Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1992 Dec;66(12):7533–7537. doi: 10.1128/jvi.66.12.7533-7537.1992

Mutational analysis of the fingers domain of human immunodeficiency virus type 1 reverse transcriptase.

P L Boyer 1, A L Ferris 1, S H Hughes 1
PMCID: PMC240464  PMID: 1279205

Abstract

Using BspMI cassette vectors, we have constructed a series of mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that cause specific amino acid substitutions within the polymerase domain. The RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant RTs were assayed. The elucidation of the structure of HIV-1 RT makes it possible to determine the locations of specific mutations in the three-dimensional structure of HIV-1 RT [E. Arnold, A. Jacobo-Molina, R. G. Nanni, R. L. Williams, X. Lu, J. Ding, A. D. Clark, Jr., A. Zhang, A. L. Ferris, P. Clark, A. Hizi, and S. H. Hughes, Nature (London) 357:85-89, 1992; L. A. Kohlstaedt, J. Wang, J. M. Friedman, P. A. Rice, and T. A. Steitz, Science 256:1783-1790, 1992]. The mutations described in this report are between amino acids 25 and 81, within the "fingers" domain of RT (Kohlstaedt et al., Science 256:1783-1790, 1992). It has been suggested that this domain may play a role in positioning the template. Although the fingers domain does not contain the active site for polymerization, several of the mutations within this domain disrupt polymerase activity without significantly affecting RNase H activity.

Full text

PDF
7537

Images in this article

7535Image
on p.7535

Selected References

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

  1. Arnold E., Jacobo-Molina A., Nanni R. G., Williams R. L., Lu X., Ding J., Clark A. D., Jr, Zhang A., Ferris A. L., Clark P. Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations. Nature. 1992 May 7;357(6373):85–89. doi: 10.1038/357085a0. [DOI] [PubMed] [Google Scholar]
  2. Barber A. M., Hizi A., Maizel J. V., Jr, Hughes S. H. HIV-1 reverse transcriptase: structure predictions for the polymerase domain. AIDS Res Hum Retroviruses. 1990 Sep;6(9):1061–1072. doi: 10.1089/aid.1990.6.1061. [DOI] [PubMed] [Google Scholar]
  3. Boyer P. L., Ferris A. L., Hughes S. H. Cassette mutagenesis of the reverse transcriptase of human immunodeficiency virus type 1. J Virol. 1992 Feb;66(2):1031–1039. doi: 10.1128/jvi.66.2.1031-1039.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gao Q., Gu Z. X., Parniak M. A., Li X. G., Wainberg M. A. In vitro selection of variants of human immunodeficiency virus type 1 resistant to 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine. J Virol. 1992 Jan;66(1):12–19. doi: 10.1128/jvi.66.1.12-19.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Hizi A., Barber A., Hughes S. H. Effects of small insertions on the RNA-dependent DNA polymerase activity of HIV-1 reverse transcriptase. Virology. 1989 May;170(1):326–329. doi: 10.1016/0042-6822(89)90389-9. [DOI] [PubMed] [Google Scholar]
  7. Hizi A., Hughes S. H., Shaharabany M. Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase. Virology. 1990 Apr;175(2):575–580. doi: 10.1016/0042-6822(90)90444-v. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Hizi A., Tal R., Hughes S. H. Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Escherichia coli. Virology. 1991 Jan;180(1):339–346. doi: 10.1016/0042-6822(91)90038-d. [DOI] [PubMed] [Google Scholar]
  10. Hostomsky Z., Hostomska Z., Fu T. B., Taylor J. Reverse transcriptase of human immunodeficiency virus type 1: functionality of subunits of the heterodimer in DNA synthesis. J Virol. 1992 May;66(5):3179–3182. doi: 10.1128/jvi.66.5.3179-3182.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hostomsky Z., Hostomska Z., Hudson G. O., Moomaw E. W., Nodes B. R. Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1148–1152. doi: 10.1073/pnas.88.4.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  13. Larder B. A., Coates K. E., Kemp S. D. Zidovudine-resistant human immunodeficiency virus selected by passage in cell culture. J Virol. 1991 Oct;65(10):5232–5236. doi: 10.1128/jvi.65.10.5232-5236.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Larder B. A., Kemp S. D. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989 Dec 1;246(4934):1155–1158. doi: 10.1126/science.2479983. [DOI] [PubMed] [Google Scholar]
  15. Larder B. A., Kemp S. D., Purifoy D. J. Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4803–4807. doi: 10.1073/pnas.86.13.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Le Grice S. F., Naas T., Wohlgensinger B., Schatz O. Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. EMBO J. 1991 Dec;10(12):3905–3911. doi: 10.1002/j.1460-2075.1991.tb04960.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Lowe D. M., Parmar V., Kemp S. D., Larder B. A. Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. FEBS Lett. 1991 May 6;282(2):231–234. doi: 10.1016/0014-5793(91)80484-k. [DOI] [PubMed] [Google Scholar]
  21. McClure M. A., Johnson M. S., Feng D. F., Doolittle R. F. Sequence comparisons of retroviral proteins: relative rates of change and general phylogeny. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2469–2473. doi: 10.1073/pnas.85.8.2469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nunberg J. H., Schleif W. A., Boots E. J., O'Brien J. A., Quintero J. C., Hoffman J. M., Emini E. A., Goldman M. E. Viral resistance to human immunodeficiency virus type 1-specific pyridinone reverse transcriptase inhibitors. J Virol. 1991 Sep;65(9):4887–4892. doi: 10.1128/jvi.65.9.4887-4892.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Prasad V. R., Goff S. P. Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain. Proc Natl Acad Sci U S A. 1989 May;86(9):3104–3108. doi: 10.1073/pnas.86.9.3104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Prasad V. R., Lowy I., de los Santos T., Chiang L., Goff S. P. Isolation and characterization of a dideoxyguanosine triphosphate-resistant mutant of human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11363–11367. doi: 10.1073/pnas.88.24.11363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Restle T., Müller B., Goody R. S. Dimerization of human immunodeficiency virus type 1 reverse transcriptase. A target for chemotherapeutic intervention. J Biol Chem. 1990 Jun 5;265(16):8986–8988. [PubMed] [Google Scholar]
  26. Shih C. K., Rose J. M., Hansen G. L., Wu J. C., Bacolla A., Griffin J. A. Chimeric human immunodeficiency virus type 1/type 2 reverse transcriptases display reversed sensitivity to nonnucleoside analog inhibitors. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9878–9882. doi: 10.1073/pnas.88.21.9878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Spanos A., Sedgwick S. G., Yarranton G. T., Hübscher U., Banks G. R. Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Apr 24;9(8):1825–1839. doi: 10.1093/nar/9.8.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. St Clair M. H., Martin J. L., Tudor-Williams G., Bach M. C., Vavro C. L., King D. M., Kellam P., Kemp S. D., Larder B. A. Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science. 1991 Sep 27;253(5027):1557–1559. doi: 10.1126/science.1716788. [DOI] [PubMed] [Google Scholar]
  29. Varmus H. Retroviruses. Science. 1988 Jun 10;240(4858):1427–1435. doi: 10.1126/science.3287617. [DOI] [PubMed] [Google Scholar]
  30. 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 Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES