Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid

Abstract

Following attachment to host receptors via σ1, reovirus particles are endocytosed and disassembled to generate infectious subvirion particles (ISVPs). ISVPs undergo conformational changes to form ISVP*, releasing σ1 and membrane-targeting peptides from the viral μ1 protein. ISVP* formation is required for delivery of the viral core into the cytoplasm for replication. We characterized the properties of T3D/T3DS1, an S1 gene monoreassortant between two laboratory isolates of prototype reovirus strain T3D: T3D and T3D T3D/T3DS1 is poorly infectious. This deficiency is a consequence of inefficient encapsidation of S1-encoded σ1 on T3D/T3DS1 virions. Additionally, compared to T3D, T3D/T3DS1 undergoes ISVP-to-ISVP* conversion more readily, revealing an unexpected role for σ1 in regulating ISVP* formation. The σ1 protein is held within turrets formed by the λ2 protein. To test if the altered properties of T3D/T3DS1 are due to a mismatch between σ1 and λ2 proteins from T3D and T3D, properties of T3D/T3DL2 and T3D/T3DS1L2, which express a T3D-derived λ2, were compared. The presence of T3D λ2 allowed more efficient σ1 incorporation, producing particles that exhibit T3D-like infectivity. Compared to T3D, T3D/T3DL2 prematurely converts to ISVP*, uncovering a role for λ2 in regulating ISVP* formation. Importantly, a virus with matching σ1 and λ2 displayed a more regulated conversion to ISVP* than either T3D/T3DS1 or T3D/T3DL2. In addition to identifying new regulators of ISVP* formation, our results highlight that protein mismatches produced by reassortment can alter virus assembly and thereby influence subsequent functions of the virus capsid. Cells coinfected with viruses that possess a multipartite or segmented genome reassort to produce progeny viruses that contain a combination of gene segments from each parent. Reassortment places new pairs of genes together, generating viruses in which mismatched proteins must function together. To test if such forced pairing of proteins that form the virus shell or capsid alters the function of the particle, we investigated properties of reovirus variants in which the σ1 attachment protein and the λ2 protein that anchors σ1 on the particle are mismatched. Our studies demonstrate that a σ1-λ2 mismatch produces particles with lower levels of encapsidated σ1, consequently decreasing virus attachment and infectivity. The mismatch between σ1 and λ2 also altered the capacity of the viral capsid to undergo conformational changes required for cell entry. These studies reveal new functions of reovirus capsid proteins and illuminate both predictable and novel implications of reassortment.

Reovirus Core Proteins λ1 and σ2 Promote Stability of Disassembly Intermediates and Influence Early Replication Events

“…T3D/T1L L3S2 and T3D ISVPs were heated to 40°C and loss of infectivity was measured by plaque assay. Consistent with previous work (20), at 40°C, most of the T3D ISVPs did not convert to ISVP* and the change in titer was minimal (Fig 2C). However, heating T3D/T1L L3S2 ISVPs to 40°C resulted in ISVP* conversion and a significant loss in infectivity (Fig 2C).…”

“…and σ2. Single gene reassortants between prototype reovirus strains T1L and T3D, which contain mismatches between outer capsid proteins, display altered capsid stability and efficiency of disassembly intermediate formation (19)(20)(21). To determine whether the core proteins λ1 and σ2 play a role in ISVP-to-ISVP* conversion efficiency, we characterized the properties of T3D/T1L L3S2.…”

Evolution of the Piscine orthoreovirus Genome Linked to Emergence of Heart and Skeletal Muscle Inflammation in Farmed Atlantic Salmon (Salmo salar)

“…We observed that segments S1 and M2 are genetically linked, which indicate that the structure and interaction of their encoded proteins are vital for virus fitness. For MRV, an in vitro forced reassortment event has been reported to alter virus infectivity and replication efficiency due to λ2 and σ1 protein mismatch [52].…”

Control of Capsid Transformations during Reovirus Entry

“…Work using reassortant viruses engineered in a laboratory indicates that σ1 and λ2, and their interactions with other outer capsid proteins, can play additional unexpected roles during entry. A reassortant virus (T3D F /T3D C S1) which contains nine gene segments from the laboratory strain T3D F and the S1 gene segment (encoding σ1) from the laboratory variant T3D C demonstrated lower stability and more efficient ISVP-to-ISVP* transition in comparison to T3D F [ 60 ]. Thus, properties of σ1 can affect the stability of ISVPs.…”