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. 1993 Feb;2(2):231–243. doi: 10.1002/pro.5560020212

Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity.

F Dib-Hajj 1, R Khan 1, D P Giedroc 1
PMCID: PMC2142354  PMID: 8443601

Abstract

The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason-Pfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC-polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 degrees C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 degrees C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer.

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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. 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]
  4. Bujalowski W., Overman L. B., Lohman T. M. Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes. Cation, anion, pH, and binding density effects. J Biol Chem. 1988 Apr 5;263(10):4629–4640. [PubMed] [Google Scholar]
  5. Chen M., Garon C. F., Papas T. S. Native ribonucleoprotein is an efficient transcriptional complex of avian myeloblastosis virus. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1296–1300. doi: 10.1073/pnas.77.3.1296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Christiansen C., Baldwin R. L. Catalysis of DNA reassociation by the Escherichia coli DNA binding protein: A polyamine-dependent reaction. J Mol Biol. 1977 Sep 25;115(3):441–454. doi: 10.1016/0022-2836(77)90164-4. [DOI] [PubMed] [Google Scholar]
  7. Churchill M. E., Travers A. A. Protein motifs that recognize structural features of DNA. Trends Biochem Sci. 1991 Mar;16(3):92–97. doi: 10.1016/0968-0004(91)90040-3. [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. Cox M. M., Lehman I. R. Renaturation of DNA: a novel reaction of histones. Nucleic Acids Res. 1981 Jan 24;9(2):389–400. doi: 10.1093/nar/9.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Davis J., Scherer M., Tsai W. P., Long C. Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA. J Virol. 1976 May;18(2):709–718. doi: 10.1128/jvi.18.2.709-718.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Delahunty M. D., South T. L., Summers M. F., Karpel R. L. Nucleic acid interactive properties of a peptide corresponding to the N-terminal zinc finger domain of HIV-1 nucleocapsid protein. Biochemistry. 1992 Jul 21;31(28):6461–6469. doi: 10.1021/bi00143a015. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Fu X. D., Katz R. A., Skalka A. M., Leis J. Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12. Mutation which affects RNA binding in vitro blocks viral replication. J Biol Chem. 1988 Feb 15;263(5):2140–2145. [PubMed] [Google Scholar]
  15. 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]
  16. Griffith J. D., Harris L. D., Register J., 3rd Visualization of SSB-ssDNA complexes active in the assembly of stable RecA-DNA filaments. Cold Spring Harb Symp Quant Biol. 1984;49:553–559. doi: 10.1101/sqb.1984.049.01.062. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Henderson L. E., Sowder R. C., Copeland T. D., Benveniste R. E., Oroszlan S. Isolation and characterization of a novel protein (X-ORF product) from SIV and HIV-2. Science. 1988 Jul 8;241(4862):199–201. doi: 10.1126/science.3388031. [DOI] [PubMed] [Google Scholar]
  19. Henderson L. E., Sowder R., Smythers G., Benveniste R. E., Oroszlan S. Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus. J Virol. 1985 Sep;55(3):778–787. doi: 10.1128/jvi.55.3.778-787.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karpel R. L., Burchard A. C. Physical studies of the interaction of a calf thymus helix-destablizing protein with nucleic acids. Biochemistry. 1980 Sep 30;19(20):4674–4682. doi: 10.1021/bi00561a021. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Karpel R. L., Miller N. S., Fresco J. R. Mechanistic studies of ribonucleic acid renaturation by a helix-destabilizing protein. Biochemistry. 1982 Apr 27;21(9):2102–2108. doi: 10.1021/bi00538a019. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Leis J., Baltimore D., Bishop J. M., Coffin J., Fleissner E., Goff S. P., Oroszlan S., Robinson H., Skalka A. M., Temin H. M. Standardized and simplified nomenclature for proteins common to all retroviruses. J Virol. 1988 May;62(5):1808–1809. doi: 10.1128/jvi.62.5.1808-1809.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lohman T. M., Overman L. B., Datta S. Salt-dependent changes in the DNA binding co-operativity of Escherichia coli single strand binding protein. J Mol Biol. 1986 Feb 20;187(4):603–615. doi: 10.1016/0022-2836(86)90338-4. [DOI] [PubMed] [Google Scholar]
  26. Lohman T. M., Overman L. B. Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl concentration. J Biol Chem. 1985 Mar 25;260(6):3594–3603. [PubMed] [Google Scholar]
  27. Morrical S. W., Alberts B. M. The UvsY protein of bacteriophage T4 modulates recombination-dependent DNA synthesis in vitro. J Biol Chem. 1990 Sep 5;265(25):15096–15103. [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. 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]
  30. Nadler S. G., Merrill B. M., Roberts W. J., Keating K. M., Lisbin M. J., Barnett S. F., Wilson S. H., Williams K. R. Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions. Biochemistry. 1991 Mar 19;30(11):2968–2976. doi: 10.1021/bi00225a034. [DOI] [PubMed] [Google Scholar]
  31. Olins D. E., Olins A. L., Von Hippel P. H. Model nucleoprotein complexes: studies on the interaction of cationic homopolypeptides with DNA. J Mol Biol. 1967 Mar 14;24(2):157–176. doi: 10.1016/0022-2836(67)90324-5. [DOI] [PubMed] [Google Scholar]
  32. Pontius B. W., Berg P. Rapid renaturation of complementary DNA strands mediated by cationic detergents: a role for high-probability binding domains in enhancing the kinetics of molecular assembly processes. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8237–8241. doi: 10.1073/pnas.88.18.8237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pontius B. W., Berg P. Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8403–8407. doi: 10.1073/pnas.87.21.8403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Porschke D. The binding of Arg- and Lys-peptides to single stranded polyribonucleotides and its effect on the polymer conformation. Biophys Chem. 1979 Jul;10(1):1–16. doi: 10.1016/0301-4622(79)80001-0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Secnik J., Wang Q., Chang C. M., Jentoft J. E. Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid. Biochemistry. 1990 Aug 28;29(34):7991–7997. doi: 10.1021/bi00486a030. [DOI] [PubMed] [Google Scholar]
  38. Sonigo P., Barker C., Hunter E., Wain-Hobson S. Nucleotide sequence of Mason-Pfizer monkey virus: an immunosuppressive D-type retrovirus. Cell. 1986 May 9;45(3):375–385. doi: 10.1016/0092-8674(86)90323-5. [DOI] [PubMed] [Google Scholar]
  39. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Sykora K. W., Moelling K. Properties of the avian viral protein p12. J Gen Virol. 1981 Aug;55(Pt 2):379–391. doi: 10.1099/0022-1317-55-2-379. [DOI] [PubMed] [Google Scholar]
  42. Weinstock G. M., McEntee K., Lehman I. R. ATP-dependent renaturation of DNA catalyzed by the recA protein of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jan;76(1):126–130. doi: 10.1073/pnas.76.1.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wetmur J. G., Davidson N. Kinetics of renaturation of DNA. J Mol Biol. 1968 Feb 14;31(3):349–370. doi: 10.1016/0022-2836(68)90414-2. [DOI] [PubMed] [Google Scholar]

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