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

Folding and particle assembly are disrupted by single‐point mutations near the autocatalytic... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Protein Science Wiley

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

Protein Science, Volume 14 (2) – Feb 1, 2005

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Publisher
Wiley
Copyright
Copyright © 2005 The Protein Society
ISSN
0961-8368
eISSN
1469-896X
DOI
10.1110/ps.041054605
Publisher site
See Article on Publisher Site

Abstract

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.

Journal

Protein ScienceWiley

Published: Feb 1, 2005

References

  • Improved methods for building protein models in electron density maps and the location of errors in these models
    Jones, Jones; Zou, Zou; Cowan, Cowan; Kjeldgaard, Kjeldgaard
  • MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures
    Kraulis, Kraulis
  • Protein splicing and autoproteolysis mechanisms
    Perler, Perler; Xu, Xu; Paulus, Paulus

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