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Ming Zhang, Brendan Marshall, Juan Mo, Sally S Atherton; Murine Cytomegalovirus Lacking the m45 Protein Does Not Induce Retinitis in the Injected Eye of Immunosuppressed Mice Following Supraciliary Inoculation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):854.
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Necroptosis, is a form of necrosis dependent on two related RHIM-containing signaling adaptors, receptor-interacting protein kinases (RIP) 1 and 3 and can be induced by virus infection. MCMV inhibits necroptosis due to the expression of the m45 protein which disrupts RIP3-RIP1 interactions. The purpose of this study was to determine if depletion of m45 would compromise virus spread and replication in the retina.
BALB/c mice, Rip3 -/- mice or Rip3+/+ C57BL/6 mice were immunosuppressed (IS) with methylprednisolone acetate (2mg per mouse, i.m. every 4 days) and inoculated with 5 × 103 PFU of either a mutant MCMV which carries mutations in the RHIM binding domain of the m45 protein (Δm45), or the parental K181 strain, via the supraciliary route. Mice were sacrificed at various times p.i. and injected eyes were collected for plaque assay, immunohistochemistry and electron microscopy.
Following inoculation of K181 into IS BALB/c mice, virus infected cells were observed in both the choroid and RPE layer at day 3 p.i., and widespread inner retinal infection and retinitis was noted from day 5 p.i. onwards. In contrast, Δm45 infection was largely confined to the RPE layer and there was very little spread throughout the retina. Significantly less replicating virus was recovered from Δm45 injected eyes than from K181 infected eyes at all time points. Electron microscopic examination revealed virus particles in the choroid and RPE of both Δm45 and K181 injected eyes. Virus infected necrotic cells, with observable virus particles exhibited marginalization of chromatin, neatly apposed to the inner nuclear membrane and marked lytic changes in both cytoplasm and organelles in both Δm45 and K181 injected eyes. However, significantly more virus infected necrotic cells were observed in Δm45 injected eyes than in K181 injected eyes. In addition, significantly less replicating virus and more virus infected necrotic cells were observed in Δm45 injected Rip3+/+ eyes than in Δm45 injected Rip3-/- eyes, K181 injected Rip3+/+ eyes or K181 injected Rip3-/- eyes.
The mutation of the MCMV m45 protein allows rapid necrotic cell death of virus infected RPE cells which limits virus spread and replication in the inner retina. Genetic ablation of RIP3 blocks this effect and restores normal virus replication to Δm45-infected retinas.
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