PROTEOLYTIC ACTIVITY OF HUMAN CYTOMEGALOVIRUS UL80 PROTEASE CLEAVAGE SITE MUTANTS

The human cytomegalovirus UL80 open reading frame encodes protease and assembly protein from its Nand C-terminal regions, respectively. We r eported previously that a 30-kDa protease is derived by autoproteolyti c tic processing of a poly-protein which is the translation product of the entire UL80 open reading frame (E. Z. Baum, G. A. Bebernitz, J. D . Hulmes, V. P. Muzithras, T. R. Jones, and Y. Gluzman, J. Virol. 67: 497-506, 1993). Three autoproteolytic cleavage sites within the UL80 p olyprotein were characterized; site 143 is within the protease domain and inactivates the protease. In this article, we report (i) expressio n analyses of UL80 in infected cells, including the processing kinetic s of the UL80 polyprotein; (ii) the existence of an additional cleavag e site (site 209) within the protease domain of the UL80 polyprotein; and (iii) the effect of mutagenesis at each of the cleavage sites upon proteolytic activity and steady-state levels of the UL80 processing p roducts. During the course of infection, UL80 poly-protein processing begins at cleavage site 643 and follows at sites 256 and 143. Cleavage at site 643 and/or 256 within the polyprotein is not a prerequisite f or efficient protease activity, since all three proteases (85-, 80-, a nd 30-kDa proteins) were equally active in cleaving the assembly prote in precursor to its mature form. Inhibition of cleavage at site 143 re sulted in a three- to sixfold increase in the steady-state level of th e 30-kDa protease, supporting the hypothesis that cleavage al this sit e may represent a mechanism by which cytomegalovirus regulates the lev el of active pretense.