Role of the DxxDxD motif in the assembly and stability of betanodavirus particles

Role of the DxxDxD motif in the assembly and stability of betanodavirus particles Piscine betanodavirus possesses a bipartite genome of single-stranded (+)RNAs. RNA2 cDNA of dragon grouper nervous necrosis virus (DGNNV) has been expressed previously to form virus-like particles (VLPs), which are highly similar to the native virion. Experiments with calcium-chelating or reducing/oxidizing reagents showed that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. The results of mutational analysis of DxxDxD, which is putatively involved in the coordination of calcium ions, showed that the D133N mutation significantly disrupted the assembly of VLPs, while D130N and D135N mutants produced heterogeneous VLPs with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of the D135N mutant were stable at a temperature of 85°C, which is slightly higher than that for wild-type, whereas VLPs of the D130N mutant could not tolerate the thermal effects at a temperature higher than 60°C. It was deduced that the three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and that, among them, the DxxD provides a more stable coordinate of calcium ligand than DxD. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Role of the DxxDxD motif in the assembly and stability of betanodavirus particles

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Publisher
Springer Journals
Copyright
Copyright © 2008 by Springer-Verlag
Subject
Biomedicine; Infectious Diseases; Medical Microbiology ; Virology
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-008-0150-6
Publisher site
See Article on Publisher Site

Abstract

Piscine betanodavirus possesses a bipartite genome of single-stranded (+)RNAs. RNA2 cDNA of dragon grouper nervous necrosis virus (DGNNV) has been expressed previously to form virus-like particles (VLPs), which are highly similar to the native virion. Experiments with calcium-chelating or reducing/oxidizing reagents showed that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. The results of mutational analysis of DxxDxD, which is putatively involved in the coordination of calcium ions, showed that the D133N mutation significantly disrupted the assembly of VLPs, while D130N and D135N mutants produced heterogeneous VLPs with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of the D135N mutant were stable at a temperature of 85°C, which is slightly higher than that for wild-type, whereas VLPs of the D130N mutant could not tolerate the thermal effects at a temperature higher than 60°C. It was deduced that the three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and that, among them, the DxxD provides a more stable coordinate of calcium ligand than DxD.

Journal

Archives of VirologySpringer Journals

Published: Sep 1, 2008

References

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