EFFECTS OF DOMAIN-SWITCHING AND SITE-DIRECTED MUTAGENESIS ON THE PROPERTIES AND FUNCTIONS OF THE VP7 PROTEINS OF 2 ORBIVIRUSES

Based on the crystal structure of the VP7 major core protein of blueto ngue virus serotype 10 (BTV-10) and that of the top domain of the VP7 protein of African horsesickness virus serotype 4 (AHSV-4), chimeras a nd site-directed mutants of the proteins were constructed and the prod ucts 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 (A BA). Further, both proteins exhibited the expected conformational epit opes of the constituent sequences. Using BAB VP7 it was demonstrated t hat residues of the upper domain of AHSV-4 VP7 contribute to the obser ved insolubility of the protein. By contrast, ABA VP7 protein was as s oluble as wild-type BTV-10 VP7. Replacement of selected amino acid res idues in the top domain (e.g., A167 by R; F209 by T) improved the solu bility 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 CL P formation. When the N-terminal region of the ABA protein (aa 1-120)w as replaced by the respective sequences of BTV VP7 (construct BBA), th e protein aggregated and did not form CLPs with coexpressed BTV VP3, m ost likely due to disruption of the required contacts between the N- a nd C-terminal regions of the bottom domain, leading to incorrect foldi ng of the chimera. (C) 1997 Academic Press.