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Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants
T R Jones1, L Sun, G A Bebernitz
1Molecular Biology Section, American Cyanamid Co., Pearl River, New York 10965.
Abstract:
The human cytomegalovirus UL80 open reading frame encodes protease and assembly protein from its N- and C-terminal regions, respectively. We reported previously that a 30-kDa protease is derived by autoproteolytic processing of a polyprotein 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 polyprotein were characterized; site 143 is within the protease domain and inactivates the protease. In this article, we report (i) expression analyses of UL80 in infected cells, including the processing kinetics of the UL80 polyprotein; (ii) the existence of an additional cleavage 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 products. During the course of infection, UL80 polyprotein 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 for efficient protease activity, since all three proteases (85-, 80-, and 30-kDa proteins) were equally active in cleaving the assembly protein precursor to its mature form. Inhibition of cleavage at site 143 resulted in a three- to sixfold increase in the steady-state level of the 30-kDa protease, supporting the hypothesis that cleavage at this site may represent a mechanism by which cytomegalovirus regulates the level of active protease.
Insights
Human cytomegalovirus UL80 polyprotein processing involves multiple cleavage sites. Inhibiting site 143 increases active protease levels, suggesting a regulatory mechanism for viral protease activity.
Area of Science:
- Virology
- Molecular Biology
- Protease Function
Background:
- The human cytomegalovirus (CMV) UL80 open reading frame produces both protease and assembly proteins.
- Previous work identified a 30-kDa protease derived from autoproteolytic processing of the UL80 polyprotein.
- Three autoproteolytic cleavage sites within the UL80 polyprotein were previously characterized, with site 143 inactivating the protease.
Purpose of the Study:
- To analyze UL80 expression and polyprotein processing kinetics in infected cells.
- To identify and characterize an additional cleavage site (site 209) within the UL80 protease domain.
- To investigate the impact of mutagenesis at cleavage sites on proteolytic activity and processing product levels.
Main Methods:
- Expression analysis of UL80 in infected cells.
- Site-directed mutagenesis of UL80 cleavage sites.
- Proteolytic activity assays.
- Analysis of steady-state levels of UL80 processing products.
Main Results:
- UL80 polyprotein processing initiates at site 643, followed by sites 256 and 143 during infection.
- Cleavage at sites 643 and/or 256 is not essential for efficient protease activity.
- Inhibition of cleavage at site 143 led to a 3- to 6-fold increase in the 30-kDa protease level.
Conclusions:
- Cleavage at site 143 within the UL80 polyprotein may serve as a regulatory mechanism controlling the level of active human cytomegalovirus protease.
- The processing pathway and regulation of UL80 protease are crucial for viral replication.