Reovirus Efficiently Reassorts Genome Segments during Coinfection and Superinfection

Thoner, Timothy W.; Meloy, Madeline M.; Long, Jacob M.; Diller, Julia R.; Slaughter, James C.; Ogden, Kristen M.

doi:10.1128/jvi.00910-22

“…To determine whether EVs can transport an infectious unit consisting of multiple reovirus particles, we used high-resolution melt (HRM) analysis to detect genotypes of individual viral particles [ 54 ]. We co-infected L cells with wild-type (WT) and genetically barcoded (BC) T3D reoviruses, collected cell culture supernatants at 24 h p.i., and enriched for large EV, medium EV, and small EV/free virus fractions ( Fig 4F ).…”

Section: Resultsmentioning

confidence: 99%

Mammalian orthoreovirus can exit cells in extracellular vesicles

Smith,

Krystofiak,

Ogden

2024

PLoS Pathog

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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.

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“…To determine whether EVs can transport an infectious unit consisting of multiple reovirus particles, we used high-resolution melt (HRM) analysis to detect genotypes of individual viral particles (54). We co-infected L cells with wild-type (WT) and genetically barcoded (BC) T3D reoviruses, collected cell culture supernatants at 24 h p.i., and enriched for large EV, medium EV, and small EV/free virus fractions ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Strain rsT3D I is a variant of the parental rsT3D prototype strain that contains a T249I mutation that renders viral attachment protein σ1 resistant to proteolytic cleavage (89). Strain rsT3D I BC is identical to rsT3D I excepting silent genetic “barcode” mutations engineered in each segment (54). Briefly, semi-confluent monolayers of BHK-T7 cells in 6 well plates were transfected with 10 plasmid constructs encoding T1L and T3D reovirus RNAs.…”

Section: Methodsmentioning

confidence: 99%

“…A total of 24 well-separated plaques per fraction per replicate were picked and amplified in L cell monolayers in 24-well plates for 2 days. RNA was extracted using TRIzol (Invitrogen), reverse transcribed using random hexamers, and genotyped using HRM, as previously described, using primers specific for the L2 segment (54).…”

Section: Negative-stain Transmission Electronmentioning

confidence: 99%

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Mammalian orthoreovirus can exit cells in extracellular vesicles

Smith,

Krystofiak,

Ogden

2023

Preprint

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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.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.

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“…Reovirus has a broad zoonotic host range 17 , infecting most mammals including humans, a high prevalence in susceptible hosts 73 , cross-species transmission capability 74 , 75 , circulation in spillover-prone bat and rodent reservoirs 74 , 76 – 80 , capacity for genetic reassortment 81 , 82 , and engagement of receptors conserved across species 24 , 26 , 29 . Combined with sporadic human cases of reovirus-associated disease 74 , 75 , 80 , 83 – 85 , these characteristics raise the possibility of emergence of more virulent strains, which may overcome the age restriction in disease development.…”

Section: Discussionmentioning

confidence: 99%

Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus

Shang

¹

,

Simpson

²

,

Taylor

³

et al. 2023

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Mammalian orthoreovirus (reovirus) infects most mammals and is associated with celiac disease in humans. In mice, reovirus infects the intestine and disseminates systemically to cause serotype-specific patterns of disease in the brain. To identify receptors conferring reovirus serotype-dependent neuropathogenesis, we conducted a genome-wide CRISPRa screen and identified paired immunoglobulin-like receptor B (PirB) as a receptor candidate. Ectopic expression of PirB allowed reovirus binding and infection. PirB extracelluar D3D4 region is required for reovirus attachment and infectivity. Reovirus binds to PirB with nM affinity as determined by single molecule force spectroscopy. Efficient reovirus endocytosis requires PirB signaling motifs. In inoculated mice, PirB is required for maximal replication in the brain and full neuropathogenicity of neurotropic serotype 3 (T3) reovirus. In primary cortical neurons, PirB expression contributes to T3 reovirus infectivity. Thus, PirB is an entry receptor for reovirus and contributes to T3 reovirus replication and pathogenesis in the murine brain.

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Reovirus Efficiently Reassorts Genome Segments during Coinfection and Superinfection

Abstract: Reassortment, or genome segment exchange between viruses, can generate novel virus genotypes and pandemic virus strains. For viruses to reassort their genome segments, they must replicate within the same physical space by coinfecting the same host cell.

Cited by 4 publications

References 81 publications

Mammalian orthoreovirus can exit cells in extracellular vesicles

Mammalian orthoreovirus can exit cells in extracellular vesicles

Mammalian orthoreovirus can exit cells in extracellular vesicles

Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus

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