Abstract
Human adenovirus proteinase (AVP), the first member of a new class of cysteine proteinases, is required for the synthesis of infectious virus. As such, it is an attractive target for proteinase inhibitors that act as antiviral agents. However, before potential inhibitors can be screened, a quick, sensitive, and quantitative assay for the enzyme is required. Here, methods for purification of a recombinant AVP expressed in Escherichia coli are presented and a fluorogenic substrate is designed, synthesized, and purified and then used in the development of a quick, sensitive, and quantitative assay for the enzyme. The reporting group in the substrate is Rhodamine 110, possibly the most detectable compound known. The substrate contains the proteinase consensus cleavage sequence (Leu-Arg-Gly-Gly). The synthesis and purification of (Leu-Arg-Gly-Gly-NH)2-Rhodamine is described. It is then used to develop assays with AVP and its various cofactors. The resultant assays are quite sensitive; enzyme activity at low nanomolar concentrations can readily be detected.
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References
Weber, J. (1976) Genetic analysis of adenovirus type 2 III. Temperaturesensitivity of processing of viral proteins. J. Virol. 17, 462ā471.
Hannan, C., Raptis, L. H., Dery, C. V., and Weber, J. (1983) Biological and structural studies with adenovirus type 2 temperature-sensitive mutant defective for uncoating. Intervirology 19, 213ā223.
Mirza, A. and Weber, J. (1980) Infectivity and uncoating of adenovirus cores. Intervirology 13, 307ā311.
Brown, M. T., McGrath, W. J., Toledo, D. L., and Mangel, W. F. (1996) Different modes of inhibition of human adenovirus proteinase, probably a cysteine proteinase, by bovine pancreatic trypsin inhibitor. FEBS Lett. 388, 233ā237.
Greber, U. F., Webster, P., Weber, J., and Helenius, A. (1996) The role of the adenovirus protease in virus entry into cells. EMBO J. 15, 1766ā1777.
Cotten, M. and Weber, J. M. (1995) The adenovirus proteinase is required for entry into host cells. Virology 213, 494ā502.
Greber, U. F. (1998) Virus assembly and disassembly: the adenovirus cysteine protease as a trigger factor. Rev. Med. Virol. 8, 213ā222.
Chen, P. H., Ornelles, D.A., and Shenk, T. (1993) The adenovirus L3 23-kilodalton proteinase cleaves the amino-terminal head domain from cytokeratin 18 and disrupts the cytokeratin network of HeLa cells. J. Virol. 67, 3507ā3514.
Brown, M. T., McBride, K. M., Baniecki, M. L., Reich, N. C., Marriott, G., and Mangel, W.F. (2002) Actin can act as a cofactor for a viral proteinase in the cleavage of the cytoskeleton. J. Biol. Chem. 277, 46,298ā46,303.
Yeh-Kai, L., Akusjarvi, G., Alestrom, P., Pettersson, U., Tremblay, M., and Weber, J. (1983) Genetic identification of an endopeptidase encoded by the adenovirus genome. J. Mol. Biol. 167, 217ā222.
Mangel, W. F., McGrath, W. J., Toledo, D.L., and Anderson, C. W. (1993) Viral DNA and a viral peptide can act as cofactors of adenovirus virion proteinase activity. Nature 361, 274ā275.
Tihanyi, K., Bourbonniere, M., Houde, A., Rancourt, C., and Weber, J. M. (1993) Isolation and properties of adenovirus type 2 proteinase. J. Biol. Chem. 268, 1780ā1785.
Anderson, C. W. (1993) Expression and purification of the adenovirus proteinase polypeptide and of a synthetic proteinase substrate. Protein Express. Purif. 4, 8ā15.
Webster, A., Hay, R.T., and Kemp, G. (1993) The adenovirus protease is activated by a virus-coded disulphide-linked peptide. Cell 72, 97ā104.
Webster, A. and Kemp, G. (1993) The active adenovirus protease is the intact L3 23K protein. J. Gen. Virol. 74, 1415ā1420.
Mangel, W. F., Toledo, D. L., Brown, M. T., Martin, J.H., and McGrath, W. J. (1996) Characterization of three components of human adenovirus proteinase activity in vitro. J. Biol. Chem. 271, 536ā543.
McGrath, W. J., Baniecki, M. L., Li, C., et al. (2001) Human adenovirus proteinase: DNA binding and stimulation of proteinase activity by DNA. Biochemistry 40, 13,237ā13,245.
Brown, M.T. and Mangel, W. F. (2004) Interaction of actin and its 11-aminoacid C-terminal peptide as cofactors with the adenovirus proteinase. FEBS 563, 213ā218.
Webster, A., Russell, S., Talbot, P., Russell, W.C., and Kemp, G. D. (1989) Characterization of the adenovirus proteinase: substrate specificity. J. Gen. Virol. 70, 3225ā3234.
Leytus, S. P., Melhado, L.L., and Mangel, W. F. (1983) Rhodamine-based compounds as fluorogenic substrates for serine proteases. Biochem. J. 209, 299ā307.
Mangel, W. F., Leytus, S.P., and Melhado, L. L. (1987) Novel Rhodamine derivatives as fluorogenic substrates. U.S. Patent 4,640,893 (February 1987).
Mangel, W. F., Leytus, S.P., and Melhado, L. L. (1985) Rhodamine derivatives as fluorogenic substrates forproteinases. U.S. Patent 4,557,862 (December 1985).
Leytus, S. P., Patterson, W.L., and Mangel, W. F. (1983) New class of sensitive, specific, and selective substrates for serine proteinases: fluorogenic, amino acid peptide derivatives of Rhodamine. Biochem. J. 215, 253ā260.
Leytus, S. P., Toledo, D.L., and Mangel, W. F. (1984) Theory and experimental method for determining individual kinetic constants for fast-acting, irreversible, protease inhibitors. Biochim. Biophys. Acta 788, 74ā86.
McGrath, W. J., Abola, A. P., Toledo, D. L., Brown, M.T., and Mangel, W. F. (1996) Characterization of human adenovirus proteinase activity in disrupted virus particles. Virology 217, 131ā138.
Baniecki, M. L., McGrath, W. J., McWhirter, S. M., et al. (2001) Interaction of the human adenovirus proteinase with its eleven amino-acid cofactor pVIc. Biochemistry 40, 12,349ā12,356.
Gill, S.G. and von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319ā326.
Bajpayee, N. S., McGrath, W.J., and Mangel, W. F. (2005) Interaction of the adenovirus proteinase with protein cofactors with high negative charge densities. Biochemistry 44, 8721ā8729.
McGrath, W. J., Ding, J., Sweet, R.M., and Mangel, W. F. (1996) Preparation and crystallization of a complex between human adenovirus serotype 2 proteinase and its 11-amino-acid cofactor pVIc. J. Struct. Biol. 117, 77ā79.
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Mangel, W.F., McGrath, W.J. (2007). Assay for the Adenovirus Proteinase. In: Wold, W.S.M., Tollefson, A.E. (eds) Adenovirus Methods and Protocols. Methods in Molecular Medicineā¢, vol 131. Humana Press. https://doi.org/10.1007/978-1-59745-277-9_19
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DOI: https://doi.org/10.1007/978-1-59745-277-9_19
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