Mammalian orthoreovirus can exit cells in extracellular vesicles

Abstract

ABSTRACTSeveral egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.AUTHOR SUMMARYThe exit strategy that many viruses use to escape cells is unknown. Reovirus is a nonenveloped human virus and an ideal model system to understand virus exit strategies and their inluence on infection. We found that two different reovirus strains, one that disrupts cell membranes and one that leaves cells largely intact, increase the release of extracellular vesicles (EVs) from cells. Both reovirus strains are released from cells as free particles and in association with EVs, which are membrane-bound structures that function in cell-to-cell communication. Depending on cell type and virus type, EVs can act like an ‘invisibility cloak’ that shields reovirus from antibodies. EVs can also bundle and ferry reovirus particles between cells. Although we used cells to examine the effects of reovirus association with EVs, it is possible that in mammalian hosts, EVs may shield reovirus from immune defenses and promote more efficient transmission and infection through a ‘strength-in-numbers’ strategy. Future work building on these findings will test the biological significance of EV-enclosed reovirus and may inform delivery strategies for oncolytic reoviruses to tumor sites. Broadly, these findings enhance our understanding of virus egress strategies and infection principles that may apply to other viruses that travel in EVs.