HUMAN CYTOMEGALOVIRUS MATURATIONAL PROTEINASE - EXPRESSION IN ESCHERICHIA-COLI, PURIFICATION, AND ENZYMATIC CHARACTERIZATION BY USING PEPTIDE SUBSTRATE MIMICS OF NATURAL CLEAVAGE SITES

The proteolytic processing of the human cytomegalovirus (HCMV) assembl y protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavag e is the amino-terminal half of the protein encoded by the U(L)80a ope n reading frame. We have obtained high expression levels of this 256-a mino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 a mino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase a nd the 15-kDa protein were purified by a two-step chromatographic proc edure utilizing anion exchange in urea and dithiothreitol and size exc lusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active prote inase. Addition of glycerol to 50% enhanced the activity. The HCMV pro teinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mi mics of the maturational (M)- and release (R)-site sequences, respecti vely, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein . The K-m value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (k(cat)) of the M-site peptide mimic substrate by the protei nase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded prot ein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor con centrations. The purified 15-kDa protein, subjected to the same activa tion conditions as the 28-kDa proteinase, had no enzymatic activity ag ainst the HCMV M- and R-site peptide substrates.