Folding and particle assembly are disrupted by single-point mutations near the autocatalytic cleavage site of Nudaurelia capensis omega virus capsid protein.
Protein subunits of several RNA viruses are known to undergo post-assembly, autocatalytic cleavage that is required for infectivity. Nudaurelia capensis omega virus (Nomega V) is one of the simplest viruses to undergo an autocatalytic cleavage, making it an excellent model to understand both assembly and the mechanism of autoproteolysis. Heterologous expression of the coat protein gene of Nomega V in a baculovirus system results in the spontaneous assembly of virus-like particles (VLPs) that remain uncleaved when purified at neutral pH. After acidification to pH 5.0, the VLPs autocatalytically cleave at residue 570, providing an in vitro control of the cleavage. The crystal structure of Nomega V displays three residues near the scissile bond that were candidates for participation in the reaction. These were changed by site-directed mutagenesis to conservative and nonconservative residues and the products analyzed. Even conservative changes at the three residues dramatically reduced cleavage when the subunits assembled properly. Unexpectedly, we discovered that these residues are not only critical to the kinetics of Nomega V autoproteolysis, but are also necessary for proper folding of subunits and, ultimately, assembly of Nomega V VLPs.
Taylor
Derek J
DJ
Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Johnson
John E
JE
D000818:Animals; D036022:Capsid Proteins / Q000737:chemistry*; D002384:Catalysis; D002460:Cell Line; D018360:Crystallography, X-Ray; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D007700:Kinetics; D008854:Microscopy, Electron; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D017354:Point Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry; D012328:RNA Viruses / Q000235:genetics; D014466:Ultraviolet Rays; D014771:Virion / Q000737:chemistry; D019065:Virus Assembly; D014780:Viruses / Q000378:metabolism*
D036022:Capsid Proteins; C092202:coat protein, Nudaurelia capensis omega virus; D012313:RNA