title: Role of the DxxDxD motif in the assembly and stability of betanodavirus particles.
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 degrees 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 degrees 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.
authors.affiliation: Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, ROC.
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: Wu
authors.forename: Chi-Hsin
authors.initials: CH
authors.lastname: Hsu
authors.forename: Chun-Hsiung
authors.initials: CH
authors.lastname: Wang
authors.forename: Wangta
authors.initials: W
authors.lastname: Liu
authors.forename: Wei-Hau
authors.initials: WH
authors.lastname: Chang
authors.forename: Chan-Shing
authors.initials: CS
authors.lastname: Lin
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D030381:Nodaviridae / Q000737:chemistry* / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D013696:Temperature; D014771:Virion / Q000737:chemistry / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D019065:Virus Assembly
chemical_list: D002118:Calcium