Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 May;86(9):3104–3108. doi: 10.1073/pnas.86.9.3104

Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain.

V R Prasad 1, S P Goff 1
PMCID: PMC287073  PMID: 2470090

Abstract

A plasmid construct expressing the p66 version of the human immunodeficiency virus reverse transcriptase as a bacterial fusion protein was subjected to in vitro mutagenesis, and the resulting variant proteins were assayed to define the locations of the two major enzymatic activities. The DNA polymerase activity was localized to the N-terminal portion of the protein; mutations altering or eliminating the C-terminal portion had little or no effect on that activity. The results suggest that, in contrast with previous reports, the p51 subunit found in virions should exhibit DNA polymerase activity. Mutations in many parts of the protein eliminated RNase H activity, suggesting that several areas are needed for proper folding and generation of that activity.

Full text

PDF
3104

Images in this article

3105Image
on p.3105
3106Image
on p.3106
3106Image
on p.3106
3106Image
on p.3106
3107Image
on p.3107

Selected References

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

  1. Barré-Sinoussi F., Chermann J. C., Rey F., Nugeyre M. T., Chamaret S., Gruest J., Dauguet C., Axler-Blin C., Vézinet-Brun F., Rouzioux C. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science. 1983 May 20;220(4599):868–871. doi: 10.1126/science.6189183. [DOI] [PubMed] [Google Scholar]
  2. Chandra P. Molecular approaches for designing antiviral and antitumor compounds. Top Curr Chem. 1974;52:99–139. doi: 10.1007/3-540-06873-2_15. [DOI] [PubMed] [Google Scholar]
  3. Chandra P., Vogel A., Gerber T. Inhibitors of retroviral DNA polymerase: their implication in the treatment of AIDS. Cancer Res. 1985 Sep;45(9 Suppl):4677s–4684s. [PubMed] [Google Scholar]
  4. Cheng Y. C., Dutschman G. E., Bastow K. F., Sarngadharan M. G., Ting R. Y. Human immunodeficiency virus reverse transcriptase. General properties and its interactions with nucleoside triphosphate analogs. J Biol Chem. 1987 Feb 15;262(5):2187–2189. [PubMed] [Google Scholar]
  5. Farmerie W. G., Loeb D. D., Casavant N. C., Hutchison C. A., 3rd, Edgell M. H., Swanstrom R. Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli. Science. 1987 Apr 17;236(4799):305–308. doi: 10.1126/science.2436298. [DOI] [PubMed] [Google Scholar]
  6. Gallo R. C., Sarin P. S., Gelmann E. P., Robert-Guroff M., Richardson E., Kalyanaraman V. S., Mann D., Sidhu G. D., Stahl R. E., Zolla-Pazner S. Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS). Science. 1983 May 20;220(4599):865–867. doi: 10.1126/science.6601823. [DOI] [PubMed] [Google Scholar]
  7. Gerard G. F., Grandgenett D. P. Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus. J Virol. 1975 Apr;15(4):785–797. doi: 10.1128/jvi.15.4.785-797.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goff S., Traktman P., Baltimore D. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J Virol. 1981 Apr;38(1):239–248. doi: 10.1128/jvi.38.1.239-248.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hansen J., Schulze T., Mellert W., Moelling K. Identification and characterization of HIV-specific RNase H by monoclonal antibody. EMBO J. 1988 Jan;7(1):239–243. doi: 10.1002/j.1460-2075.1988.tb02805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hansen J., Schulze T., Moelling K. RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. J Biol Chem. 1987 Sep 15;262(26):12393–12396. [PubMed] [Google Scholar]
  11. Hizi A., McGill C., Hughes S. H. Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1218–1222. doi: 10.1073/pnas.85.4.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hoffman A. D., Banapour B., Levy J. A. Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology. 1985 Dec;147(2):326–335. doi: 10.1016/0042-6822(85)90135-7. [DOI] [PubMed] [Google Scholar]
  13. Johnson M. S., McClure M. A., Feng D. F., Gray J., Doolittle R. F. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7648–7652. doi: 10.1073/pnas.83.20.7648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Larder B. A., Purifoy D. J., Powell K. L., Darby G. Site-specific mutagenesis of AIDS virus reverse transcriptase. 1987 Jun 25-Jul 1Nature. 327(6124):716–717. doi: 10.1038/327716a0. [DOI] [PubMed] [Google Scholar]
  15. Larder B., Purifoy D., Powell K., Darby G. AIDS virus reverse transcriptase defined by high level expression in Escherichia coli. EMBO J. 1987 Oct;6(10):3133–3137. doi: 10.1002/j.1460-2075.1987.tb02623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Le Grice S. F., Beuck V., Mous J. Expression of biologically active human T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis. Gene. 1987;55(1):95–103. doi: 10.1016/0378-1119(87)90252-6. [DOI] [PubMed] [Google Scholar]
  17. Le Grice S. F., Zehnle R., Mous J. A single 66-kilodalton polypeptide processed from the human immunodeficiency virus type 2 pol polyprotein in Escherichia coli displays reverse transcriptase activity. J Virol. 1988 Jul;62(7):2525–2529. doi: 10.1128/jvi.62.7.2525-2529.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lightfoote M. M., Coligan J. E., Folks T. M., Fauci A. S., Martin M. A., Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol. 1986 Nov;60(2):771–775. doi: 10.1128/jvi.60.2.771-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moelling K. Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex. J Virol. 1976 May;18(2):418–425. doi: 10.1128/jvi.18.2.418-425.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mölling K., Bolognesi D. P., Bauer H., Büsen W., Plassmann H. W., Hausen P. Association of viral reverse transcriptase with an enzyme degrading the RNA moiety of RNA-DNA hybrids. Nat New Biol. 1971 Dec 22;234(51):240–243. doi: 10.1038/newbio234240a0. [DOI] [PubMed] [Google Scholar]
  21. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  22. Rey M. A., Spire B., Dormont D., Barre-Sinoussi F., Montagnier L., Chermann J. C. Characterization of the RNA dependent DNA polymerase of a new human T-lymphotropic retrovirus (lymphadenopathy associated virus). Biochem Biophys Res Commun. 1984 May 31;121(1):126–133. doi: 10.1016/0006-291x(84)90696-x. [DOI] [PubMed] [Google Scholar]
  23. Rucheton M., Lelay M. N., Jeanteur P. Evidence from direct visualization after denaturing gel electrophoresis that RNase H is associated with MSV-MuLV reverse transcripase. Virology. 1979 Aug;97(1):221–223. doi: 10.1016/0042-6822(79)90392-1. [DOI] [PubMed] [Google Scholar]
  24. Starnes M. C., Gao W. Y., Ting R. Y., Cheng Y. C. Enzyme activity gel analysis of human immunodeficiency virus reverse transcriptase. J Biol Chem. 1988 Apr 15;263(11):5132–5134. [PubMed] [Google Scholar]
  25. Tanese N., Goff S. P. Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1777–1781. doi: 10.1073/pnas.85.6.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tanese N., Prasad V. R., Goff S. P. Structural requirements for bacterial expression of stable, enzymatically active fusion proteins containing the human immunodeficiency virus reverse transcriptase. DNA. 1988 Jul-Aug;7(6):407–416. doi: 10.1089/dna.1.1988.7.407. [DOI] [PubMed] [Google Scholar]
  27. Tanese N., Roth M., Goff S. P. Expression of enzymatically active reverse transcriptase in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4944–4948. doi: 10.1073/pnas.82.15.4944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tanese N., Sodroski J., Haseltine W. A., Goff S. P. Expression of reverse transcriptase activity of human T-lymphotropic virus type III (HTLV-III/LAV) in Escherichia coli. J Virol. 1986 Sep;59(3):743–745. doi: 10.1128/jvi.59.3.743-745.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tisdale M., Ertl P., Larder B. A., Purifoy D. J., Darby G., Powell K. L. Characterization of human immunodeficiency virus type 1 reverse transcriptase by using monoclonal antibodies: role of the C terminus in antibody reactivity and enzyme function. J Virol. 1988 Oct;62(10):3662–3667. doi: 10.1128/jvi.62.10.3662-3667.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Verma I. M. Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H. J Virol. 1975 Apr;15(4):843–854. doi: 10.1128/jvi.15.4.843-854.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Verma I. M. The reverse transcriptase. Biochim Biophys Acta. 1977 Mar 21;473(1):1–38. doi: 10.1016/0304-419x(77)90005-1. [DOI] [PubMed] [Google Scholar]
  32. Xiong Y., Eickbush T. H. The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons. Mol Cell Biol. 1988 Jan;8(1):114–123. doi: 10.1128/mcb.8.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. di Marzo Veronese F., Copeland T. D., DeVico A. L., Rahman R., Oroszlan S., Gallo R. C., Sarngadharan M. G. Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. Science. 1986 Mar 14;231(4743):1289–1291. doi: 10.1126/science.2418504. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES