title: Recombinant full-length human cytomegalovirus protease has lower activity than recombinant processed protease domain in purified enzyme and cell-based assays.
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.
authors.affiliation: Pharmacia Discovery Research, 700 Chesterfield Village Parkway North, St. Louis, MO 63198, USA.
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Wittwer
authors.forename: Christy L
authors.initials: CL
authors.lastname: Funckes-Shippy
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hippenmeyer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D015533:Transcriptional Activation; D014709:Vero Cells; D014764:Viral Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D001616:beta-Galactosidase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C071910:assemblin