Investigation into the catalytic role of threonine 712 in VP4 protease from the blotched snakehead virus

The blotched snakehead virus (BSNV) is a member of the Birnaviridae family; it is characterized by a single-shelled capsid and a bisegmented, double-stranded RNA genome. One segment of its genome, segment A, encodes the BSNV polyprotein, NH3+-pVP2-X-VP4-VP3-COO-, which includes VP4 - a peptidase that uses a 'nonclassical', Ser-Lys catalytic dyad mechanism. VP4 cleaves the polyprotein at specific recognition sites to release four peptides and four polypeptides. The polypeptides include the capsid protein, VP2, protein X (whose function is unknown), the multifunctional protein VP3, and also VP4. Previous site-directed mutagenesis and crystallographic studies of BSNV VP4 have identified the serine nucleophile and lysine general base, however the potential roles played by other residues within the active site have yet to be experimentally investigated. One conserved active site residue of interest is Thr712, which is appropriately positioned, according to the BSNV VP4 crystal structure (PDB ID 2GEF), to form a hydrogen bond with the active site's general base, Lys729. Herein, we present data supporting Thr712's significance to VP4-mediated catalysis from experiments using site-directed mutagenesis and time-course cleavage assays. We also describe the crystallization of a truncated, active site mutant construct - K729A - of BSNV VP4.