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Folding and particle assembly are disrupted by single-point mutations near the autocatalytic cleavage site of Nudaurelia capensis virus capsid protein

¹ Department of Chemistry and Biochemistry, University of California–San Diego, La Jolla, California 92093, USA
² Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA

(RECEIVED August 19, 2004; FINAL REVISION October 19, 2004; ACCEPTED October 19, 2004)

Protein subunits of several RNA viruses are known to undergo post-assembly, autocatalytic cleavage that is required for infectivity. Nudaurelia capensis virus (N 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 N 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 N 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 N V autoproteolysis, but are also necessary for proper folding of subunits and, ultimately, assembly of N V VLPs.

Keywords: autocatalysis; autoproteolysis; virus maturation; Nudaurelia capensis virus; tetravirus; nodavirus

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041054605.

Reprint requests to: John E. Johnson, Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: jackj{at}scripps.edu; fax: (858) 784-8660.

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