Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

Chun Hsiung Wang, Chi Hsin Hsu, Yi Min Wu, Yu Chun Luo, Mei Hui Tu, Wei Hau Chang, R. Holland Cheng, Chan Shing Lin

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S-S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles.

Original languageEnglish (US)
Pages (from-to)73-80
Number of pages8
JournalVirus Genes
Volume41
Issue number1
DOIs
StatePublished - Aug 2010

Fingerprint

Nodaviridae
Virion
Cysteine
Capsid
Necrosis
Mercaptoethanol
Hot Temperature
Egtazic Acid
Sulfhydryl Compounds
Disulfides
Viruses
Viral Structures
Mutation
Electron Microscopy
Ions
Calcium
Peptides

Keywords

  • Disulfide linkages
  • Nervous necrosis virus
  • Reassembly
  • Virus-like particle

ASJC Scopus subject areas

  • Virology
  • Genetics
  • Molecular Biology

Cite this

Wang, C. H., Hsu, C. H., Wu, Y. M., Luo, Y. C., Tu, M. H., Chang, W. H., ... Lin, C. S. (2010). Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles. Virus Genes, 41(1), 73-80. https://doi.org/10.1007/s11262-010-0488-1

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles. / Wang, Chun Hsiung; Hsu, Chi Hsin; Wu, Yi Min; Luo, Yu Chun; Tu, Mei Hui; Chang, Wei Hau; Cheng, R. Holland; Lin, Chan Shing.

In: Virus Genes, Vol. 41, No. 1, 08.2010, p. 73-80.

Research output: Contribution to journalArticle

Wang, CH, Hsu, CH, Wu, YM, Luo, YC, Tu, MH, Chang, WH, Cheng, RH & Lin, CS 2010, 'Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles', Virus Genes, vol. 41, no. 1, pp. 73-80. https://doi.org/10.1007/s11262-010-0488-1
Wang, Chun Hsiung ; Hsu, Chi Hsin ; Wu, Yi Min ; Luo, Yu Chun ; Tu, Mei Hui ; Chang, Wei Hau ; Cheng, R. Holland ; Lin, Chan Shing. / Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles. In: Virus Genes. 2010 ; Vol. 41, No. 1. pp. 73-80.
@article{4c65247df086414c9920e3e4d5f4f944,
title = "Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles",
abstract = "The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S-S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles.",
keywords = "Disulfide linkages, Nervous necrosis virus, Reassembly, Virus-like particle",
author = "Wang, {Chun Hsiung} and Hsu, {Chi Hsin} and Wu, {Yi Min} and Luo, {Yu Chun} and Tu, {Mei Hui} and Chang, {Wei Hau} and Cheng, {R. Holland} and Lin, {Chan Shing}",
year = "2010",
month = "8",
doi = "10.1007/s11262-010-0488-1",
language = "English (US)",
volume = "41",
pages = "73--80",
journal = "Virus Genes",
issn = "0920-8569",
publisher = "Springer Netherlands",
number = "1",

}

TY - JOUR

T1 - Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

AU - Wang, Chun Hsiung

AU - Hsu, Chi Hsin

AU - Wu, Yi Min

AU - Luo, Yu Chun

AU - Tu, Mei Hui

AU - Chang, Wei Hau

AU - Cheng, R. Holland

AU - Lin, Chan Shing

PY - 2010/8

Y1 - 2010/8

N2 - The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S-S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles.

AB - The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S-S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles.

KW - Disulfide linkages

KW - Nervous necrosis virus

KW - Reassembly

KW - Virus-like particle

UR - http://www.scopus.com/inward/record.url?scp=77953870682&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77953870682&partnerID=8YFLogxK

U2 - 10.1007/s11262-010-0488-1

DO - 10.1007/s11262-010-0488-1

M3 - Article

C2 - 20446029

AN - SCOPUS:77953870682

VL - 41

SP - 73

EP - 80

JO - Virus Genes

JF - Virus Genes

SN - 0920-8569

IS - 1

ER -