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Folding and particle assembly are disrupted by single‐point mutations near the autocatalytic cleavage site of Nudaurelia capensis ω virus capsid protein
Author(s) -
Taylor Derek J.,
Johnson John E.
Publication year - 2005
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.041054605
Subject(s) - scissile bond , capsid , cleavage (geology) , autocatalysis , biophysics , chemistry , protein folding , rna , biology , biochemistry , active site , gene , enzyme , paleontology , fracture (geology) , catalysis
Protein subunits of several RNA viruses are known to undergo post‐assembly, autocatalytic cleavage that is required for infectivity. Nudaurelia capensis ω virus (Nω 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 Nω 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 Nω 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 Nω V autoproteolysis, but are also necessary for proper folding of subunits and, ultimately, assembly of Nω V VLPs.