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. 1992 May;1(5):563–574. doi: 10.1002/pro.5560010502

Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1.

M F Summers 1, L E Henderson 1, M R Chance 1, J W Bess Jr 1, T L South 1, P R Blake 1, I Sagi 1, G Perez-Alvarado 1, R C Sowder 3rd 1, D R Hare 1, et al.
PMCID: PMC2142235  PMID: 1304355

Abstract

All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys-1His array (CCHC = C-X2-C-X4-H-X4-C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc-binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc-edge extended X-ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus-bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840-847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys-1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV-1 NC protein and have determined its three-dimensional solution-state structure. The CCHC arrays of HIV-1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV-1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid-binding results provide a basis for the rational design of new NC-targeted antiviral agents and vaccines for the control of AIDS.

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Selected References

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  1. 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]
  2. Bess J. W., Jr, Powell P. J., Issaq H. J., Schumack L. J., Grimes M. K., Henderson L. E., Arthur L. O. Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses. J Virol. 1992 Feb;66(2):840–847. doi: 10.1128/jvi.66.2.840-847.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolognesi D. P., Montelaro R. C., Frank H., Schäfer W. Assembly of type C oncornaviruses: a model. Science. 1978 Jan 13;199(4325):183–186. doi: 10.1126/science.202022. [DOI] [PubMed] [Google Scholar]
  4. Chen Z. G., Stauffacher C., Li Y., Schmidt T., Bomu W., Kamer G., Shanks M., Lomonossoff G., Johnson J. E. Protein-RNA interactions in an icosahedral virus at 3.0 A resolution. Science. 1989 Jul 14;245(4914):154–159. doi: 10.1126/science.2749253. [DOI] [PubMed] [Google Scholar]
  5. Copeland T. D., Morgan M. A., Oroszlan S. Complete amino acid sequence of the basic nucleic acid binding protein of feline leukemia virus. Virology. 1984 Feb;133(1):137–145. doi: 10.1016/0042-6822(84)90432-x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  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. 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]
  9. 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]
  10. Gadhavi P. L., Davis A. L., Povey J. F., Keeler J., Laue E. D. Polypeptide-metal cluster connectivities in Cd(II) GAL4. FEBS Lett. 1991 Apr 9;281(1-2):223–226. doi: 10.1016/0014-5793(91)80398-m. [DOI] [PubMed] [Google Scholar]
  11. Gadhavi P. L., Raine A. R., Alefounder P. R., Laue E. D. Complete assignment of the 1H NMR spectrum and secondary structure of the DNA binding domain of GAL4. FEBS Lett. 1990 Dec 10;276(1-2):49–53. doi: 10.1016/0014-5793(90)80504-c. [DOI] [PubMed] [Google Scholar]
  12. Gardner K. H., Pan T., Narula S., Rivera E., Coleman J. E. Structure of the binuclear metal-binding site in the GAL4 transcription factor. Biochemistry. 1991 Nov 26;30(47):11292–11302. doi: 10.1021/bi00111a015. [DOI] [PubMed] [Google Scholar]
  13. Giedroc D. P., Giu H. W., Khan R., King G. C., Chen K. Zn(II) coordination domain mutants of T4 gene 32 protein. Biochemistry. 1992 Jan 28;31(3):765–774. doi: 10.1021/bi00118a018. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Gorelick R. J., Nigida S. M., Jr, Bess J. W., Jr, Arthur L. O., Henderson L. E., Rein A. Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA. J Virol. 1990 Jul;64(7):3207–3211. doi: 10.1128/jvi.64.7.3207-3211.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Gronenborn A. M., Filpula D. R., Essig N. Z., Achari A., Whitlow M., Wingfield P. T., Clore G. M. A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science. 1991 Aug 9;253(5020):657–661. doi: 10.1126/science.1871600. [DOI] [PubMed] [Google Scholar]
  19. Henderson L. E., Bowers M. A., Sowder R. C., 2nd, Serabyn S. A., Johnson D. G., Bess J. W., Jr, Arthur L. O., Bryant D. K., Fenselau C. Gag proteins of the highly replicative MN strain of human immunodeficiency virus type 1: posttranslational modifications, proteolytic processings, and complete amino acid sequences. J Virol. 1992 Apr;66(4):1856–1865. doi: 10.1128/jvi.66.4.1856-1865.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Henderson L. E., Copeland T. D., Sowder R. C., Smythers G. W., Oroszlan S. Primary structure of the low molecular weight nucleic acid-binding proteins of murine leukemia viruses. J Biol Chem. 1981 Aug 25;256(16):8400–8406. [PubMed] [Google Scholar]
  21. Henderson L. E., Sowder R. C., Copeland T. D., Oroszlan S., Benveniste R. E. Gag precursors of HIV and SIV are cleaved into six proteins found in the mature virions. J Med Primatol. 1990;19(3-4):411–419. [PubMed] [Google Scholar]
  22. Härd T., Kellenbach E., Boelens R., Maler B. A., Dahlman K., Freedman L. P., Carlstedt-Duke J., Yamamoto K. R., Gustafsson J. A., Kaptein R. Solution structure of the glucocorticoid receptor DNA-binding domain. Science. 1990 Jul 13;249(4965):157–160. doi: 10.1126/science.2115209. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Katz R. A., Jentoft J. E. What is the role of the cys-his motif in retroviral nucleocapsid (NC) proteins? Bioessays. 1989 Dec;11(6):176–181. doi: 10.1002/bies.950110605. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Lee M. S., Gippert G. P., Soman K. V., Case D. A., Wright P. E. Three-dimensional solution structure of a single zinc finger DNA-binding domain. Science. 1989 Aug 11;245(4918):635–637. doi: 10.1126/science.2503871. [DOI] [PubMed] [Google Scholar]
  27. Méric C., Darlix J. L., Spahr P. F. It is Rous sarcoma virus protein P12 and not P19 that binds tightly to Rous sarcoma virus RNA. J Mol Biol. 1984 Mar 15;173(4):531–538. doi: 10.1016/0022-2836(84)90396-6. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Navia M. A., Fitzgerald P. M., McKeever B. M., Leu C. T., Heimbach J. C., Herber W. K., Sigal I. S., Darke P. L., Springer J. P. Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1. Nature. 1989 Feb 16;337(6208):615–620. doi: 10.1038/337615a0. [DOI] [PubMed] [Google Scholar]
  30. Omichinski J. G., Clore G. M., Appella E., Sakaguchi K., Gronenborn A. M. High-resolution three-dimensional structure of a single zinc finger from a human enhancer binding protein in solution. Biochemistry. 1990 Oct 9;29(40):9324–9334. doi: 10.1021/bi00492a004. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Párraga G., Horvath S. J., Eisen A., Taylor W. E., Hood L., Young E. T., Klevit R. E. Zinc-dependent structure of a single-finger domain of yeast ADR1. Science. 1988 Sep 16;241(4872):1489–1492. doi: 10.1126/science.3047872. [DOI] [PubMed] [Google Scholar]
  33. Rajavashisth T. B., Taylor A. K., Andalibi A., Svenson K. L., Lusis A. J. Identification of a zinc finger protein that binds to the sterol regulatory element. Science. 1989 Aug 11;245(4918):640–643. doi: 10.1126/science.2562787. [DOI] [PubMed] [Google Scholar]
  34. Roberts W. J., Pan T., Elliott J. I., Coleman J. E., Williams K. R. p10 single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39. Biochemistry. 1989 Dec 26;28(26):10043–10047. doi: 10.1021/bi00452a024. [DOI] [PubMed] [Google Scholar]
  35. Sato S. M., Sargent T. D. Localized and inducible expression of Xenopus-posterior (Xpo), a novel gene active in early frog embryos, encoding a protein with a 'CCHC' finger domain. Development. 1991 Jul;112(3):747–753. doi: 10.1242/dev.112.3.747. [DOI] [PubMed] [Google Scholar]
  36. Schiff L. A., Nibert M. L., Fields B. N. Characterization of a zinc blotting technique: evidence that a retroviral gag protein binds zinc. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4195–4199. doi: 10.1073/pnas.85.12.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith B. J., Bailey J. M. The binding of an avian myeloblastosis virus basic 12,000 dalton protein to nucleic acids. Nucleic Acids Res. 1979 Dec 11;7(7):2055–2072. doi: 10.1093/nar/7.7.2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. Summers M. F. Zinc finger motif for single-stranded nucleic acids? Investigations by nuclear magnetic resonance. J Cell Biochem. 1991 Jan;45(1):41–48. doi: 10.1002/jcb.240450110. [DOI] [PubMed] [Google Scholar]
  42. Wlodawer A., Miller M., Jaskólski M., Sathyanarayana B. K., Baldwin E., Weber I. T., Selk L. M., Clawson L., Schneider J., Kent S. B. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science. 1989 Aug 11;245(4918):616–621. doi: 10.1126/science.2548279. [DOI] [PubMed] [Google Scholar]

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