Effects of domain-switching and site-directed mutagenesis on the properties and functions of the VP7 proteins of two orbiviruses.

摘要

The soluble structural protein, VP7, which forms trimers in vivo, has been crystallized from the orbivirus, bluetongue virus serotype 10 (BTV-10) and has been found to consist of 2 domains. The upper domain of the homologous (insoluble) protein fromAfrican horse sickness virus serotype 4 (AHSV-4) has also been crystallized. Chimeric genes were prepared and expressed in order to determine the effects of domain-switching on solubility, folding and trimerization. The chimeric protein, BAB, was composed of the upper domain of AHSV-4 VP7, and the lower domain of BTV-10. The converse construct, ABA, was made up of the upper domain of BTV-10 and the lower domain of AHSV-4. Both chimeras formed trimers and displayed the conformational epitopes of their constituent proteins. ABA VP7 was as soluble as BTV-10 VP7, but BAB VP7 was insoluble, indicating that it is the upper domain of AHSV-4 VP7 which confers its insolubility. The replacement of selected amino acids in the upper domain by site-directed mutagenesis improved the solubility of BAB VP7 but not AHSV-4 VP7. Wild type VP7 forms core-like particles (CLPs) with another structural protein, VP3. Neither chimera formed CLPs when coexpressed with VP3. The N-terminal region of ABA VP7 was replaced by thatof BTV-10 VP7, but CPLs were still not formed when the new chimera was expressed with VP3. This was thought to be due to incorrect folding.