Point mutation in calcium-binding domain of mouse polyomavirus VP1 protein does not prevent virus-like particle formation, but changes VP1 interactions with cell structures

Adamec, T; Palková, Zdena; Velková, K; Štokrová, Jitka; Forstová, Jitka

doi:10.1016/j.femsyr.2004.10.012

Cited by 6 publications

(3 citation statements)

References 29 publications

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“…The removal of Ca 2ϩ from capsids could be particularly crucial for viruses that depend on metal ions for assembly and stability, such as rotavirus, mouse polyomavirus, and dragon grouper nervous necrosis virus (DGNNV) (1,39,43). A few studies have reported that mutations at the metal-binding sites decrease virus infectivity by impairing the early stages of virus-host interaction and genome release (23,24).…”

Section: Divalent Metal Ions Are Components Of Numerous Icosahedral Vmentioning

confidence: 99%

Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate

Banerjee

Speir

Kwan³

et al. 2010

J Virol

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Divalent metal ions are components of numerous icosahedral virus capsids. Flock House virus (FHV), a small RNA virus of the family Nodaviridae, was utilized as an accessible model system with which to address the effects of metal ions on capsid structure and on the biology of virus-host interactions. Mutations at the calcium-binding sites affected FHV capsid stability and drastically reduced virus infectivity, without altering the overall architecture of the capsid. The mutations also altered the conformation of gamma, a membranedisrupting, virus-encoded peptide usually sequestered inside the capsid, by increasing its exposure under neutral pH conditions. Our data demonstrate that calcium binding is essential for maintaining a pH-based control on gamma exposure and host membrane disruption, and they reveal a novel rationale for the metal ion requirement during virus entry and infectivity. In the light of the phenotypes displayed by a calcium site mutant of FHV, we suggest that this mutant corresponds to an early entry intermediate formed in the endosomal pathway.During cellular entry, the capsids of nonenveloped viruses undergo conformational changes triggered by various host factors. The transitions include the exposure of membrane-active, hydrophobic viral polypeptides and the destabilization and disassembly of the capsid, culminating in the delivery of the viral genome to the cytoplasm. A detailed, stepwise pathway of disassembly is unavailable for most viruses and requires molecular characterization of the intermediates formed during this process. Replication of disassembly intermediates in vitro necessitates careful treatment of native virions to simulate conditions likely to be encountered inside host cells. Receptor binding induces conformational changes in poliovirus, and the poliovirus entry intermediates, the 135S and 80S particles, can be generated in vitro by treating the native virion with the purified receptor, or by heating (6,10,19). Reovirus, which requires cathepsin-mediated proteolysis in the late endosomes in order to undergo entry-related changes, produces infectious subvirion particles (ISVP) and core particles as disassembly intermediates upon treatment with proteases in vitro (11,14).Apart from receptors and cellular proteases, other host determinants affecting viruses include low pH and Ca 2ϩ depletion in specialized cellular compartments. The removal of Ca 2ϩ from capsids could be particularly crucial for viruses that depend on metal ions for assembly and stability, such as rotavirus, mouse polyomavirus, and dragon grouper nervous necrosis virus (DGNNV) (1,39,43). A few studies have reported that mutations at the metal-binding sites decrease virus infectivity by impairing the early stages of virus-host interaction and genome release (23,24). When Ca 2ϩ is removed from the capsids of plant viruses such as cowpea chlorotic mottle virus (CCMV), tomato bushy stunt virus (TBSV), and turnip crinkle virus (TCV) by metal chelators (2, 33, 35), the virion swells by almost 10% and becomes suscept...

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Section: Divalent Metal Ions Are Components Of Numerous Icosahedral Vmentioning

confidence: 99%

Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate

Banerjee

Speir

Kwan³

et al. 2010

J Virol

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“…VLP samples were suspended in buffer with bromophenol blue and 10% v/v glycerol (final concentration) to aid loading. EDTA was avoided in buffers as polyomavirus VLPs are known to be stabilized by interactions with calcium ions. , Nucleic acid staining was performed by soaking the gel in 1× GelRed (Biotium #41003) in TA buffer for 1 h at room temperature, with gentle shaking. 0.5 μg of DNA ladder (Thermo Scientific #SM0311) was loaded as a positive control.…”

Section: Methodsmentioning

confidence: 99%

Artificial Self-assembling Nanocompartment for Organizing Metabolic Pathways in Yeast

et al. 2021

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Metabolic pathways are commonly organized by sequestration into discrete cellular compartments. Compartments prevent unfavorable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme/pathway behavior and for metabolic engineering. Here, we expand the intracellular compartmentalization toolbox for budding yeast ( Saccharomyces cerevisiae ) with Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilized green fluorescent protein (GFP) fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. An engineered VP1 variant displayed improved cargo capture properties and differential subcellular localization compared to wild-type VP1. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in d -glucaric acid biosynthesis. Strains with encapsulated MIOX produced ∼20% more d -glucaric acid compared to controls expressing “free” MIOX—despite accumulating dramatically less expressed protein—and also grew to higher cell densities. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.

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“…VLP samples were suspended in buffer with bromophenol blue and 10% v/v glycerol (final concentration) to aid loading. EDTA was avoided in buffers as polyomavirus VLPs are known to be stabilised by interactions with calcium ions 30,55 . Nucleic acid staining was performed by soaking the gel in 1X GelRed (Biotium #41003) in TA buffer for 1 hour at room temperature, with gentle shaking.…”

Section: Methodsmentioning

confidence: 99%

An artificial self-assembling nanocompartment for organising metabolic pathways in yeast

Cheah

Stark

Adamson

et al. 2021

Preprint

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Metabolic pathways are commonly organised by sequestration into discrete cellular compartments. Compartments prevent unfavourable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme/pathway behaviour and for metabolic engineering. Here, we expand the intracellular compartmentalisation toolbox for budding yeast (Saccharomyces cerevisiae) with engineered Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell; and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilised GFP fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. In order to access metabolites of native and engineered yeast metabolism, VLP-based nanocompartments were directed to assemble in the cytosol by removal of the VP1 nuclear localisation signal. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in D-glucaric acid biosynthesis. Strains with encapsulated MIOX produced ~20% more D-glucaric acid compared to controls expressing ‘free’ MIOX - despite accumulating dramatically less expressed protein - and also grew to higher cell densities. These effects were linked to enzyme stabilisation and mitigation of cellular toxicity by the engineered compartment. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.

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Point mutation in calcium-binding domain of mouse polyomavirus VP1 protein does not prevent virus-like particle formation, but changes VP1 interactions with cell structures

Cited by 6 publications

References 29 publications

Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate

Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate

Artificial Self-assembling Nanocompartment for Organizing Metabolic Pathways in Yeast

An artificial self-assembling nanocompartment for organising metabolic pathways in yeast

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