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Recombinant full-length human cytomegalovirus protease has lower activity than recombinant processed protease domain
Arthur J Wittwer1, Christy L Funckes-Shippy, Paul J Hippenmeyer
1Pharmacia Discovery Research, 700 Chesterfield Village Parkway North, St. Louis, MO 63198, USA.
Abstract:
Herpesviruses encode a protease that is essential for virus replication. The protease undergoes cleavage to a processed form during capsid maturation. A recombinant 75 kDa form of the protease from human cytomegalovirus was purified and compared with the recombinant 29 kDa processed form. Modification with an active site titrant suggested that most of each recombinant protease preparation was active (66 and 86%, respectively). Protease activity was compared using a low-molecular weight peptide substrate and the native substrate, capsid assembly protein. In addition, a cell-based assay for both enzymes was developed in which the target sequence of the protease has been fused inframe into the herpes simplex virus VP16 molecule. Cleavage of the fusion protein by the protease releases the carboxyl terminal transactivation domain, resulting in a decrease in the ability of the fusion molecule to transactivate a target promoter linked to a reporter gene in mammalian cells. Results suggest that the 75 kDa form of the enzyme is significantly less active than the 29 kDa form by all criteria.
Insights
Human cytomegalovirus protease's 75 kDa form is less active than its 29 kDa processed form. This difference in viral protease activity was observed using various substrates and a novel cell-based assay.
Area of Science:
- Virology
- Enzymology
Background:
- Herpesviruses utilize essential proteases for replication.
- Viral proteases undergo cleavage into processed forms during capsid maturation.
Purpose of the Study:
- To compare the activity of the unprocessed (75 kDa) and processed (29 kDa) forms of human cytomegalovirus (HCMV) protease.
- To characterize the enzymatic properties of different HCMV protease forms.
Main Methods:
- Purification of recombinant 75 kDa and 29 kDa HCMV protease forms.
- Active site titration to determine enzyme activity.
- Enzyme assays using peptide and native substrates (capsid assembly protein).
- Development of a cell-based assay involving a fusion protein with herpes simplex virus VP16.
Main Results:
- Both recombinant protease forms showed significant active site occupancy (66% for 75 kDa, 86% for 29 kDa).
- The 75 kDa protease form exhibited significantly lower activity compared to the 29 kDa form across all tested assays.
- The cell-based assay demonstrated reduced transactivation upon cleavage by the 75 kDa protease compared to the 29 kDa form.
Conclusions:
- The processed 29 kDa form of HCMV protease is substantially more active than the 75 kDa unprocessed form.
- Enzyme processing is critical for achieving optimal viral protease function in human cytomegalovirus replication.
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