Mammalian orthoreovirus can exit cells in extracellular vesicles.

Several 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

Reovirus nonstructural proteins associate with EV fractions in a strain-specific manner.

(A-C) L cells were adsorbed with three individual clones (C1-C3) of T1L or T3D reovirus at an MOI of 1 PFU/cell for 72 h. Reovirus nonstructural protein association with large EV, medium EV, and small EV/free virus fractions was quantified following SDS-PAGE and immunoblotting (A) for T1L σNS (B) or T3D σNS (C). Error bars indicate SD. n = 3. *, P (TIF)</p

Reovirus infection enhances EV release.

Representative confocal images described in Fig 6G–6H are displayed for a single field of view, which is made up of an 8 x 8 tile imaging structure under 63X oil immersion. (TIF)</p

Reovirus infection enhances EV release compared to uninfected cells.

L cells were adsorbed with media (mock; M) or with three individual clones of T1L or T3D reovirus at an MOI of 1 PFU/cell for 72 h. (A-C) Fractions enriched in large, medium, and small EVs were harvested from supernatants using sequential centrifugation, as previously described, then lysed. Equal lysate volumes were resolved by SDS-PAGE and Coomassie staining (A), three independent experiments were quantified (B), and they were normalized by dividing the average virus-infected value by the average mock-infected value (C). (D-F) Fractions enriched in large, medium, and small EVs were harvested from supernatants using sequential centrifugation, as previously described. Then, EVs were immunoprecipitated using annexin V nanobeads, which bind to phosphatidylserine. Equal volumes of immunoprecipitated material were resolved by SDS-PAGE and Coomassie staining (D), three independent experiments were quantified (E), and normalized by dividing the average virus-infected value by the average mock-infected value (F). (G-H) Fractions enriched in large, medium, and small EVs were harvested from supernatants using sequential centrifugation, as previously described. Each sample was resuspended in an equal volume of salt-balanced buffer, allowed to interact with a fluorescent lipid dye, loaded into the well of a Mattek dish, covered with a sterile glass cover slip, and imaged using confocal microscopy. EVs were counted in 10 random fields of view, each representing an 8 x 8 tile imaging structure (G) and normalized by dividing the average virus-infected value by the average mock-infected value (H). Error bars indicate SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons.</p

Reovirus infection does not significantly alter whole cell protein expression.

L cells were adsorbed with medium (mock) or with three individual clones of T1L or T3D reovirus at an MOI of 1 PFU/cell for 72 h. (A-C) Cells were lysed in RIPA buffer, and lysates were resolved by SDS-PAGE and Coomassie staining (A), three independent experiments were quantified (B), and normalized by dividing the average virus-infected value by the average mock-infected value (C). Error bars indicate SD, n = 3. Comparisons by one-way ANOVA with Tukey’s multiple comparisons. (TIF)</p

Reovirus nonstructural protein association with EV fractions is cell type dependent.

Caco-2 cells were adsorbed with three individual clones (C1-C3) of T1L or T3D reovirus at an MOI of 5 PFU/cell for 72 h. Reovirus nonstructural protein association with large EV, medium EV, and small EV/free virus fractions was quantified following SDS-PAGE and immunoblotting for T1L σNS or T3D σNS. (TIF)</p

Reovirus protein co-fractionates with EV-enriched fractions released from cells regardless of plasma membrane integrity phenotype.

L cells were adsorbed with three individual clones of T1L or T3D reovirus at an MOI of 1 PFU/cell. (A) Every 24 h, cell lysates were collected, and virus in the supernatant was quantified by FFA. Error bars indicate SD. n = 3. *, P n = 3. *, P Biorender.com. (D-G) Infected-cell supernatants were collected every 24 h for 96 h. Mock-infected supernatant was collected at 96 h, but reovirus protein was not detected. Reovirus protein association with large EV, medium EV, and small EV/free virus fractions was quantified following SDS-PAGE and immunoblotting. Representative immunoblots probed using reovirus antiserum for T1L (D) and T3D (F) and graphs showing results quantified from three independent immunoblots for T1L (E) and T3D (G) are shown. Asterisk denotes the reovirus λ3 protein band used for quantitation. Error bars indicate SD. n = 3. *, P n = 3. **, P < 0.01 by two-way ANOVA with Tukey’s multiple comparisons.</p

Reovirus plasma membrane disruption is strain-specific in Caco-2 cells.

Caco-2 cells were adsorbed with medium (mock) or with three individual clones of T1L or T3D reovirus at an MOI of 5 PFU/cell. Cell membrane disruption was quantified for T1L-, T3D-, and mock-infected cells every 24 h for 96 h using an LDH assay. A medium-only negative control and a kit-specific positive control quantified in triplicate at 96 h are shown. Error bars indicate SD. n = 3. ***, P (TIF)</p

Multiple reovirus particles can be transported by protective, medium-sized EVs and non-protective large EVs.

(A) L cells were adsorbed with three individual clones of reovirus strains T1L or T3D at an MOI of 1 PFU/cell. After 72 h, EV-associated and free reovirus particles were enriched using sequential centrifugation and iodixanol gradient separation, as previously described, then incubated with σ1-specific reovirus antiserum (treated) or with diluent (untreated). Infectious units were quantified by plaque assay. Created using Biorender.com. (B-E) Plaque titer (B, D) and percent relative infectivity, quantified by dividing the treated infectious units by mock-treated infectious units and multiplying by 100 (C, E), for each sample are shown. Error bars indicate SD. “n.d.” = not detected. n = two titers per sample in each of three independent experiments. *, P Biorender.com. (G) Normalized melt curves for control RNA from WT (red), BC (blue), and 2:1, 1:1, and 1:2 mixtures of WT and BC (green) RNA are shown. (H) Genotype quantitation for the large EV, medium EV, and small EV/free virus fractions as a percentage of total plaques analyzed. Error bars indicate SD. n = 24 plaques represented by each data point in four independent experiments. **, P < 0.01; ***, P < 0.001 by Pearson’s chi-squared analysis with pairwise comparisons.</p

EV-mediated reovirus egress is consistent with microvesicle biogenesis.

Transmission electron microscopy of T1L-infected (A) or T3D-infected (B) L cells at 24 h p.i. Arrows point to viral particles observed near bleb-like structures budding from the plasma membrane in or around cells. Scale bar = 200 nm. (TIF)</p