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. 1991 Feb;65(2):922–930. doi: 10.1128/jvi.65.2.922-930.1991

Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein.

R J Tritch 1, Y E Cheng 1, F H Yin 1, S Erickson-Viitanen 1
PMCID: PMC239833  PMID: 1987379

Abstract

The virally encoded protease of human immunodeficiency virus (HIV) is responsible for specific cleavage events leading to the liberation of the enzymes reverse transcriptase, integrase, ribonuclease H, and the core proteins from the gag-pol and gag polyprotein precursors. Utilizing gag polyprotein synthesized in vitro, we have shown that this substrate is sequentially cleaved by purified HIV protease to yield products that on the basis of their sizes and immunoreactivities correspond to p15, p6, p7, p17, and finally mature p24. We have placed unique restriction sites flanking the p17-p24 domain in order to facilitate replacement of cleavage site sequences by utilizing oligonucleotide cassettes. Replacement of the rapidly cleaved methionine-methionine bond at the p24-p15 junction with tyrosine-proline or replacement of the tyrosine-proline bond at the p17-p24 junction with methionine-methionine results in sites that cannot be efficiently cleaved. A basic amino acid at the p17-p24 scissile bond is not tolerated. Replacement of this cleavage site with an inverted repeat amino acid sequence gives intermediate rates of cleavage. In an attempt to convert the p17-p24 domain into a p24-p15 domain, residues flanking the scissile bond were exchanged in an expanding iterative fashion. When four residues flanking the scissile bond had been replaced, the rate of cleavage relative to that of the native p17-p24 sequence was increased fourfold. The cleavage rate of the native p24-p15 sequence is still some 10-fold greater than that of the p17-p24 sequence, suggesting that more-distant residues significantly affect the cleavage rate.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Billich S., Knoop M. T., Hansen J., Strop P., Sedlacek J., Mertz R., Moelling K. Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease. J Biol Chem. 1988 Dec 5;263(34):17905–17908. [PubMed] [Google Scholar]
  2. Cheng Y. S., McGowan M. H., Kettner C. A., Schloss J. V., Erickson-Viitanen S., Yin F. H. High-level synthesis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies. Gene. 1990 Mar 15;87(2):243–248. doi: 10.1016/0378-1119(90)90308-e. [DOI] [PubMed] [Google Scholar]
  3. Copeland T. D., Oroszlan S. Genetic locus, primary structure, and chemical synthesis of human immunodeficiency virus protease. Gene Anal Tech. 1988 Nov-Dec;5(6):109–115. doi: 10.1016/0735-0651(88)90010-6. [DOI] [PubMed] [Google Scholar]
  4. Darke P. L., Nutt R. F., Brady S. F., Garsky V. M., Ciccarone T. M., Leu C. T., Lumma P. K., Freidinger R. M., Veber D. F., Sigal I. S. HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins. Biochem Biophys Res Commun. 1988 Oct 14;156(1):297–303. doi: 10.1016/s0006-291x(88)80839-8. [DOI] [PubMed] [Google Scholar]
  5. Debouck C., Gorniak J. G., Strickler J. E., Meek T. D., Metcalf B. W., Rosenberg M. Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8903–8906. doi: 10.1073/pnas.84.24.8903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dreyer G. B., Metcalf B. W., Tomaszek T. A., Jr, Carr T. J., Chandler A. C., 3rd, Hyland L., Fakhoury S. A., Magaard V. W., Moore M. L., Strickler J. E. Inhibition of human immunodeficiency virus 1 protease in vitro: rational design of substrate analogue inhibitors. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9752–9756. doi: 10.1073/pnas.86.24.9752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Erickson-Viitanen S., Manfredi J., Viitanen P., Tribe D. E., Tritch R., Hutchison C. A., 3rd, Loeb D. D., Swanstrom R. Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease. AIDS Res Hum Retroviruses. 1989 Dec;5(6):577–591. doi: 10.1089/aid.1989.5.577. [DOI] [PubMed] [Google Scholar]
  8. Gowda S. D., Stein B. S., Engleman E. G. Identification of protein intermediates in the processing of the p55 HIV-1 gag precursor in cells infected with recombinant vaccinia virus. J Biol Chem. 1989 May 25;264(15):8459–8462. [PubMed] [Google Scholar]
  9. Graves M. C., Lim J. J., Heimer E. P., Kramer R. A. An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2449–2453. doi: 10.1073/pnas.85.8.2449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hostomsky Z., Appelt K., Ogden R. C. High-level expression of self-processed HIV-1 protease in Escherichia coli using a synthetic gene. Biochem Biophys Res Commun. 1989 Jun 30;161(3):1056–1063. doi: 10.1016/0006-291x(89)91350-8. [DOI] [PubMed] [Google Scholar]
  11. Kay J., Dunn B. M. Viral proteinases: weakness in strength. Biochim Biophys Acta. 1990 Jan 30;1048(1):1–18. doi: 10.1016/0167-4781(90)90015-t. [DOI] [PubMed] [Google Scholar]
  12. Kohl N. E., Emini E. A., Schleif W. A., Davis L. J., Heimbach J. C., Dixon R. A., Scolnick E. M., Sigal I. S. Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4686–4690. doi: 10.1073/pnas.85.13.4686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kotler M., Katz R. A., Danho W., Leis J., Skalka A. M. Synthetic peptides as substrates and inhibitors of a retroviral protease. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4185–4189. doi: 10.1073/pnas.85.12.4185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kräusslich H. G., Ingraham R. H., Skoog M. T., Wimmer E., Pallai P. V., Carter C. A. Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides. Proc Natl Acad Sci U S A. 1989 Feb;86(3):807–811. doi: 10.1073/pnas.86.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lapatto R., Blundell T., Hemmings A., Overington J., Wilderspin A., Wood S., Merson J. R., Whittle P. J., Danley D. E., Geoghegan K. F. X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes. Nature. 1989 Nov 16;342(6247):299–302. doi: 10.1038/342299a0. [DOI] [PubMed] [Google Scholar]
  18. Loeb D. D., Hutchison C. A., 3rd, Edgell M. H., Farmerie W. G., Swanstrom R. Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases. J Virol. 1989 Jan;63(1):111–121. doi: 10.1128/jvi.63.1.111-121.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Louis J. M., Wondrak E. M., Copeland T. D., Smith C. A., Mora P. T., Oroszlan S. Chemical synthesis and expression of the HIV-1 protease gene in E. coli. Biochem Biophys Res Commun. 1989 Feb 28;159(1):87–94. doi: 10.1016/0006-291x(89)92408-x. [DOI] [PubMed] [Google Scholar]
  20. Margolin N., Heath W., Osborne E., Lai M., Vlahos C. Substitutions at the P2' site of gag p17-p24 affect cleavage efficiency by HIV-1 protease. Biochem Biophys Res Commun. 1990 Mar 16;167(2):554–560. doi: 10.1016/0006-291x(90)92060-d. [DOI] [PubMed] [Google Scholar]
  21. Meek T. D., Lambert D. M., Dreyer G. B., Carr T. J., Tomaszek T. A., Jr, Moore M. L., Strickler J. E., Debouck C., Hyland L. J., Matthews T. J. Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues. Nature. 1990 Jan 4;343(6253):90–92. doi: 10.1038/343090a0. [DOI] [PubMed] [Google Scholar]
  22. Mervis R. J., Ahmad N., Lillehoj E. P., Raum M. G., Salazar F. H., Chan H. W., Venkatesan S. The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors. J Virol. 1988 Nov;62(11):3993–4002. doi: 10.1128/jvi.62.11.3993-4002.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miller M., Schneider J., Sathyanarayana B. K., Toth M. V., Marshall G. R., Clawson L., Selk L., Kent S. B., Wlodawer A. Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution. Science. 1989 Dec 1;246(4934):1149–1152. doi: 10.1126/science.2686029. [DOI] [PubMed] [Google Scholar]
  24. Moore M. L., Bryan W. M., Fakhoury S. A., Magaard V. W., Huffman W. F., Dayton B. D., Meek T. D., Hyland L., Dreyer G. B., Metcalf B. W. Peptide substrates and inhibitors of the HIV-1 protease. Biochem Biophys Res Commun. 1989 Mar 15;159(2):420–425. doi: 10.1016/0006-291x(89)90008-9. [DOI] [PubMed] [Google Scholar]
  25. Navia M. A., Fitzgerald P. M., McKeever B. M., Leu C. T., Heimbach J. C., Herber W. K., Sigal I. S., Darke P. L., Springer J. P. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature. 1989 Feb 16;337(6208):615–620. doi: 10.1038/337615a0. [DOI] [PubMed] [Google Scholar]
  26. Pearl L. H., Taylor W. R. A structural model for the retroviral proteases. Nature. 1987 Sep 24;329(6137):351–354. doi: 10.1038/329351a0. [DOI] [PubMed] [Google Scholar]
  27. Peng C., Ho B. K., Chang T. W., Chang N. T. Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity. J Virol. 1989 Jun;63(6):2550–2556. doi: 10.1128/jvi.63.6.2550-2556.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roberts N. A., Martin J. A., Kinchington D., Broadhurst A. V., Craig J. C., Duncan I. B., Galpin S. A., Handa B. K., Kay J., Kröhn A. Rational design of peptide-based HIV proteinase inhibitors. Science. 1990 Apr 20;248(4953):358–361. doi: 10.1126/science.2183354. [DOI] [PubMed] [Google Scholar]
  29. Schneider J., Kent S. B. Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease. Cell. 1988 Jul 29;54(3):363–368. doi: 10.1016/0092-8674(88)90199-7. [DOI] [PubMed] [Google Scholar]
  30. Veronese F. D., Rahman R., Copeland T. D., Oroszlan S., Gallo R. C., Sarngadharan M. G. Immunological and chemical analysis of P6, the carboxyl-terminal fragment of HIV P15. AIDS Res Hum Retroviruses. 1987 Fall;3(3):253–264. doi: 10.1089/aid.1987.3.253. [DOI] [PubMed] [Google Scholar]
  31. Weber I. T., Miller M., Jaskólski M., Leis J., Skalka A. M., Wlodawer A. Molecular modeling of the HIV-1 protease and its substrate binding site. Science. 1989 Feb 17;243(4893):928–931. doi: 10.1126/science.2537531. [DOI] [PubMed] [Google Scholar]
  32. Wlodawer A., Miller M., Jaskólski M., Sathyanarayana B. K., Baldwin E., Weber I. T., Selk L. M., Clawson L., Schneider J., Kent S. B. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science. 1989 Aug 11;245(4918):616–621. doi: 10.1126/science.2548279. [DOI] [PubMed] [Google Scholar]
  33. Wolfe H. R., Wilk R. R. The RaMPS system: simplified peptide synthesis for life science researchers. Pept Res. 1989 Sep-Oct;2(5):352–356. [PubMed] [Google Scholar]

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