CHARACTERIZATION OF A SOLUBLE STABLE HUMAN CYTOMEGALOVIRUS PROTEASE AND INHIBITION BY M-SITE PEPTIDE MIMICS

The human cytomegalovirus (HCMV) protease is a potential target for an tiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studie s. Using peptides encompassing the sequence of the natural M-site subs trate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitut ion of glycine for valine at the P3 position in the substrate abrogate s processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of th e two major internal processing sites, positions 143 and 209, in the m ature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was pro duced almost exclusively as the 30-kDa full-length protein. The full-l ength V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammo nium sulfate precipitation followed by DEAE ion-exchange chromatograph y, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indisting uishable from the wild-type recombinant protease, exhibiting K-m and c atalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlin ed residues indicate additions to or substitutions from peptides deriv ed from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted matu ration site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and th ese analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCM V protease.