Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Sep 15;24(18):3568–3575. doi: 10.1093/nar/24.18.3568

Interaction of retroviral nucleocapsid proteins with transfer RNAPhe: a lead ribozyme and 1H NMR study.

R Khan 1, H O Chang 1, K Kaluarachchi 1, D P Giedroc 1
PMCID: PMC146139  PMID: 8836184

Abstract

In the initiation of reverse transcription in retroviruses, nucleocapsid (NC) protein accelerates the rate of annealing of transfer RNA replication primer to a complementary sequence on the genomic RNA. In this report, we have probed the conformational changes induced by HIV-1 NC protein and domain deletion mutants in a structurally well-characterized transfer RNA, yeast tRNAPhe, as a model for the natural primer. One molar equivalent of recombinant 71 amino acid HIV-1 nucleocapsid protein (NC 1-71) is sufficient to completely inhibit the Pb2(+)-ribozyme activity of tRNAPhe at 25 degrees C, pH 7.0 and 15 mM MgCl2, Zn2 HIV-1 NC proteins which lack one or both flexible terminal domains also inhibit the ribozyme activity. 1H NMR spectra acquired for Mg(2+)-tRNAPhe suggest that NC 1-71 and NC 12-55 (lacking residues 1-11 and 56-71) inhibit the lead-ribozyme activity by only modestly altering the active site region rather than inducing large-scale unfolding of the molecule. In the absence of Mg2+, the extent of destabilization of tRNAPhe is greater but appears to be confined to internal regions of the acceptor and T psi C helices, as evidenced by the selectively enhanced exchange rates for imino protons associated with these base pairs. These findings show that NC destabilizes the folded form of tRNAPhe and by extension, other complex RNAs, in tertiary and secondary structural regions most susceptible to thermally-induced denaturation.

Full Text

The Full Text of this article is available as a PDF (162.5 KB).

Selected References

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

  1. Allain B., Lapadat-Tapolsky M., Berlioz C., Darlix J. L. Transactivation of the minus-strand DNA transfer by nucleocapsid protein during reverse transcription of the retroviral genome. EMBO J. 1994 Feb 15;13(4):973–981. doi: 10.1002/j.1460-2075.1994.tb06342.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barat C., Lullien V., Schatz O., Keith G., Nugeyre M. T., Grüninger-Leitch F., Barré-Sinoussi F., LeGrice S. F., Darlix J. L. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J. 1989 Nov;8(11):3279–3285. doi: 10.1002/j.1460-2075.1989.tb08488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barat C., Schatz O., Le Grice S., Darlix J. L. Analysis of the interactions of HIV1 replication primer tRNA(Lys,3) with nucleocapsid protein and reverse transcriptase. J Mol Biol. 1993 May 20;231(2):185–190. doi: 10.1006/jmbi.1993.1273. [DOI] [PubMed] [Google Scholar]
  4. Behlen L. S., Sampson J. R., DiRenzo A. B., Uhlenbeck O. C. Lead-catalyzed cleavage of yeast tRNAPhe mutants. Biochemistry. 1990 Mar 13;29(10):2515–2523. doi: 10.1021/bi00462a013. [DOI] [PubMed] [Google Scholar]
  5. Bieth E., Gabus C., Darlix J. L. A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro. Nucleic Acids Res. 1990 Jan 11;18(1):119–127. doi: 10.1093/nar/18.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown R. S., Dewan J. C., Klug A. Crystallographic and biochemical investigation of the lead(II)-catalyzed hydrolysis of yeast phenylalanine tRNA. Biochemistry. 1985 Aug 27;24(18):4785–4801. doi: 10.1021/bi00339a012. [DOI] [PubMed] [Google Scholar]
  7. Cornille F., Mely Y., Ficheux D., Savignol I., Gerard D., Darlix J. L., Fournie-Zaluski M. C., Roques B. P. Solid phase synthesis of the retroviral nucleocapsid protein NCp10 of Moloney murine leukaemia virus and related "zinc-fingers" in free SH forms. Influence of zinc chelation on structural and biochemical properties. Int J Pept Protein Res. 1990 Dec;36(6):551–558. [PubMed] [Google Scholar]
  8. Dannull J., Surovoy A., Jung G., Moelling K. Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues. EMBO J. 1994 Apr 1;13(7):1525–1533. doi: 10.1002/j.1460-2075.1994.tb06414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Darlix J. L., Lapadat-Tapolsky M., de Rocquigny H., Roques B. P. First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. J Mol Biol. 1995 Dec 8;254(4):523–537. doi: 10.1006/jmbi.1995.0635. [DOI] [PubMed] [Google Scholar]
  10. De Rocquigny H., Ficheux D., Gabus C., Allain B., Fournie-Zaluski M. C., Darlix J. L., Roques B. P. Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity. Nucleic Acids Res. 1993 Feb 25;21(4):823–829. doi: 10.1093/nar/21.4.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. De Rocquigny H., Gabus C., Vincent A., Fournié-Zaluski M. C., Roques B., Darlix J. L. Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6472–6476. doi: 10.1073/pnas.89.14.6472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dib-Hajj F., Khan R., Giedroc D. P. Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity. Protein Sci. 1993 Feb;2(2):231–243. doi: 10.1002/pro.5560020212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Déméné H., Dong C. Z., Ottmann M., Rouyez M. C., Jullian N., Morellet N., Mely Y., Darlix J. L., Fournié-Zaluski M. C., Saragosti S. 1H NMR structure and biological studies of the His23-->Cys mutant nucleocapsid protein of HIV-1 indicate that the conformation of the first zinc finger is critical for virus infectivity. Biochemistry. 1994 Oct 4;33(39):11707–11716. doi: 10.1021/bi00205a006. [DOI] [PubMed] [Google Scholar]
  14. Förster C., Limmer S., Ribeiro S., Hilgenfeld R., Sprinzl M. Ternary complex between elongation factor Tu.GTP and Phe-tRNA(Phe). Biochimie. 1993;75(12):1159–1166. doi: 10.1016/0300-9084(93)90015-k. [DOI] [PubMed] [Google Scholar]
  15. Gelfand C. A., Wang Q., Randall S., Jentoft J. E. Interactions of avian myeloblastosis virus nucleocapsid protein with nucleic acids. J Biol Chem. 1993 Sep 5;268(25):18450–18456. [PubMed] [Google Scholar]
  16. Giedroc D. P., Khan R., Barnhart K. Site-specific 1,N6-ethenoadenylated single-stranded oligonucleotides as structural probes for the T4 gene 32 protein-ssDNA complex. Biochemistry. 1991 Aug 20;30(33):8230–8242. doi: 10.1021/bi00247a020. [DOI] [PubMed] [Google Scholar]
  17. Heerschap A., Haasnoot C. A., Hilbers C. W. Nuclear magnetic resonance studies on yeast tRNAPhe I. Assignment of the iminoproton resonances of the acceptor and D stem by means of Nuclear Overhauser Effect experiments at 500 MHz. Nucleic Acids Res. 1982 Nov 11;10(21):6981–7000. doi: 10.1093/nar/10.21.6981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heerschap A., Haasnoot C. A., Hilbers C. W. Nuclear magnetic resonance studies on yeast tRNAPhe. II. Assignment of the iminoproton resonances of the anticodon and T stem by means of nuclear Overhauser effect experiments at 500 MHz. Nucleic Acids Res. 1983 Jul 11;11(13):4483–4499. doi: 10.1093/nar/11.13.4483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heerschap A., Haasnoot C. A., Hilbers C. W. Nuclear magnetic resonance studies on yeast tRNAPhe. III. Assignments of the iminoproton resonances of the tertiary structure by means of nuclear Overhauser effect experiments at 500 MHz. Nucleic Acids Res. 1983 Jul 11;11(13):4501–4520. doi: 10.1093/nar/11.13.4501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heerschap A., Mellema J. R., Janssen H. G., Walters J. A., Haasnoot C. A., Hilbers C. W. Imino-proton resonances of yeast tRNAPhe studied by two-dimensional nuclear Overhauser enhancement spectroscopy. Eur J Biochem. 1985 Jun 18;149(3):649–655. doi: 10.1111/j.1432-1033.1985.tb08973.x. [DOI] [PubMed] [Google Scholar]
  21. Heerschap A., Walters J. A., Hilbers C. W. Influence of the polyamines spermine and spermidine on yeast tRNAPhe as revealed from its imino proton NMR spectrum. Nucleic Acids Res. 1986 Jan 24;14(2):983–998. doi: 10.1093/nar/14.2.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heerschap A., Walters J. A., Mellema J. R., Hilbers C. W. Study of the interaction between uncharged yeast tRNAPhe and elongation factor Tu from Bacillus stearothermophilis. Biochemistry. 1986 May 6;25(9):2707–2713. doi: 10.1021/bi00357a064. [DOI] [PubMed] [Google Scholar]
  23. Herschlag D. RNA chaperones and the RNA folding problem. J Biol Chem. 1995 Sep 8;270(36):20871–20874. doi: 10.1074/jbc.270.36.20871. [DOI] [PubMed] [Google Scholar]
  24. Johnston P. D., Redfield A. G. Study of transfer ribonucleic acid unfolding by dynamic nuclear magnetic resonance. Biochemistry. 1981 Jul 7;20(14):3996–4006. doi: 10.1021/bi00517a008. [DOI] [PubMed] [Google Scholar]
  25. Karpel R. L., Henderson L. E., Oroszlan S. Interactions of retroviral structural proteins with single-stranded nucleic acids. J Biol Chem. 1987 Apr 15;262(11):4961–4967. [PubMed] [Google Scholar]
  26. Khan R., Giedroc D. P. Nucleic acid binding properties of recombinant Zn2 HIV-1 nucleocapsid protein are modulated by COOH-terminal processing. J Biol Chem. 1994 Sep 9;269(36):22538–22546. [PubMed] [Google Scholar]
  27. Khan R., Giedroc D. P. Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA. J Biol Chem. 1992 Apr 5;267(10):6689–6695. [PubMed] [Google Scholar]
  28. Kim S. H. Three-dimensional structure of transfer RNA and its functional implications. Adv Enzymol Relat Areas Mol Biol. 1978;46:279–315. doi: 10.1002/9780470122914.ch4. [DOI] [PubMed] [Google Scholar]
  29. Lapadat-Tapolsky M., De Rocquigny H., Van Gent D., Roques B., Plasterk R., Darlix J. L. Interactions between HIV-1 nucleocapsid protein and viral DNA may have important functions in the viral life cycle. Nucleic Acids Res. 1993 Feb 25;21(4):831–839. doi: 10.1093/nar/21.4.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lapadat-Tapolsky M., Pernelle C., Borie C., Darlix J. L. Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1. Nucleic Acids Res. 1995 Jul 11;23(13):2434–2441. doi: 10.1093/nar/23.13.2434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morellet N., Jullian N., De Rocquigny H., Maigret B., Darlix J. L., Roques B. P. Determination of the structure of the nucleocapsid protein NCp7 from the human immunodeficiency virus type 1 by 1H NMR. EMBO J. 1992 Aug;11(8):3059–3065. doi: 10.1002/j.1460-2075.1992.tb05377.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mély Y., de Rocquigny H., Sorinas-Jimeno M., Keith G., Roques B. P., Marquet R., Gérard D. Binding of the HIV-1 nucleocapsid protein to the primer tRNA(3Lys), in vitro, is essentially not specific. J Biol Chem. 1995 Jan 27;270(4):1650–1656. doi: 10.1074/jbc.270.4.1650. [DOI] [PubMed] [Google Scholar]
  33. Omichinski J. G., Clore G. M., Sakaguchi K., Appella E., Gronenborn A. M. Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy. FEBS Lett. 1991 Nov 4;292(1-2):25–30. doi: 10.1016/0014-5793(91)80825-n. [DOI] [PubMed] [Google Scholar]
  34. Pan T., Gutell R. R., Uhlenbeck O. C. Folding of circularly permuted transfer RNAs. Science. 1991 Nov 29;254(5036):1361–1364. doi: 10.1126/science.1720569. [DOI] [PubMed] [Google Scholar]
  35. Pan T., Uhlenbeck O. C. In vitro selection of RNAs that undergo autolytic cleavage with Pb2+. Biochemistry. 1992 Apr 28;31(16):3887–3895. doi: 10.1021/bi00131a001. [DOI] [PubMed] [Google Scholar]
  36. Peliska J. A., Balasubramanian S., Giedroc D. P., Benkovic S. J. Recombinant HIV-1 nucleocapsid protein accelerates HIV-1 reverse transcriptase catalyzed DNA strand transfer reactions and modulates RNase H activity. Biochemistry. 1994 Nov 22;33(46):13817–13823. doi: 10.1021/bi00250a036. [DOI] [PubMed] [Google Scholar]
  37. Prats A. C., Housset V., de Billy G., Cornille F., Prats H., Roques B., Darlix J. L. Viral RNA annealing activities of the nucleocapsid protein of Moloney murine leukemia virus are zinc independent. Nucleic Acids Res. 1991 Jul 11;19(13):3533–3541. doi: 10.1093/nar/19.13.3533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Prats A. C., Sarih L., Gabus C., Litvak S., Keith G., Darlix J. L. Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO J. 1988 Jun;7(6):1777–1783. doi: 10.1002/j.1460-2075.1988.tb03008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Priel E., Aflalo E., Seri I., Henderson L. E., Arthur L. O., Aboud M., Segal S., Blair D. G. DNA binding properties of the zinc-bound and zinc-free HIV nucleocapsid protein: supercoiled DNA unwinding and DNA-protein cleavable complex formation. FEBS Lett. 1995 Mar 27;362(1):59–64. doi: 10.1016/0014-5793(95)00208-q. [DOI] [PubMed] [Google Scholar]
  40. Privalov P. L., Filimonov V. V. Thermodynamic analysis of transfer RNA unfolding. J Mol Biol. 1978 Jul 15;122(4):447–464. doi: 10.1016/0022-2836(78)90421-7. [DOI] [PubMed] [Google Scholar]
  41. Qiu H., Kodadek T., Giedroc D. P. Zinc-free and reduced T4 gene 32 protein binds single-stranded DNA weakly and fails to stimulate UvsX-catalyzed homologous pairing. J Biol Chem. 1994 Jan 28;269(4):2773–2781. [PubMed] [Google Scholar]
  42. Robillard G. T., Tarr C. E., Vosman F., Reid B. R. A nuclear magnetic resonance study of secondary and tertiary structure in yeast tRNAPhe. Biochemistry. 1977 Nov 29;16(24):5261–5273. doi: 10.1021/bi00643a016. [DOI] [PubMed] [Google Scholar]
  43. Roy S., Redfield A. G. Assignment of imino proton spectra of yeast phenylalanine transfer ribonucleic acid. Biochemistry. 1983 Mar 15;22(6):1386–1390. doi: 10.1021/bi00275a010. [DOI] [PubMed] [Google Scholar]
  44. Schmalzbauer E., Strack B., Dannull J., Guehmann S., Moelling K. Mutations of basic amino acids of NCp7 of human immunodeficiency virus type 1 affect RNA binding in vitro. J Virol. 1996 Feb;70(2):771–777. doi: 10.1128/jvi.70.2.771-777.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. South T. L., Summers M. F. Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR structure of the complex with the Psi-site analog, dACGCC. Protein Sci. 1993 Jan;2(1):3–19. doi: 10.1002/pro.5560020102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Summers M. F., Henderson L. E., Chance M. R., Bess J. W., Jr, South T. L., Blake P. R., Sagi I., Perez-Alvarado G., Sowder R. C., 3rd, Hare D. R. Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1. Protein Sci. 1992 May;1(5):563–574. doi: 10.1002/pro.5560010502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tsuchihashi Z., Brown P. O. DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein. J Virol. 1994 Sep;68(9):5863–5870. doi: 10.1128/jvi.68.9.5863-5870.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tsuchihashi Z., Khosla M., Herschlag D. Protein enhancement of hammerhead ribozyme catalysis. Science. 1993 Oct 1;262(5130):99–102. doi: 10.1126/science.7692597. [DOI] [PubMed] [Google Scholar]
  49. You J. C., McHenry C. S. Human immunodeficiency virus nucleocapsid protein accelerates strand transfer of the terminally redundant sequences involved in reverse transcription. J Biol Chem. 1994 Dec 16;269(50):31491–31495. [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES