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. 1992 Aug;11(8):3059–3065. doi: 10.1002/j.1460-2075.1992.tb05377.x

Determination of the structure of the nucleocapsid protein NCp7 from the human immunodeficiency virus type 1 by 1H NMR.

N Morellet 1, N Jullian 1, H De Rocquigny 1, B Maigret 1, J L Darlix 1, B P Roques 1
PMCID: PMC556789  PMID: 1639074

Abstract

The retroviral gag nucleocapsid protein NCp7 (72 amino acids) of HIV-1 (LAV strain), which contains two successive zinc fingers of the Cys-X2-Cys-X4-His-X4-Cys form linked by a stretch of basic residues, promotes viral RNA dimerization and encapsidation and activates annealing of the primer tRNA to the initiation site of reverse transcription. The structure of NCp7 and other shorter fragments was studied by 600 MHz 1H nuclear magnetic resonance (NMR) in aqueous solution to account for its various biological properties. Complete sequence specific 1H NMR assignments of the 13-51 residues of NCp7 encompassing the two zinc fingers was achieved by two-dimensional NMR experiments and the three-dimensional structure of (13-51)NCp7 was deduced from DIANA calculations, using nuclear Overhauser effects as constraints. The structure of the zinc complexed form of NCp7 is characterized by a kink at the Pro31 level in the basic Arg29-Ala-Pro-Arg-Lys-Lys-Gly35 RNA binding linker leading to a proximity of the Lys14-Cys18 to the Gly35-Cys39 sequences, which belong to the folded proximal and distal zinc fingers, respectively. Accordingly, the aromatic residues Phe16 and Trp37 were found to be spatially close. The Lys33 and Lys34 side-chains involved in viral RNA dimerization were solvent exposed. The N- and C-terminal sequences of NCp7 behave as flexible independent domains. The proposed structure of NCp7 might be used to rationally design new anti-viral agents aimed at inhibiting its functions.

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Selected References

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

  1. Aldovini A., Young R. A. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990 May;64(5):1920–1926. doi: 10.1128/jvi.64.5.1920-1926.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arseniev A., Schultze P., Wörgötter E., Braun W., Wagner G., Vasák M., Kägi J. H., Wüthrich K. Three-dimensional structure of rabbit liver [Cd7]metallothionein-2a in aqueous solution determined by nuclear magnetic resonance. J Mol Biol. 1988 Jun 5;201(3):637–657. doi: 10.1016/0022-2836(88)90644-4. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Braun W., Go N. Calculation of protein conformations by proton-proton distance constraints. A new efficient algorithm. J Mol Biol. 1985 Dec 5;186(3):611–626. doi: 10.1016/0022-2836(85)90134-2. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Covey S. N. Amino acid sequence homology in gag region of reverse transcribing elements and the coat protein gene of cauliflower mosaic virus. Nucleic Acids Res. 1986 Jan 24;14(2):623–633. doi: 10.1093/nar/14.2.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Darlix J. L., Gabus C., Nugeyre M. T., Clavel F., Barré-Sinoussi F. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1. J Mol Biol. 1990 Dec 5;216(3):689–699. doi: 10.1016/0022-2836(90)90392-Y. [DOI] [PubMed] [Google Scholar]
  11. Dupraz P., Oertle S., Meric C., Damay P., Spahr P. F. Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein. J Virol. 1990 Oct;64(10):4978–4987. doi: 10.1128/jvi.64.10.4978-4987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fitzgerald D. W., Coleman J. E. Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions. Biochemistry. 1991 May 28;30(21):5195–5201. doi: 10.1021/bi00235a012. [DOI] [PubMed] [Google Scholar]
  13. Gorelick R. J., Henderson L. E., Hanser J. P., Rein A. Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8420–8424. doi: 10.1073/pnas.85.22.8420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Green L. M., Berg J. M. A retroviral Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys peptide binds metal ions: spectroscopic studies and a proposed three-dimensional structure. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4047–4051. doi: 10.1073/pnas.86.11.4047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Green L. M., Berg J. M. Retroviral nucleocapsid protein-metal ion interactions: folding and sequence variants. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6403–6407. doi: 10.1073/pnas.87.16.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Güntert P., Braun W., Wüthrich K. Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA. J Mol Biol. 1991 Feb 5;217(3):517–530. doi: 10.1016/0022-2836(91)90754-t. [DOI] [PubMed] [Google Scholar]
  17. Kochoyan M., Havel T. F., Nguyen D. T., Dahl C. E., Keutmann H. T., Weiss M. A. Alternating zinc fingers in the human male associated protein ZFY: 2D NMR structure of an even finger and implications for "jumping-linker" DNA recognition. Biochemistry. 1991 Apr 9;30(14):3371–3386. doi: 10.1021/bi00228a004. [DOI] [PubMed] [Google Scholar]
  18. Kumar A., Ernst R. R., Wüthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980 Jul 16;95(1):1–6. doi: 10.1016/0006-291x(80)90695-6. [DOI] [PubMed] [Google Scholar]
  19. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  20. Méric C., Goff S. P. Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein. J Virol. 1989 Apr;63(4):1558–1568. doi: 10.1128/jvi.63.4.1558-1568.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Méric C., Spahr P. F. Rous sarcoma virus nucleic acid-binding protein p12 is necessary for viral 70S RNA dimer formation and packaging. J Virol. 1986 Nov;60(2):450–459. doi: 10.1128/jvi.60.2.450-459.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Prats A. C., Roy C., Wang P. A., Erard M., Housset V., Gabus C., Paoletti C., Darlix J. L. cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA. J Virol. 1990 Feb;64(2):774–783. doi: 10.1128/jvi.64.2.774-783.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Rance M., Sørensen O. W., Bodenhausen G., Wagner G., Ernst R. R., Wüthrich K. Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. Biochem Biophys Res Commun. 1983 Dec 16;117(2):479–485. doi: 10.1016/0006-291x(83)91225-1. [DOI] [PubMed] [Google Scholar]
  27. Roberts M. M., Copeland T. D., Oroszlan S. In situ processing of a retroviral nucleocapsid protein by the viral proteinase. Protein Eng. 1991 Aug;4(6):695–700. doi: 10.1093/protein/4.6.695. [DOI] [PubMed] [Google Scholar]
  28. South T. L., Blake P. R., Hare D. R., Summers M. F. C-terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain. Biochemistry. 1991 Jun 25;30(25):6342–6349. doi: 10.1021/bi00239a036. [DOI] [PubMed] [Google Scholar]
  29. South T. L., Blake P. R., Sowder R. C., 3rd, Arthur L. O., Henderson L. E., Summers M. F. The nucleocapsid protein isolated from HIV-1 particles binds zinc and forms retroviral-type zinc fingers. Biochemistry. 1990 Aug 28;29(34):7786–7789. doi: 10.1021/bi00486a002. [DOI] [PubMed] [Google Scholar]
  30. Summers M. F., South T. L., Kim B., Hare D. R. High-resolution structure of an HIV zinc fingerlike domain via a new NMR-based distance geometry approach. Biochemistry. 1990 Jan 16;29(2):329–340. doi: 10.1021/bi00454a005. [DOI] [PubMed] [Google Scholar]
  31. Veronese F. D., Rahman R., Copeland T. D., Oroszlan S., Gallo R. C., Sarngadharan M. G. Immunological and chemical analysis of P6, the carboxyl-terminal fragment of HIV P15. AIDS Res Hum Retroviruses. 1987 Fall;3(3):253–264. doi: 10.1089/aid.1987.3.253. [DOI] [PubMed] [Google Scholar]
  32. Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell. 1985 Jan;40(1):9–17. doi: 10.1016/0092-8674(85)90303-4. [DOI] [PubMed] [Google Scholar]
  33. Williamson M. P., Havel T. F., Wüthrich K. Solution conformation of proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry. J Mol Biol. 1985 Mar 20;182(2):295–315. doi: 10.1016/0022-2836(85)90347-x. [DOI] [PubMed] [Google Scholar]
  34. Wüthrich K., Billeter M., Braun W. Pseudo-structures for the 20 common amino acids for use in studies of protein conformations by measurements of intramolecular proton-proton distance constraints with nuclear magnetic resonance. J Mol Biol. 1983 Oct 5;169(4):949–961. doi: 10.1016/s0022-2836(83)80144-2. [DOI] [PubMed] [Google Scholar]
  35. de Rocquigny H., Ficheux D., Gabus C., Fournié-Zaluski M. C., Darlix J. L., Roques B. P. First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form. Biochem Biophys Res Commun. 1991 Oct 31;180(2):1010–1018. doi: 10.1016/s0006-291x(05)81166-0. [DOI] [PubMed] [Google Scholar]

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