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Journal of Virology, September 2000, p. 8658-8669, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Functional Dissection of the Major Structural Protein of Bluetongue Virus: Identification of Key Residues within VP7 Essential for Capsid Assembly

Chang-Kwang Limn,1 Norbert Staeuber,2,3 Katherine Monastyrskaya,2,3 Patrice Gouet,4 and Polly Roy1,2,3,*

Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294,1 and NERC Institute of Virology and Environmental Microbiology, Oxford OX1 3SR,2 and Department of Biochemistry3 and Laboratory of Molecular Biophysics,4 University of Oxford, Oxford OX1 3QU, United Kingdom

Received 28 February 2000/Accepted 8 June 2000

A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.

* Corresponding author. Mailing address: NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, United Kingdom. Phone: 44 1865 281640. Fax: 44 1865 281696. E-mail: por{at}wpo.nerc.ac.uk.

Journal of Virology, September 2000, p. 8658-8669, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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