Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1994 Sep;68(9):5721–5729. doi: 10.1128/jvi.68.9.5721-5729.1994

Contributions of DNA polymerase subdomains to the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase.

J S Smith 1, K Gritsman 1, M J Roth 1
PMCID: PMC236975  PMID: 7520094

Abstract

Previous studies showed that an isolated human immunodeficiency virus type 1 (HIV-1) RNase H domain expressed as a fusion protein is highly active in Mn2+, but activity was dependent on a hexahistidine tag located at either the carboxyl or amino terminus of the fusion protein (J. Smith and M. Roth, J. Virol. 67:4037-4049, 1993). It was postulated that a histidine tag can somehow provide a function normally associated with the DNA polymerase domain of HIV-1 reverse transcriptase. To determine the contributions of the DNA polymerase subdomains of HIV-1 reverse transcriptase to its RNase H activity, we have characterized the activity of isolated RNase H domains which include either portions of the connection, the entire connection, or both the thumb and connection as N-terminal extensions. Including increasing lengths of these domains at the N terminus of the RNase H resulted in a progressive increase in Mn(2+)-dependent RNase H activity that was independent of a histidine tag. Activity of the isolated RNase H domains was also stimulated by the addition of independently purified polymerase subdomains. Further, this stimulation was shown to be a result of direct physical interactions between the thumb, connection, and RNase H domains. The connection and thumb subdomains were shown to contribute to substrate binding. The fingers and palm subdomains were found to be essential for Mg(2+)-dependent RNase H activity.

Full text

PDF
5721

Images in this article

5723Image
on p.5723
5724Image
on p.5724
5725Image
on p.5725
5727Image
on p.5727

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. Basu A., Ahluwalia K. K., Basu S., Modak M. J. Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase. Biochemistry. 1992 Jan 21;31(2):616–623. doi: 10.1021/bi00117a045. [DOI] [PubMed] [Google Scholar]
  3. Becerra S. P., Clore G. M., Gronenborn A. M., Karlström A. R., Stahl S. J., Wilson S. H., Wingfield P. T. Purification and characterization of the RNase H domain of HIV-1 reverse transcriptase expressed in recombinant Escherichia coli. FEBS Lett. 1990 Sep 17;270(1-2):76–80. doi: 10.1016/0014-5793(90)81238-j. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Boyer P. L., Ferris A. L., Hughes S. H. Mutational analysis of the fingers domain of human immunodeficiency virus type 1 reverse transcriptase. J Virol. 1992 Dec;66(12):7533–7537. doi: 10.1128/jvi.66.12.7533-7537.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cirino N. M., Kalayjian R. C., Jentoft J. E., Le Grice S. F. Fluorimetric analysis of recombinant p15 HIV-1 ribonuclease H. J Biol Chem. 1993 Jul 15;268(20):14743–14749. [PubMed] [Google Scholar]
  7. Davies J. F., 2nd, Hostomska Z., Hostomsky Z., Jordan S. R., Matthews D. A. Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase. Science. 1991 Apr 5;252(5002):88–95. doi: 10.1126/science.1707186. [DOI] [PubMed] [Google Scholar]
  8. DeStefano J. J., Bambara R. A., Fay P. J. Parameters that influence the binding of human immunodeficiency virus reverse transcriptase to nucleic acid structures. Biochemistry. 1993 Jul 13;32(27):6908–6915. doi: 10.1021/bi00078a014. [DOI] [PubMed] [Google Scholar]
  9. Evans D. B., Brawn K., Deibel M. R., Jr, Tarpley W. G., Sharma S. K. A recombinant ribonuclease H domain of HIV-1 reverse transcriptase that is enzymatically active. J Biol Chem. 1991 Nov 5;266(31):20583–20585. [PubMed] [Google Scholar]
  10. Furfine E. S., Reardon J. E. Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNA(Lys3)-primer excision. Biochemistry. 1991 Jul 23;30(29):7041–7046. doi: 10.1021/bi00243a001. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Hoffmann A., Roeder R. G. Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res. 1991 Nov 25;19(22):6337–6338. doi: 10.1093/nar/19.22.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Jacobo-Molina A., Ding J., Nanni R. G., Clark A. D., Jr, Lu X., Tantillo C., Williams R. L., Kamer G., Ferris A. L., Clark P. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6320–6324. doi: 10.1073/pnas.90.13.6320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kanaya S., Katsuda-Nakai C., Ikehara M. Importance of the positive charge cluster in Escherichia coli ribonuclease HI for the effective binding of the substrate. J Biol Chem. 1991 Jun 25;266(18):11621–11627. [PubMed] [Google Scholar]
  17. Katayanagi K., Miyagawa M., Matsushima M., Ishikawa M., Kanaya S., Ikehara M., Matsuzaki T., Morikawa K. Three-dimensional structure of ribonuclease H from E. coli. Nature. 1990 Sep 20;347(6290):306–309. doi: 10.1038/347306a0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Krug M. S., Berger S. L. Reverse transcriptase from human immunodeficiency virus: a single template-primer binding site serves two physically separable catalytic functions. Biochemistry. 1991 Nov 5;30(44):10614–10623. doi: 10.1021/bi00108a003. [DOI] [PubMed] [Google Scholar]
  20. Kumar A., Kim H. R., Sobol R. W., Becerra S. P., Lee B. J., Hatfield D. L., Suhadolnik R. J., Wilson S. H. Mapping of nucleic acid binding in proteolytic domains of HIV-1 reverse transcriptase. Biochemistry. 1993 Jul 27;32(29):7466–7474. doi: 10.1021/bi00080a018. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Powers R., Clore G. M., Bax A., Garrett D. S., Stahl S. J., Wingfield P. T., Gronenborn A. M. Secondary structure of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase in solution using three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy. J Mol Biol. 1991 Oct 20;221(4):1081–1090. doi: 10.1016/0022-2836(91)90920-2. [DOI] [PubMed] [Google Scholar]
  23. Powers R., Clore G. M., Stahl S. J., Wingfield P. T., Gronenborn A. Analysis of the backbone dynamics of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase using 15N relaxation measurements. Biochemistry. 1992 Sep 29;31(38):9150–9157. doi: 10.1021/bi00153a006. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Pullen K. A., Ishimoto L. K., Champoux J. J. Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. J Virol. 1992 Jan;66(1):367–373. doi: 10.1128/jvi.66.1.367-373.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reardon J. E., Furfine E. S., Cheng N. Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding. J Biol Chem. 1991 Jul 25;266(21):14128–14134. [PubMed] [Google Scholar]
  27. Restle T., Müller B., Goody R. S. RNase H activity of HIV reverse transcriptases is confined exclusively to the dimeric forms. FEBS Lett. 1992 Mar 23;300(1):97–100. doi: 10.1016/0014-5793(92)80172-d. [DOI] [PubMed] [Google Scholar]
  28. Roth M. J., Tanese N., Goff S. P. Gene product of Moloney murine leukemia virus required for proviral integration is a DNA-binding protein. J Mol Biol. 1988 Sep 5;203(1):131–139. doi: 10.1016/0022-2836(88)90097-6. [DOI] [PubMed] [Google Scholar]
  29. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  30. Smith J. S., Roth M. J. Purification and characterization of an active human immunodeficiency virus type 1 RNase H domain. J Virol. 1993 Jul;67(7):4037–4049. doi: 10.1128/jvi.67.7.4037-4049.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smith J. S., Roth M. J. Specificity of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H in removal of the minus-strand primer, tRNA(Lys3). J Biol Chem. 1992 Jul 25;267(21):15071–15079. [PubMed] [Google Scholar]
  32. Sobol R. W., Suhadolnik R. J., Kumar A., Lee B. J., Hatfield D. L., Wilson S. H. Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding. Biochemistry. 1991 Nov 5;30(44):10623–10631. doi: 10.1021/bi00108a004. [DOI] [PubMed] [Google Scholar]
  33. Stammers D. K., Tisdale M., Court S., Parmar V., Bradley C., Ross C. K. Rapid purification and characterisation of HIV-1 reverse transcriptase and RNaseH engineered to incorporate a C-terminal tripeptide alpha-tubulin epitope. FEBS Lett. 1991 Jun 3;283(2):298–302. doi: 10.1016/0014-5793(91)80613-8. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Telesnitsky A., Blain S. W., Goff S. P. Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H. J Virol. 1992 Feb;66(2):615–622. doi: 10.1128/jvi.66.2.615-622.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Telesnitsky A., Goff S. P. RNase H domain mutations affect the interaction between Moloney murine leukemia virus reverse transcriptase and its primer-template. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1276–1280. doi: 10.1073/pnas.90.4.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Whitcomb J. M., Hughes S. H. Retroviral reverse transcription and integration: progress and problems. Annu Rev Cell Biol. 1992;8:275–306. doi: 10.1146/annurev.cb.08.110192.001423. [DOI] [PubMed] [Google Scholar]
  38. Yang W., Hendrickson W. A., Crouch R. J., Satow Y. Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein. Science. 1990 Sep 21;249(4975):1398–1405. doi: 10.1126/science.2169648. [DOI] [PubMed] [Google Scholar]
  39. 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