Functional determinants of the Epstein-Barr virus protease

Herpesvirus proteases are essential for the production of progeny virus. Th ey cleave the assembly protein that fills the immature capsid in order to m ake place for the viral DNA. The recombinant protease of the human gamma-he rpesvirus Epstein-Barr virus (EBV) was expressed in Escherichia coli and pu rified. Circular dichroism indicated that the protein was properly folded w ith a secondary structure content similar to that of other herpesvirus prot eases. Gel filtration and sedimentation analysis indicated a fast monomer-d imer equilibrium of the protease with a K-d of about 60 muM. This value was not influenced by glycerol but was lowered to 1.7 muM in the presence of 0 .5 M sodium citrate. We also developed an HPLC-based enzymatic assay using a 20 amino acid residue synthetic peptide substrate derived from one of the viral target sequences for the protease. We found that conditions that sta bilised the dimer also led to a higher enzymatic activity. Through sequenti al deletion of an-Lino acid residues from either side of the cleavage site, the minimal peptide substrate for the protease was determined as P5-P2'. T his minimal sequence is shorter than that for other herpesvirus proteases. The implications of our findings are discussed with reference to the viral life-cycle. These results are the first ever published on the EBV protease and represent a first step towards the development of protease inhibitors. (C) 2001 Academic Press.