Recombinant Human Cytomegalovirus Protease with a C-Terminal (His)6Extension: Purification, Autocatalytic Release of the Mature Enzyme, and Biochemical Characterization

Tomasselli, A. G.; Paddock, Donna J.; Curry, Kimberly A.; Garlick, Robert; Leone, Joseph W.; Lull, June M.; Mutchler, Veronica T.; Baker, Carolyn A.; Cavey, Gregory S.; Mathews, W R; Shelly, John A.; Finzel, B.C.; Baldwin, Eric T.; Wells, Peter A.; Tomich, C.‐S. C.

doi:10.1006/prep.1998.0958

Cited by 3 publications

(1 citation statement)

References 29 publications

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“…Following DNA packaging and C capsid formation, additional viral proteins are added to the outer portion of the nucleocapsid and the nucleocapsid leaves the nucleus. Baum et al (1993) Burck et al (1994) LaFemina et al (1996 Tomasselli et al (1998) Simian CMV Human, insect Welch et al (1993) Hall & Gibson (1996 VZV E. coli Qiu et al (1997)…”

Section: Herpesvirus Protease Catalysis In Viral Replicationmentioning

confidence: 99%

The Herpesvirus Proteases as Targets for Antiviral Chemotherapy

Waxman

Darke

2000

Antivir Chem Chemother

117

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Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure-activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

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Section: Herpesvirus Protease Catalysis In Viral Replicationmentioning

confidence: 99%

The Herpesvirus Proteases as Targets for Antiviral Chemotherapy

Waxman

Darke

2000

Antivir Chem Chemother

117

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Production of Active Mammalian and Viral Proteases in Bacterial Expression Systems

Babé¹,

Linnevers²,

Schmidt

2000

Biotechnology and Genetic Engineering Reviews

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Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency

Kapust

Tőzsér

Fox

et al. 2001

Protein Engineering, Design and Selection

767

653

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Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.

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Recombinant Human Cytomegalovirus Protease with a C-Terminal (His)6Extension: Purification, Autocatalytic Release of the Mature Enzyme, and Biochemical Characterization

Cited by 3 publications

References 29 publications

The Herpesvirus Proteases as Targets for Antiviral Chemotherapy

The Herpesvirus Proteases as Targets for Antiviral Chemotherapy

Production of Active Mammalian and Viral Proteases in Bacterial Expression Systems

Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency

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