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, beta-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 beta-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.

Fig. 1

VLP formations from the DGNNV capsid protein with cysteine mutations. a The intensity profiles of wild-type and cysteine mutants determined by sucrose density gradient centrifugation. Bold line: WT (no mutation); thin line: C115A; dotted line: C201A. b Bold line: C331A; thin line: C187A; dotted line: C187A/C331A. Fractions from #5 to #15 are denoted as I-f5-15 and fraction #24 is denoted as II-f24. c, d Micrographs from fractions #5 to #15 and fraction #24 of the cysteine mutants. The arrow indicates the broken particles with irregular shape. e Control: native DGNNV virus. Bar: 50 nm

Fig. 2

Thermal stability of WT-VLPs, β-ME-VLPs, and cysteine-mutant VLPs. a The amounts of VLPs in fraction #24 were calculated for the treatments at 40–85°C. b Arrhenius plots of the thermal decay rate constants for the VLPs

Fig. 3

Micrographs from electron microscopy of VLPs treated at different temperatures. a WT-VLPs; b β-ME-VLPs; c cysteine-mutant VLPs. Bar: 50 nm

Fig. 4

Disassembly and reassembly of DGNNV VLPs. a WT-VLPs; b β-mercaptoethanol-treated WT-VLPs. c Reassembly of mutant VLPs of C187A, C331A, and C187A/C331A. Bar: 50 nm