Genetically Engineered Bacteria to Identify and Produce Anti-Viral Agents

  • Robert H. Grafstrom
  • Katherine Zachariasewycz
  • Richard A. Brigandi
  • Timothy M. Block
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 312)

Summary

We have prepared a strain of Escherichia coli that expresses both the HIV protease and a Tet protein which has been modified to contain the HIV protease recognition sequence. When the protease is expressed, the bacteria will not grow in the presence of tetracycline. However, when the protease is inhibited the bacteria can grow in tetracycline containing media (Block and Grafstrom 1990). We have selected spontaneously arising Tet resistant mutants and have screened them for those that could be producing an inhibitor of HIV protease. The problems in the construction of this strain and the characterization of the various Teti mutants are discussed.

Keywords

Human Immunodeficiency Virus Antiviral Agent Tetracycline Resistance Tetracycline Resistance Gene Mutant Bacterium 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Ames, B.N., McCann, J., and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Research, 31:347–364.PubMedCrossRefGoogle Scholar
  2. Ashby, J., and Tennant R.W., 1988, Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogens among 222 chemicals tested on rodents by the U.S. NCl/NTP, Mutation Research 204:17–115.PubMedCrossRefGoogle Scholar
  3. Backman, K. and Boyer, H.W., 1983, Tetracycline resistance determined by pBR322 is mediated by 1 polypeptide, Gene 26:197–203.PubMedCrossRefGoogle Scholar
  4. Barany, F., 1985, Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides, Proc. Natl. Acad. Sci. USA 82:4202–4206.PubMedCrossRefGoogle Scholar
  5. Baum, E.Z., Bebernitz, G.Z., and Gluzman, Y., 1990a, Beta galactosidase containing a human immunodeficiency virus protease cleavage site is cleaved and inactivated by human immunodeficiency protease, Proc. Natl. Acad. Sci. USA 87:10023–10027.CrossRefGoogle Scholar
  6. Baum, E.Z., Bebernitz, G.A., and Gluzman, Y., 1990b, Isolation of mutants of human immunodeficiency virus protease based on the toxicity of the enzyme in Escherichia coli, Proc. Natl. Acad.Sci. USA 87:5573–5577.CrossRefGoogle Scholar
  7. Bjornsti, M.A., Benedetti, GA., Viglianti, A., and Wang, J.C., 1989, Expression of human DNA topoisomerase I in yeast cells lacking yeast DNA topoisomerase I: Restoration of sensitivity of the cells to the antitumor drug camptothecin, Cancer Research 49:6318–6323.PubMedGoogle Scholar
  8. Block, T.M., and Grafstrom R.H., 1990, Novel bacteriological assay for the detection of potential antiviral agents, Antimicrob. Agents and Chemo. 34:2337–2341.CrossRefGoogle Scholar
  9. Borel, J.F., Feurer, C., Magnee, C., and Stahelin, H., 1977, Effects of the new anti-lymphocytic peptide cyclosporin A in animals, Immunol. 32:1017–1025.Google Scholar
  10. Cairns, J., Overbaugh, J., and Miller, S., 1988, The origin of mutants, Nature 335:142–145.PubMedCrossRefGoogle Scholar
  11. Curiale, M.S., McMurry L.M., and Levy, S.B., 1984, Intracistronic complementation of the tetracycline resistance membrane protein of Tn10, J. Bacteriol. 157:211–217.PubMedGoogle Scholar
  12. Cwirla, S.E., Peters, E.A., Barrett, R.W., and Dower, W.J., 1990, Peptides on phage: A vast library of peptides for identifying ligands, Proc. Natl. Acad. Sci. USA 87:6378–6382.PubMedCrossRefGoogle Scholar
  13. Debouck, C., Gorniak, J.G., Strickler, J.E., Meek, T.D., Metcalf, B.W., and Rosenberg, M., 1987, Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor, Proc. Natl. Acad.Sci. USA 84:8903–8906.PubMedCrossRefGoogle Scholar
  14. DeClercq, KE., 1990, Targets and strategies for the antiviral chemotherapy of AIDS, TIPS 11:198–205.Google Scholar
  15. Dreyer G.B., Metcalf, B.W., Tomaszek, T.A., Jr., Carr, T.J., Chandler, A.C., III, Hyland, L., Fakhouryk, S.A., Magnard, V.W., Moore, M.L., Strickler, J.F., Debouck, C., and Meek, T.D.1989, Inhibition of HIV-1 protease in vitro: rational design of substrate analogue inhibitors, Proc. Natl.Acad. Sci. USA 86:9752–9756.PubMedCrossRefGoogle Scholar
  16. Evnin, L.B., Vazquez, J.R., and Craik, C.S., 1990, Substrate specificity of trypsin investigated by using a genetic selection, Proc. Natl. Acad. Sci. USA 87:6659–6663.PubMedCrossRefGoogle Scholar
  17. Hall, B., 1990, Spontaneous point mutations that occur more often when advantageous than when neutral, Genetics 126:5–16.PubMedGoogle Scholar
  18. Hansen, J., Billich, S., Schulze, T., Sukrow, S., and Moelling, K., 1988, Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody, EMBO J. 7:1785–1791.PubMedGoogle Scholar
  19. Harley, C.B., Laurie, J., Betlack, M., Crea, R., Boyer H.W., and Hedgpeth, J., 1988, Transcription initiation a the tet promoter and effect of mutations, Nucleic Acids Res. 16:7269–7285.PubMedCrossRefGoogle Scholar
  20. Harley, C.B., Laurie, J., Boyer, H.W., and Hedgpeth, J., 1990, Reiterative copying by the E. coli RNA polymerase during transcription initiation of mutant pBR322 tet promoters, Nucleic Acids Res. 18:547–552.PubMedCrossRefGoogle Scholar
  21. Huse, W.D., Sastry, L., Iverson, S.A., Kong, A.S., Alting, S., Mees, M., Burton, D.R., Benkovic, Si., and Lerner, R.A.,1989, Generation of a large combinational library of the immunoglobulin repertoire in phage lambda, Science 246:1275–1281.PubMedCrossRefGoogle Scholar
  22. Ivanoff, L.A., Towatari, T., Ray, J., Korant, B.D., and Petteway, S.R., 1986, Expression and site specific mutagenesis of the poliovirus 3C protease in Escherichia coli, Proc. Natl. Acad. Sci. USA 83:5392–5396.PubMedCrossRefGoogle Scholar
  23. Kadonaga, J.T., Carner, K.R., Masiarz, F.R., and Tijan, R., 1987, Isolation of cDNA encoding transcription factor Spl and functional analysis of the DNA binding domain, Cell 51:1079–1090.PubMedCrossRefGoogle Scholar
  24. Kholberg, R., 1991, Critics call for a smarter way to screen for drugs, J. NIH Res. 3:25–26.Google Scholar
  25. Kohl, N.E., Emini, E.A., Schleif, W.A., Davis, L.J., Heimbach, J.C., Dixon, R.A.F., Scolnick, E.M., and Sigal, I.S., 1988, Active immunodeficiency virus protease is required for viral infectivity, Proc. Natl. Acad. Sci. USA 85:4686–4690.PubMedCrossRefGoogle Scholar
  26. Korant, B., 1990, AIDS Research and Reference Reagent Program Catalog, U.S. Department of Health and Human Services, p.51 (January).Google Scholar
  27. Krausslich, H.G., Schneider, H., Zybarth, G., Carter, C.A., and Wimmer, E., 1988, Processing of in vitro synthesized gag precursor proteins of human immunodeficiency virus (HIV) type-1 by HIV protease generated in Escherichia coli, J. Virol. 62:4393–397.PubMedGoogle Scholar
  28. Legrice, S.F.J., Mills, J., and Mous, J., 1988, Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation, EMBO J. 7:2547–2553.Google Scholar
  29. Marshall, B., Morrissey, S., Flynn, P., and Levy, S.B., 1986, A new tetracycline resistant determinant, class E isolated from Enterobacteriaceae, Gene 50:111–117.PubMedCrossRefGoogle Scholar
  30. McCann, J., Choi, E., Yamasaki, E., and Ames, B.N., 1975,Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. USA 72:5135–5139.PubMedCrossRefGoogle Scholar
  31. McMurry, L., Petrucci, R.E., and Levy, S.B., 1980, Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants of Escherichia coli, Proc. Natl. Acad. Sci. USA 77:3974–3977.PubMedCrossRefGoogle Scholar
  32. McQuade, T.J., Tomasselli, A.G., Liu, L., Karacostas V., Moss, B., Sawyer, T.K., Heinrikson, R.L., Tarpley, W.G., 1990, A synthetic HIV-protease inhibitor with antiviral activity arrests HIV-like particle maturation, Science 247:454–456.PubMedCrossRefGoogle Scholar
  33. Middleman, E., Luce, J., and Frei, E., 1971, Clinical trials with adriamycin, Cancer 28:844–850.PubMedCrossRefGoogle Scholar
  34. Miller, M., Schneider, J., Sathyanarayana, B.K., Toth, M.V., Markshall, G.R., Clawson, L., Selk, L., Kent, S.B.H., and Wlodawer, A.,1989, Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution, Science 246:1149–1151.PubMedCrossRefGoogle Scholar
  35. Mitsuya, H., Yarchoan, R., and Broder, S., 1990, Molecular targets for AIDS therapy, Science 249:1533–1544.PubMedCrossRefGoogle Scholar
  36. Moore, M.L., Bryan, W.M., Fakhoury, S.A., and Maagard, V.M., Huffman, W.F., Dayton, B.D., Meek, T.D., Hyland, L., Dreyer, G.B., Metcalf, B.W., Strickler, J.E., Gorniak, J.G., and Debouck, C., 1989, Peptide substrates and inhibitors of the HIV-1 protease, Biochem. Biophys. Res. Comm. 159:420–425.PubMedCrossRefGoogle Scholar
  37. Pauwels, R., Andries, K., Desmyter, J., Schols, D., Kukla, M.J., Breslin, H.J., Raeymaeckers, A., Van Gelder, J., Woestenborghs, R., Heykants, J., Schellekens, K., Janssen, M.A.C., DeClerq, E., and Janssen, P.A.J., 1990, Potent and selective inhibition of HIV-1 replication in vitro by a novel series of TIBO derivatives, Nature 343:470–474.PubMedCrossRefGoogle Scholar
  38. Roberts, N.A., Martin, J.A., Kinchington, D., Broadhurst, A.V., Craig, J.C., Duncan, I.B., Galpin, S.A., Handa, B.K., Krohn, J.K.A., Lambert, R.W., Merrett, J.H., Mills, J.S., Parkes, K.B.B., Redshaw, S., Ritchie, A.J., Taylor, D.L., Thomas, G.J., Machin, P.J., 1990, Rational design of peptide-based HIV proteinase inhibitors, Science 248:358–361.PubMedCrossRefGoogle Scholar
  39. Rubin, R.A., and Levy, S., 1990, Interdomain hybrid Tet proteins confer tetracycline resistance only when they are derived from closely related members of the tet gene family, J. Bacteriol. 172:2303–2312.PubMedGoogle Scholar
  40. Seelmeir, S., Schmidt, H., Turk, V., and von der Helm, K., 1988, Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A, Proc. Natl. Acad. Sci. USA 85:6612–6616.CrossRefGoogle Scholar
  41. Sidwell, R.W., Dixon, G.J., Schabel, F.M., 1968, Antiviral activity of 9-beta-Darabinofuranosyl adenine, Antimicrob. Agent. Chemo. 8:148–154.Google Scholar
  42. Yang, H.L., Zubay, kG., and Levy, S., 1976, Synthesis of a R plasmid associated with tetracycline resistance is negatively regulated, Proc. Natl. Acad. Sci. USA 73:1509–1512.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1992

Authors and Affiliations

  • Robert H. Grafstrom
    • 1
  • Katherine Zachariasewycz
    • 1
  • Richard A. Brigandi
    • 1
  • Timothy M. Block
    • 1
  1. 1.Department of Microbiology and Immunology Jefferson Medical CollegeThomas Jefferson UniversityPhiladelphiaUSA

Personalised recommendations