CYTOMEGALOVIRUS ASSEMBLIN - THE AMINO AND CARBOXYL DOMAINS OF THE PROTEINASE FORM ACTIVE ENZYME WHEN SEPARATELY CLONED AND COEXPRESSED IN EUKARYOTIC CELLS

The cytomegalovirus (CMV) serine proteinase assemblin is synthesized a s a precursor that undergoes three principal autoproteolytic cleavages . Two of these are common to the assemblin homologs of all herpes grou p viruses: one at the maturational site near the carboxyl end of the p recursor and another at the release site near the midpoint of the prec ursor. Release-site cleavage frees the proteolytic amino domain, assem blin, from the nonproteolytic carboxyl domain of the precursor. In CMV , a third autoproteolytic cleavage at an internal site divides assembl in into an amino subunit (A(n)) and a carboxyl subunit (A(c)) of appro ximately the same size that remain associated as an active ''two-chain '' enzyme. We have cloned the sequences encoding A(n) and A(c) as sepa rate genes and expressed them by transfecting human cells with recombi nant plasmids and by infecting insect cells with recombinant baculovir uses. When A(n) and A(c) from either simian CMV or human CMV were coex pressed in human or insect cells, active two-chain assemblin was forme d. This finding demonstrates that A(n) and A(c) do not require synthes is as single-chain assemblin to fold and associate correctly in these eukaryotic systems, and it suggests that they may be structurally, if not functionally, distinct domains. An interaction between the indepen dently expressed A(n) and A(c) subunits was demonstrated by coimmunopr ecipitation experiments, and efforts to disrupt the complex indicate t hat the subunit interaction is hydrophobic. Cell-based cleavage assays of the two-chain assemblin formed from independently expressed A(n) a nd A(c) also indicate that (i) its specificity for both CMV and herpes simplex virus native substrates is similar to that of single-chain as semblin, (ii) R-site cleavage is not essential for the activity of two -chain recombinant assemblin, and (iii) the human CMV and simian CMV A (n) and A(c) recombinant subunits are functionally interchangeable.