HERPESVIRUS PROTEINASE - SITE-DIRECTED MUTAGENESIS USED TO STUDY MATURATIONAL, RELEASE, AND INACTIVATION CLEAVAGE SITES OF PRECURSOR AND TOIDENTIFY A POSSIBLE CATALYTIC SITE SERINE AND HISTIDINE

The cytomegalovirus maturational proteinase is synthesized as a precur sor that undergoes at least three processing cleavages. Two of these w ere predicted to be at highly conserved consensus sequences-one near t he carboxyl end of the precursor, called the maturational (M) site, an d the other near the middle of the precursor, called the release (R) s ite. A third less-well-conserved cleavage site, called the inactivatio n (I) site, was also identified near the middle of the human cytomegal ovirus 28-kDa assemblin homolog. We have used site-directed mutagenesi s to verify all three predicted sequences in the simian cytomegaloviru s proteinase, and have shown that the proteinase precursor is active w ithout cleavage at these sites. We have also shown that the P4 tyrosin e and the P2 lysine of the R site were more sensitive to substitution than the other R- and M-site residues tested: substitution of alanine for P4 tyrosine at the R site severely reduced cleavage at that site b ut not at the M site, and substitution of asparagine for lysine at P2 of the R site reduced M-site cleavage and nearly eliminated I-site cle avage but had little effect on R-site cleavage. With the exception of P1' serine, all R-site mutations hindered I-site cleavage, suggesting a role for the carboxyl end of assemblin in I-site cleavage. Pulse-cha se radiolabeling and site-directed mutagenesis indicated that assembli n is metabolically unstable and is degraded by cleavage at its I site. Fourteen amino acid substitutions were also made in assemblin, the en zymatic amino half of the proteinase precursor. Among those tested, on ly 2 amino acids were identified as essential for activity: the single absolutely conserved serine and one of the two absolutely conserved h istidines. When the highly conserved glutamic acid (Glu22) was substit uted, the proteinase was able to cleave at the M and I sites but not a t the R site, suggesting either a direct (e.g., substrate recognition) or indirect (e.g., protein conformation) role for this residue in det ermining substrate specificity.