1887

Abstract

Sequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (Ad2) protease. Primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis. All conserved serine and cysteine residues were mutated separately and following expression in their activity in a synthetic peptide assay was compared to that of wild-type recombinant protease. Mutants containing altered serine residues were active while mutations to cysteine-104 and cysteine-122 reduced activity by more than 95 %. These results taken together with the known inhibition profile of the adenovirus protease confirm that it is a cysteine protease and suggest that one of these residues provides the active site nucleophile while the other is a part of the thiol-disulphide interchange mechanism previously reported to be involved in its activation.

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1994-10-01
2024-04-25
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References

  1. Anderson C. W. 1990; The proteinase polypeptide of adenovirus serotype 2 virions. Virology 177:259–272
    [Google Scholar]
  2. Anderson C. W., Baum P. R., Gesteland R. F. 1973; Processing of adenovirus 2 induced proteins. Journal of Virology 12:241–252
    [Google Scholar]
  3. Anderson C. W., Young M. E., Flint S. J. 1989; Characterization of the adenovirus 2 virion protein, mu. Virology 172:506–512
    [Google Scholar]
  4. Atherton E., Cameron L. R., Sheppard R. C. 1988; Peptide synthesis. 10. Use of pentafluorophenyl esters of fluorenyl methoxycarbonylamino acids in solid phase peptide synthesis. Tetrahedron 44:843–857
    [Google Scholar]
  5. Cai F., Weber J. M. 1993; Organization of the avian adenovirus genome and the structure of its endopeptidase. Virology 196:358–362
    [Google Scholar]
  6. Cai F., Tang D., Weber J. M. 1992; Primary structure of the murine adenovirus type 1 proteinase. Biochimica et Biophysica Acta 1129:339–341
    [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12:387–396
    [Google Scholar]
  8. Houde A., Weber L. M. 1990; Adenovirus proteinases: comparison of amino acid sequences and expression of the cloned cDNA in Escherichia coli . Gene 88:269–213
    [Google Scholar]
  9. Landt O., Grunert H. P., Hahn U. 1990; A general method for rapid site directed mutagenesis using the polymerase chain reaction. Gene 96:125–128
    [Google Scholar]
  10. Mangel W. F., Mcgrath W. J., Toledo D., Anderson C. W. 1993; Viral DNA and a viral peptide are cofactors of adenovirus virion proteinase activity. Nature; London: 361274–275
    [Google Scholar]
  11. Orr D. C., Long A. C., Kay J., Dunn B. M., Cameron J. M. 1989; Hydrolysis of a series of synthetic peptide substrates by the human rhino virus 14 3C proteinase, cloned and expressed in Escherichia coli . Journal of General Virology 70:2931–2942
    [Google Scholar]
  12. Rancourt C., Tihanyi K., Bourbonniere M., Weber J. M. 1994; Identification of active site residues of the adenovirus endopeptidase. Proceedings of the National Academy of Sciences, U.S.A 91:844–847
    [Google Scholar]
  13. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. 1987; Vectors for selective expression of cloned DNAs by T7 polymerase. Gene 56:125–135
    [Google Scholar]
  14. Temperley S. M., Hay R. T. 1991; Replication of adenovirus type 4 DNA by a purified fraction from infected cells. Nucleic Acids Research 19:3243–3249
    [Google Scholar]
  15. Tremblay M. L., Dery C. V., Talbot B. G., Weber J. 1983; In vitro cleavage specificity of the adenovirus type 2 proteinase. Biochimica et Biophysica Acta 74:3239–3245
    [Google Scholar]
  16. Udenfriend S., Stein S., Bohlen P., Dairman W., Leimgruber W., Weigele M. 1972; Fluorescamine: a reagent for assay of amino acids peptides, proteins and primary amines in the picomole range. Science 178:871–872
    [Google Scholar]
  17. Weber J. 1976; Genetic analysis of adenovirus type 1. III. Temperature sensitivity of processing of viral proteins. Journal of Virology 17:462–471
    [Google Scholar]
  18. Webster A., Kemp G. 1993; The active adenovirus protease is the intact L3 23K protein. Journal of General Virology 74:1415–1420
    [Google Scholar]
  19. Webster A., Hay R. T., Kemp G. 1993; The adenovirus protease is activated by a virus-coded disulphide-linked peptide. Cell 72:97–104
    [Google Scholar]
  20. Webster A., Russell W. C., Kemp G. D. 1989a; Characterization of the adenovirus proteinase: development and use of a specific peptide assay. Journal of General Virology 70:3215–3223
    [Google Scholar]
  21. Webster A., Russell S., Talbot P., Russell W. C., Kemp G. D. 1989b; Characterization of the adenovirus proteinase: substrate specificity. Journal of General Virology 70:3225–3234
    [Google Scholar]
  22. Yeh-Kai L., Akusjarvi G., Alestrom P., Pettersson U., Tremblay M., Weber J. 1983; Genetic identification of an endoproteinase encoded by the adenovirus genome. Journal of Molecular Biology 167:217–222
    [Google Scholar]
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