseases, and infections. The applicants have recently shown that neurotropic reoviruses induce apoptolis in cultured cells, and that viral strains differ in this capacity. Using reassortant viruses containing different combinations of genes derived from apoptosis-inducing (AP0+) and non-inducing (AP0-) reovirus strains it has been possible to identify the reovirus S1 gene and its encoded viral outer surface protein, sigma 1, as the principal determinant of this process. Apoptosis does not require viral replication, and can be induced by purified sigma 1 protein. Apoptosis also occurs in neuroblastoma cells in culture, and in neurons following reovirus infection of the murine central nervous system (CNS) in vivo. The combined availability of in vitro and in vivo models of apoptosis induced by naturally occurring viral strains is a unique feature of this experimental system. The indification of individual viral genes and proteins involved in this process is another key aspect of this experimental system that will facilitate subsequent analysis of molecular mechanism(s) of apoptosis induction and regulation. In the proposed studies, the applicants will use experimental reovirus infection of cultured cells and mice as a odel system to identify at a molecular level: (1) regions of the reovirus sigma 1 protein required for induction of apoptosis, (2) cellular factors involved in viral apoptosis, and (3) the role of apoptosis in viral injury to the CNS in vivo, and the mechanisms by which this occurs and can be controlled. The applicants believe that reovirus-induced apoptosis is triggered by the interaction of sigma 1 with cell surface receptors. This event results in alterations in mitogen activated protein kinase (MAPK) signal transduction pathways, and rapid changes in intracellular calcium concentrations in reovirus infected cells. Changes in calcium may, in turn, activate calcium dependent proteases including calpains, which they have implicated in reovirus-induced apoptosis. Their hypothesis is that apoptosis playsa critical role in CNS cell death and tissue injury during viral infection and that identification of host and viral factors involved in this process and their subsequent manipulation will decrease the magnitude of cellular and tissue injury and enhance survival. Identification of the molecular mechanisms of apoptosis and determination of the ways in which apoptosis can be modified to prevent cell death will allow the development of strategies designed to prevent and treat degenerative and inflammatory diseases of the nervous system.
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