Amino acids substitutions in σ1 and μ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly

Sandekian, Véronique; Lemay, Guy

doi:10.1016/j.virusres.2014.11.002

Cited by 13 publications

(20 citation statements)

References 57 publications

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“…Indeed, we have previously observed that a 1 variant contains different levels of particle-associated 1 (76). Another study has also suggested that compatibility between 1 and 1 confers optimal 1 encapsidation and function on the particle (70). The difference in 1 encapsidation between T3D F and T3D C may be governed by the function of 1 or yet another protein.…”

Section: Discussionmentioning

confidence: 91%

“…Since both the attachment and ISVP* phenotype of T3D F /T3D C S1 could be explained by 1-2 interaction, we decided to determine whether a mismatch between T3D C 1 and T3D F 2 accounts for the phenotypes observed. The 2 proteins of T3D F and T3D C differ at residue 504, resulting in a glycine-to-glutamate change, and at residue 509, resulting in a glycine-to-arginine change (70). Thus, to test our idea about the contribution of 1-2 mismatch, we generated two additional viruses, T3D F /T3D C L2 (a monoreassortant that contains an L2 gene segment from strain T3D C in an otherwise T3D F background) and T3D F /T3D C S1L2 (a double reassortant that contains both the S1 and L2 gene segments from strain T3D C in an otherwise T3D F background).…”

Section: Figmentioning

confidence: 99%

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Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid

Thete

Danthi

2018

J Virol

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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.

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Section: Discussionmentioning

confidence: 91%

Section: Figmentioning

confidence: 99%

Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid

Thete

Danthi

2018

J Virol

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“…Finally, reovirus is a promising contender for virus-mediated tumor oncolysis. We and others have found that genetic modification of reovirus can improve oncolytic activity in vitro and in animal cancer models (87)(88)(89)(90)(91). Reovirus also provides a potential vaccine vector (92,93).…”

Section: Discussionmentioning

confidence: 99%

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

Eaton

Kobayashi

Dermody

et al. 2017

J Virol

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Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

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“…For example, many reovirus protein functions have been characterized by comparing phenotypes among natural reovirus variants, reovirus genome reassortants, and laboratory derived reovirus mutants10111213141516171819. In conjunction with reverse genetics approaches for these viruses202122232425, it is now possible to design copious virus mutants, purify them in-slurry, and make rapid comparisons of virus proteins, virion structure, and virus replication.…”

Section: Discussionmentioning

confidence: 99%

Novel High-throughput Approach for Purification of Infectious Virions

James

Cooney²,

Agopsowicz

et al. 2016

Sci Rep

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Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents. Existing methods to purify viruses such as gradient ultracentrifugation or chromatography have limitations, for example demand for technical expertise or specialized equipment, high time consumption, and restricted capacity. Our laboratory explores mutations in oncolytic reovirus that could improve oncolytic activity, and makes routine use of numerous virus variants, genome reassortants, and reverse engineered mutants. Our research pace was limited by the lack of high-throughput virus purification methods that efficiently remove confounding cellular contaminants such as cytokines and proteases. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700 kDa. Capto. Core 700 chromatography produced virion purity and infectivity indistinguishable from CsCl density gradient ultracentrifugation as determined by electron microscopy, gel electrophoresis analysis and plaque titration. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. The in-slurry purification approach offered substantially increased virus purity over crude cell lysates, media, or high-spin preparations and would be especially useful for high-throughput virus screening applications where density gradient ultracentrifugation is not feasible.

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Amino acids substitutions in σ1 and μ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly

Cited by 13 publications

References 57 publications

Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid

Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

Novel High-throughput Approach for Purification of Infectious Virions

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