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Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid.

ABSTRACT: Following attachment to host receptors via ?1, reovirus particles are endocytosed and disassembled to generate infectious subvirion particles (ISVPs). ISVPs undergo conformational changes to form ISVP*, releasing ?1 and membrane-targeting peptides from the viral ?1 protein. ISVP* formation is required for delivery of the viral core into the cytoplasm for replication. We characterized the properties of T3DF/T3DCS1, an S1 gene monoreassortant between two laboratory isolates of prototype reovirus strain T3D: T3DF and T3DC T3DF/T3DCS1 is poorly infectious. This deficiency is a consequence of inefficient encapsidation of S1-encoded ?1 on T3DF/T3DCS1 virions. Additionally, compared to T3DF, T3DF/T3DCS1 undergoes ISVP-to-ISVP* conversion more readily, revealing an unexpected role for ?1 in regulating ISVP* formation. The ?1 protein is held within turrets formed by the ?2 protein. To test if the altered properties of T3DF/T3DCS1 are due to a mismatch between ?1 and ?2 proteins from T3DF and T3DC, properties of T3DF/T3DCL2 and T3DF/T3DCS1L2, which express a T3DC-derived ?2, were compared. The presence of T3DC ?2 allowed more efficient ?1 incorporation, producing particles that exhibit T3DF-like infectivity. Compared to T3DF, T3DF/T3DCL2 prematurely converts to ISVP*, uncovering a role for ?2 in regulating ISVP* formation. Importantly, a virus with matching ?1 and ?2 displayed a more regulated conversion to ISVP* than either T3DF/T3DCS1 or T3DF/T3DCL2. 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.IMPORTANCE 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 ?1 attachment protein and the ?2 protein that anchors ?1 on the particle are mismatched. Our studies demonstrate that a ?1-?2 mismatch produces particles with lower levels of encapsidated ?1, consequently decreasing virus attachment and infectivity. The mismatch between ?1 and ?2 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.

SUBMITTER: Thete D

PROVIDER: S-EPMC6158412 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid.

Thete Deepti D Danthi Pranav P

Journal of virology 20180926 20

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<sup>F</sup>/T3D<sup>C</sup>S1, an S1 gene monoreassortant between two laboratory isolates o ...[more]

PMID: 30068646

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Project description:Reovirus is an enteric virus comprising eight structural proteins that form a double-layered capsid. During reovirus entry into cells, the outermost capsid layer (composed of proteins sigma3 and mu1C) is proteolytically processed to generate an infectious subviral particle (ISVP) that is subsequently uncoated to produce the transcriptionally active core particle. Kinetic studies suggest that protein sigma3 is rapidly removed from virus particles and then protein mu1C is cleaved. Initial cleavage of mu1C has been well described and generates an amino (N)-terminal delta peptide and a carboxyl (C)-terminal phi peptide. However, cleavage and removal of sigma3 is an extremely rapid event that has not been well defined. We have treated purified reovirus serotype 1 Lang virions with a variety of endoproteases. Time-course digestions with chymotrypsin, Glu-C, pepsin, and trypsin resulted in the initial generation of two peptides that were resolved in SDS-PAGE and analyzed by in-gel tryptic digestion and MALDI-Qq-TOFMS. Most tested proteases cut sigma3 within a "hypersensitive" region between amino acids 217 and 238. In addition, to gain a better understanding of the sequence of subsequent proteolytic events that result in generation of reovirus subviral particles, time-course digestions of purified particles were performed under physiologic salt conditions and released peptide fragments ranging from 500 to 5000 Da were directly analyzed by MALDI-Qq-TOFMS. Trypsin digestion initially released a peptide that corresponded to the C-terminus of mu1C, followed by a peptide that corresponded to amino acids 214-236 of the sigma3 protein. Other regions of mu1C were not observed until protein sigma3 was completely digested. Similar experiments with Glu-C indicated the hypersensitive region of sigma3 was cut first when virions were treated at pH values of 4.5 or 7.4, but treatment of virions with pepsin at pH 3.0 released different sigma3 peptides, suggesting acid-induced conformational changes in this outer capsid protein. These studies also revealed that the N-terminus of sigma3 is acetylated.

Project description:The mammalian orthoreovirus (reovirus) outer capsid is composed of 200 ?1-?3 heterohexamers and a maximum of 12 ?1 trimers. During cell entry, ?3 is degraded by luminal or intracellular proteases to generate the infectious subviral particle (ISVP). When ISVP formation is prevented, reovirus fails to establish a productive infection, suggesting proteolytic priming is required for entry. ISVPs are then converted to ISVP*s, which is accompanied by ?1 rearrangements. The ?1 and ?3 proteins confer resistance to inactivating agents; however, neither the impact on capsid properties nor the mechanism (or basis) of inactivation is fully understood. Here, we utilized T1L/T3D M2 and T3D/T1L S4 to investigate the determinants of reovirus stability. Both reassortants encode mismatched subunits. When ?1-?3 were derived from different strains, virions resembled wild-type particles in structure and protease sensitivity. T1L/T3D M2 and T3D/T1L S4 ISVPs were less thermostable than wild-type ISVPs. In contrast, virions were equally susceptible to heating. Virion associated ?1 adopted an ISVP*-like conformation concurrent with inactivation; ?3 preserves infectivity by preventing ?1 rearrangements. Moreover, thermostability was enhanced by a hyperstable variant of ?1. Unlike the outer capsid, the inner capsid (core) was highly resistant to elevated temperatures. The dual layered architecture allowed for differential sensitivity to inactivating agents.IMPORTANCE Nonenveloped and enveloped viruses are exposed to the environment during transmission to a new host. Protein-protein and/or protein-lipid interactions stabilize the particle and protect the viral genome. Mammalian orthoreovirus (reovirus) is composed of two concentric, protein shells. The ?1 and ?3 proteins form the outer capsid; contacts between neighboring subunits are thought to confer resistance to inactivating agents. We further investigated the determinants of reovirus stability. The outer capsid was disrupted concurrent with the loss of infectivity; virion associated ?1 rearranged into an altered conformation. Heat sensitivity was controlled by ?3; however, particle integrity was enhanced by a single ?1 mutation. In contrast, the inner capsid (core) displayed superior resistance to heating. These findings reveal structural components that differentially contribute to reovirus stability.

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Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid.

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Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid.

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