Cleavage of RT/RNase H by HIV-1 Protease and Analysis of Substrate Cleavage Sites in vitro

  • K. Moelling
  • M. Nawrath
  • T. Schulze
  • L. Pavlitzkova
  • M. Soucek
  • K.-H. Budt
  • L. H. Pearl
  • M.-T. Knoop
  • J. Kay
  • V. Kruft
Chapter

Abstract

The pol-gene of HIV-1 expressed in bacteria undergoes autocatalytic processing which results in the generation of the p66 reverse transcriptase (RT)/RN ase H, the p32 endonuclease and the p9 protease. The partially purified protease generates in vitro from the p66 molecule a p66/p51 heterodimer, typical of the normally observed RT/RNase H, and a p15 carboxyterminal fragment. A synthetic peptide AETF’YVD derived from the p51/p15 junction is cleaved by the protease in vitro and may represent the natural cleavage site. The RT/RNase H activities associated with p66 and p51 and p66/p51 heterodimers were determined after renaturation of these proteins from Polyacrylamide gels. The p66/p51 heterodimers exhibit about eightfold higher RT and RNase H activities than each of the individual subunits alone suggesting that heterodimers are of biological relevance. Protease activity was monitored by three assays, by cleavage of denatured ovalbumin, of the MS2-gag fusion protein or synthetic peptides. Several synthetic peptides representing various poten tial protease cleavage sites and modifications thereof were analyzed in vitro for their efficiency of cleavage by the protease. One synthetic peptide representing the natural amino-terminal cleavage site of the protease was modified by several amino acid substitutions in order to characterize the specificity of the protease. Several of the peptides as well as cerulenin and acetyl-pepstatin were analyzed as protease inhibitors.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Billich, S., Knoop, M.-T., Hansen, J., Strop, P., Sedlacek, J., Mertz, R., and Moelling, K. J.Biol.Chem., 263, 17905–17908, (1988).PubMedGoogle Scholar
  2. Cleveland, D.W., Fischer, S.G., Kirchner, M.W., and Laemmli, U.K. J.Biol.Chem., 252, 1102–1106, (1977).PubMedGoogle Scholar
  3. D’Angelo, G., Rosenfeld, I.S., Awaya, J., Omura, S. and Vagelos, P.R. Biochim.Biophys.Acta, 326, 155–166, (1973).CrossRefGoogle Scholar
  4. Di Marzo Veronese, F., Copeland, T.D., DeVico, A.L., Rahman, R., Orozlan, S., Gallo, R.C. Science, 231, 1281–1291, (1986).CrossRefGoogle Scholar
  5. Dittmar, K.J. and Moelling, K. J. Virol., 28, 106–118, (1978).PubMedCentralPubMedGoogle Scholar
  6. Farmerie, W.G., Loeb, D.H., Casavant, N.C., Hutchinson III, C.A, Edgell, M.H. and Swanstrom, R. Science, 263, 305–308, (1987).CrossRefGoogle Scholar
  7. Goldfine, H., Harley, J.B. and Wyke, J.A. Biochim.Biophys.Acta, 152, 229–240, (1978).CrossRefGoogle Scholar
  8. Hager, D.A. and Burgess, R.R. Anal. Biochem., 109, 76–86, (1980).PubMedCrossRefGoogle Scholar
  9. Hansen, J., Schulze, T., and Moelling, K. J.Biol.Chem., 262, 12393–12396, (1987).PubMedGoogle Scholar
  10. Hansen, J., Billich, S., Schulze, T., Sukrow, S., and Moelling, K. EMBO J., 7, 1785–1791, (1988a).PubMedCentralPubMedGoogle Scholar
  11. Hansen, J., Schulze, T., Meliert, W. and Moelling, K. EMBO J., 7, 239–243, (1988b).PubMedCentralPubMedGoogle Scholar
  12. Ikuta, K. and Luftig, R.B. Virology, 154, 195–206, (1986).PubMedCrossRefGoogle Scholar
  13. Ikuta, K., Coward, J. and Luftig, R.B. Virology, 154, 207–213, (1986).PubMedCrossRefGoogle Scholar
  14. James, M.N.G., Hsu, I.-N. and Delabaere, L.T.J. Nature, 267, 808–813, (1977).PubMedCrossRefGoogle Scholar
  15. Johnson, M.S., McClure, M.A., Feng, D.F., Gray, J. and Doolittle, R.F. Proc.Natl.Acad.Sci. USA, 83, 7648–7652, (1986).PubMedCentralPubMedCrossRefGoogle Scholar
  16. Kay, J., Jupp, R.A., Norey, C.G., Richards, A.D., Reid, W.A., Taggart, R.T., Samloff, J.M. and Dunn, B.M. in ‘Proteases: Potential Role in Health and Disease’ (Heidland, A. and Horl, W.H., eds.). Plenum Press, (1987).Google Scholar
  17. Larder, B., Purifoy, D., Powell, K. and Darby, G. EMBO J., 6, 3133–3137, (1987).PubMedCentralPubMedGoogle Scholar
  18. LeGrice, S.F.J., Beuck, V. and Mous, J. Gene, 55, 95–103, (1987).CrossRefGoogle Scholar
  19. Lightfoote, M.M., Coligan, J.E., Folks, T.M., Fanci, A.S., Martin, M.A. and Venkatesan, S. J.Virol., 60, 771–775, (1986).PubMedCentralPubMedGoogle Scholar
  20. Lori, F., Scovassi, A.I., Zella, R., Achilli, G., Cattaneo, E., Casoli, C. and Bertazzoni, U. AIDS Res. Hum. Retro., 4, 393–398, (1988).CrossRefGoogle Scholar
  21. Lowe, D.M., Aitken, A., Bradley, C., Darby, G.K., Larder, B.A., Powell, K.L. Purifoy, D.J.M., Tisdale, M. and Stammers, D.K. Biochemistry, 27, 8884–8889, (1988).PubMedCrossRefGoogle Scholar
  22. Moelling, K. Cold Spring Harbor Symp.Quant. Biol., 39, 969–973, (1974a).CrossRefGoogle Scholar
  23. Moelling, K. Virology, 62, 46–59, (1974b).PubMedCrossRefGoogle Scholar
  24. Moelling, K., Knoop, M.-T., Billich, S., Blaha, J., Pavlickova, L. and Soucek, M. in ‘Proteases and Retroviruses’. Proceedings of the 14th Int. Congress of Biochemistry, (V. Kostka, ed.), W. de Gruyter and Co., Berlin, p.155–164, (1989).Google Scholar
  25. Moelling, K., Shulze, T., Knoop, M.-T., Kay, J., Jupp, R., Nicolaou, G. and Pearl, L.H. FEBS Lett., in press, (1990).Google Scholar
  26. Pal, R., Gallo, R.C. and Sarngadharen, M.G. Proc.Natl.Acad.Sci. USA, 85, 9283–9286, (1988).PubMedCentralPubMedCrossRefGoogle Scholar
  27. Pearl, L.H. and Taylor, W.R. Nature, 328, 482, (1987).Google Scholar
  28. Prasad, V.R. and Goff, S.P. Proc.Natl.Acad.Sci. USA, 86, 3104–3108, (1989).PubMedCentralPubMedCrossRefGoogle Scholar
  29. Schlesinger, and Malfer, J.Biol.Chem., 257, 9887–9890, (1982).PubMedGoogle Scholar
  30. Tanese, N., Prasad, V.R. and Goff, S.P. DNA, 7, 407–416, (1988).PubMedCrossRefGoogle Scholar
  31. Tang, J. J.Biol.Chem., 246, 4510–4517, (1971).PubMedGoogle Scholar

Copyright information

© Macmillan Publishers Limited 1990

Authors and Affiliations

  • K. Moelling
  • M. Nawrath
  • T. Schulze
  • L. Pavlitzkova
  • M. Soucek
  • K.-H. Budt
  • L. H. Pearl
  • M.-T. Knoop
  • J. Kay
  • V. Kruft

There are no affiliations available

Personalised recommendations