Effects of domain-switching and site-directed mutagenesis on the properties and functions of the VP7 proteins of two orbiviruses.
Based on the crystal structure of the VP7 major core protein of bluetongue virus serotype 10 (BTV-10) and that of the top domain of the VP7 protein of African horsesickness virus serotype 4 (AHSV-4), chimeras and site-directed mutants of the proteins were constructed and the products analyzed with respect to their properties and functions. Chimeras with the central upper domain of BTV-10 VP7 replaced by that of AHSV-4 VP7 (construct BAB) formed trimers, as did the converse construct (ABA). Further, both proteins exhibited the expected conformational epitopes of the constituent sequences. Using BAB VP7 it was demonstrated that residues of the upper domain of AHSV-4 VP7 contribute to the observed insolubility of the protein. By contrast, ABA VP7 protein was as soluble as wild-type BTV-10 VP7. Replacement of selected amino acid residues in the top domain (e.g., A167 by R; F209 by T) improved the solubility of BAB VP7. Since the trimeric BAB and ABA VP7 proteins did not form core-like particles (CLPs) when coexpressed with BTV VP3, it was concluded that trimerization of chimeric VP7 is not sufficient for CLP formation. When the N-terminal region of the ABA protein (aa 1-120) was replaced by the respective sequences of BTV VP7 (construct BBA), the protein aggregated and did not form CLPs with coexpressed BTV VP3, most likely due to disruption of the required contacts between the N- and C-terminal regions of the bottom domain, leading to incorrect folding of the chimera.
Monastyrskaya
K
K
Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom.
Staeuber
N
N
Sutton
G
G
Roy
P
P
D019943:Amino Acid Substitution; D000956:Antigens, Viral / Q000235:genetics; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D015967:Gene Expression Regulation, Viral; D016297:Mutagenesis, Site-Directed; D018163:Orbivirus / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics
D000956:Antigens, Viral; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; C050779:VP7 protein, Rotavirus; D014758:Viral Core Proteins; C069264:VP7 major core antigen, African horsesickness virus