Herpesvirus Protease Assays

  • Peter Ertl
  • Linda Russell
  • Jane Angier
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 24)

Abstract

Herpesviruses encode a serine protease that is essential for the maturation of viral capsids (1,2). The protease is expressed as part of a polyprotein. The catalytic domain is contained within the N-terminal third of the protein, and the remainder comprises a structural “scaffold” protein. The scaffold protein is independently expressed in excess to the polyprotein from an internal initiation codon. The protease cleaves the polyprotein at two sites: one at the c-terminus of the protease catalytic domain, the release or R-site, and the other close to the c-terminus of the scaffold protein, the maturation or M-site (Fig. 1). Cleavage of the M-site follows assembly of the viral procapsids and precedes packaging of the viral DNA. The M-site sequence is conserved among the herpesviruses and has a consensus sequence (V/L)-X-A-S, with cleavage between A-S (3). Structural studies have shown that the herpesvirus proteases have a novel structure, and their essential role in capsid maturation makes them a potential target for antiviral intervention.
Fig. 1.

Cartoon illustration of the structure of the herpesvirus protease/scaffold polyprotein showing the position of the protease catalytic domain, the scafold protein, and the release and maturation cleavage sites.

Keywords

Zinc Toxicity HPLC Glycerol Codon 

References

  1. 1.
    Preston, V. G., Coates, J. A., and Rixon, F. J. (1983) Identification and characterisation of a herpes simplex virus gene product required for encapsidation of virus DNA. J. Virol. 45, 1056–1064.PubMedGoogle Scholar
  2. 2.
    Gao, M., Matusick-Kumar, L., Hurlburt, W., DiTusa, S. F., Newcombe, W. W., Brown, J. C., McCann P. J., III, Deckman, I., and Colonno, R. J. (1994) The protease of herpes simplex virus type 1 is essential for functional capsid formation and viral growth. J. Virol. 68, 3702–3712.PubMedGoogle Scholar
  3. 3.
    Welch, A. R., Woods, A. S., McNally, L. M., Cotter, R. J., and Gibson, W. (1991) A Herpesvirus maturational protease, Assemblin: identification of its gene, putative active site domain and cleavage site. Proc. Natl. Acad. Sci. USA 88, 10,792–10,796.CrossRefPubMedGoogle Scholar
  4. 4.
    Sardana, V. V., Wolfgang, J. A., Veloski, C. A., Long, W. J., LeGrow, K., Wolanski, B., Emini, E. A., and LaFemina, R. L. (1994) Peptide substrate cleavage specificity of human cytomegalovirus protease. J. Biol. Chem. 269, 14,337–14,340.PubMedGoogle Scholar
  5. 5.
    Hall, D. L. and Darke, P. L. (1995) Activation of the herpes simplex virus type 1 protease. J. Biol. Chem. 270, 22,697–22,700.CrossRefPubMedGoogle Scholar
  6. 6.
    LaFemina, R. I., Bakshi, K. P., Long, W. J., Pramanik, B., Veloski, C. A., Wolanski, B. S., Marcy, A. I., and Hazuda, D. L. (1996) Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics. J. Virol. 70, 4819–4824.PubMedGoogle Scholar
  7. 7.
    Baum, E. Z., Johnston, S. H., Bebernitz, G. A., and Gluzman, Y. (1996) Development of a scintillation proximity assay for human cytomegalovirus protease using 33Phosphorous. Anal. Biochem. 237, 129–134.CrossRefPubMedGoogle Scholar
  8. 8.
    Handa B. K., Keech, E., Conway, E. A., Broadhurst, A., and Ritchie, A. (1995) Design and synthesis of a quenched fluorogenic peptide substrate for human cytomegalovirus proteinase. Antiviral Chem. Chemother. 6(4), 255–261.Google Scholar
  9. 9.
    Holskin B. P., Bukhtiyarova, M., Dunn, B. M., Baur, P., de Chastonay, J., and Pennington, M. W. (1995) A continuous fluorescence-based assay of human cytomegalovirus protease using a peptide substrate. Anal. Biochem. 226, 148–155.CrossRefGoogle Scholar
  10. 10.
    Darke, P. L., Cole, J. L., Waxman, L., Hall, D. L., Sardana, M. K., and Kuo, L. C. (1996) Active human cytomegalovirus protease is a dimer. J. Biol. Chem. 271, 7445–7449.CrossRefPubMedGoogle Scholar
  11. 11.
    Cole, J. L. (1996) Characterization of human cytomegalovirus protease dimerisation by analytical centrifugation. Biochemistry 35, 15,601–15,610.CrossRefPubMedGoogle Scholar
  12. 12.
    Margosiak, S. A., Vanderpool, D. L., Sisson, W., Pinko, C., and Kan, C-C. (1996) Dimerisation of the human cytomegalovirus protease: kinetic and biochemical characterization of the catalytic homodimer. Biochemistry 35, 5300–5307.CrossRefPubMedGoogle Scholar
  13. 13.
    Schmidt, U. and Darke, P. L. (1997) Dimerisation and activation of the herpes simplex virus type 1 protease. J. Biol. Chem. 272, 7732–7735.CrossRefPubMedGoogle Scholar
  14. 14.
    Welch, A. R. and Villarreal, E. C. (1995) Cytomegalovirus protein substrates are not cleaved by herpes simplex virus type 1 proteinase. J. Virol. 69, 341–347.PubMedGoogle Scholar
  15. 15.
    Donaghy, G. and Jupp, R. (1995) Characterization of the Epstein-Barr virus proteinase and comparison with the human cytomegalovirus proteinase. J. Virol. 69, 1265–1270.PubMedGoogle Scholar
  16. 16.
    Mapelli, C., Dilanni, C. L., Tsao, J. F., Stevens, J. T., Weinheimer, S. P., O’Boyle, D. R., Drier, D. A., and Meyers, C. A. (1994) Substrate requirement of the HSV-l and CMV proteases in vitro using peptides derived from their maturation and release sites in “Peptides 1994” (Maia, H. L. S., ed.), Proceedings of the 23rd European Peptide Symposium.Google Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Peter Ertl
  • Linda Russell
  • Jane Angier

There are no affiliations available

Personalised recommendations