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Juan Mo, Ming Zhang, Brendan Marshall, Sally S. Atherton; Rapamycin Treatment Shifts Balance of Autophagy and Apoptosis during Murine Cytomegalovirus (MCMV) Infection of the Retina or RPE Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2785. doi: https://doi.org/.
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The contribution of apoptosis and autophagy to the pathogenesis of cytomegalovirus infection has not been explored. The purpose of this study was to determine if MCMV infection affects apoptosis and autophagy during MCMV retinitis.
In vitro, RPE cells were seeded and mock-infected or infected with MCMV at an MOI of 0.1 PFU/cell and treated with 10-6 M of rapamycin. Fluorescent images were obtained by transfecting GFP-LC3 to MCMV-infected RPE cells. In vivo, female BALB/c mice were inoculated with 5×103 PFU of MCMV via a supraciliary injection. Mice were untreated (infected control) or treated with rapamycin (5x10-7 M), an inhibitor of mTOR. Inoculated eyes were collected at several times p.i., examined by MCMV staining for morphology and location of MCMV infected cells, by TUNEL assay for apoptotic cells, and by immunoblot to monitor the levels of processed LC3B (autophagy) and cleaved caspase 3 (apoptosis).
In vivo, at days 5, 7 and 10 p.i., most of the apoptotic cells in the retina were uninfected whereas most of the infected cells were not apoptotic. In cultured MCMV-infected RPE cells, infection at an MOI of 0.1 induced caspase 3-mediated apoptosis. Rapamycin treatment decreased the levels of cleaved caspase-3 at days 2, 3 and 4 p.i. in vitro and at days 5 and 7 p.i. in vivo. Rapamycin treatment increased the levels of LC3B-II, a marker of autophagy, at days 2 and 4 p.i. in vitro and at days 5, 7 and 10 p.i. in vivo. More diffuse staining for LC3B was observed in the cytoplasm of cultured RPE cells treated with rapamycin whereas punctuate staining was observed in normal untreated RPE. The levels of phosphorylation of mTOR’s substrates, 4EBP1 (in vitro) and P70S6K (in vivo), were increased during MCMV infection and were down-regulated by rapamycin compared to mock-injected controls.
The results of these studies suggest that during MCMV infection of the retina or of cultured RPE cells, the balance between autophagy and apoptosis favors apoptosis. However, if mTOR is inhibited by rapamycin, the balance shifts toward autophagy rather than apoptosis.
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