Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1995 Aug;69(8):5113–5116. doi: 10.1128/jvi.69.8.5113-5116.1995

Reversion of a Moloney murine leukemia virus RNase H mutant at a second site restores enzyme function and infectivity.

S W Blain 1, W A Hendrickson 1, S P Goff 1
PMCID: PMC189329  PMID: 7541847

Abstract

The reverse transcriptase of retroviruses contains an RNase H activity essential for the proper synthesis of the viral DNA copy of the RNA genome. We have previously characterized a number of point mutations altering the RNase domain of the Moloney murine leukemia virus reverse transcriptase (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). One such mutation, Y586F (a Y-to-F change at position 586), reduced RNase H activity, as assayed by in situ gel analysis, to about 5% of the wild-type level and prevented viral replication. We have now recovered a revertant virus with near-normal infectivity and in vitro enzymatic activity. The revertant contains a single substitution, N613H, distant in the primary sequence of the protein, but modeling with the Escherichia coli RNase H structure suggests that the reverted residue is close in space to the original substituted residue. Examination of the structure permits some suggestions as to how this second-site revertant restores enzyme activity.

Full Text

The Full Text of this article is available as a PDF (494.5 KB).

Selected References

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

  1. Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970 Jun 27;226(5252):1209–1211. doi: 10.1038/2261209a0. [DOI] [PubMed] [Google Scholar]
  2. Ben-Artzi H., Zeelon E., Gorecki M., Panet A. Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):927–931. doi: 10.1073/pnas.89.3.927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blain S. W., Goff S. P. Effects on DNA synthesis and translocation caused by mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase. J Virol. 1995 Jul;69(7):4440–4452. doi: 10.1128/jvi.69.7.4440-4452.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blain S. W., Goff S. P. Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities. J Biol Chem. 1993 Nov 5;268(31):23585–23592. [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. Colicelli J., Goff S. P. Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988 Jan 5;199(1):47–59. doi: 10.1016/0022-2836(88)90378-6. [DOI] [PubMed] [Google Scholar]
  7. Crouch R. J. Ribonuclease H: from discovery to 3D structure. New Biol. 1990 Sep;2(9):771–777. [PubMed] [Google Scholar]
  8. 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]
  9. Dudding L. R., Mizrahi V. Rapid kinetic analysis of a point mutant of HIV-1 reverse transcriptase lacking ribonuclease H activity. Biochemistry. 1993 Jun 15;32(23):6116–6120. doi: 10.1021/bi00074a025. [DOI] [PubMed] [Google Scholar]
  10. Fedoroff OYu, Salazar M., Reid B. R. Structure of a DNA:RNA hybrid duplex. Why RNase H does not cleave pure RNA. J Mol Biol. 1993 Oct 5;233(3):509–523. doi: 10.1006/jmbi.1993.1528. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  13. Hostomsky Z., Hudson G. O., Rahmati S., Hostomska Z. RNase D, a reported new activity associated with HIV-1 reverse transcriptase, displays the same cleavage specificity as Escherichia coli RNase III. Nucleic Acids Res. 1992 Nov 11;20(21):5819–5824. doi: 10.1093/nar/20.21.5819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hostomsky Z., Hughes S. H., Goff S. P., Le Grice S. F. Redesignation of the RNase D activity associated with retroviral reverse transcriptase as RNase H. J Virol. 1994 Mar;68(3):1970–1971. doi: 10.1128/jvi.68.3.1970-1971.1994. [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. Kanaya S., Katsuda C., Kimura S., Nakai T., Kitakuni E., Nakamura H., Katayanagi K., Morikawa K., Ikehara M. Stabilization of Escherichia coli ribonuclease H by introduction of an artificial disulfide bond. J Biol Chem. 1991 Apr 5;266(10):6038–6044. [PubMed] [Google Scholar]
  18. Kanaya S., Kohara A., Miura Y., Sekiguchi A., Iwai S., Inoue H., Ohtsuka E., Ikehara M. Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis. J Biol Chem. 1990 Mar 15;265(8):4615–4621. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
  22. Mizrahi V., Brooksbank R. L., Nkabinde N. C. Mutagenesis of the conserved aspartic acid 443, glutamic acid 478, asparagine 494, and aspartic acid 498 residues in the ribonuclease H domain of p66/p51 human immunodeficiency virus type I reverse transcriptase. Expression and biochemical analysis. J Biol Chem. 1994 Jul 29;269(30):19245–19249. [PubMed] [Google Scholar]
  23. Nakamura H., Katayanagi K., Morikawa K., Ikehara M. Structural models of ribonuclease H domains in reverse transcriptases from retroviruses. Nucleic Acids Res. 1991 Apr 25;19(8):1817–1823. doi: 10.1093/nar/19.8.1817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nakamura H., Oda Y., Iwai S., Inoue H., Ohtsuka E., Kanaya S., Kimura S., Katsuda C., Katayanagi K., Morikawa K. How does RNase H recognize a DNA.RNA hybrid? Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11535–11539. doi: 10.1073/pnas.88.24.11535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Salazar M., Fedoroff O. Y., Miller J. M., Ribeiro N. S., Reid B. R. The DNA strand in DNA.RNA hybrid duplexes is neither B-form nor A-form in solution. Biochemistry. 1993 Apr 27;32(16):4207–4215. doi: 10.1021/bi00067a007. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Tanese N., Telesnitsky A., Goff S. P. Abortive reverse transcription by mutants of Moloney murine leukemia virus deficient in the reverse transcriptase-associated RNase H function. J Virol. 1991 Aug;65(8):4387–4397. doi: 10.1128/jvi.65.8.4387-4397.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Temin H. M., Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature. 1970 Jun 27;226(5252):1211–1213. doi: 10.1038/2261211a0. [DOI] [PubMed] [Google Scholar]
  30. Volkmann S., Wöhrl B. M., Tisdale M., Moelling K. Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleases H. J Biol Chem. 1993 Feb 5;268(4):2674–2683. [PubMed] [Google Scholar]
  31. Wintersberger U. Ribonucleases H of retroviral and cellular origin. Pharmacol Ther. 1990;48(2):259–280. doi: 10.1016/0163-7258(90)90083-e. [DOI] [PubMed] [Google Scholar]
  32. 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]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES