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. 1994 Sep;68(9):5863–5870. doi: 10.1128/jvi.68.9.5863-5870.1994

DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein.

Z Tsuchihashi 1, P O Brown 1
PMCID: PMC236991  PMID: 8057466

Abstract

Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.

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Selected References

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  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agard D. A. To fold or not to fold.... Science. 1993 Jun 25;260(5116):1903–1904. doi: 10.1126/science.8100365. [DOI] [PubMed] [Google Scholar]
  3. Alberts B. M., Frey L. T4 bacteriophage gene 32: a structural protein in the replication and recombination of DNA. Nature. 1970 Sep 26;227(5265):1313–1318. doi: 10.1038/2271313a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Aronoff R., Hajjar A. M., Linial M. L. Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs. J Virol. 1993 Jan;67(1):178–188. doi: 10.1128/jvi.67.1.178-188.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berkowitz R. D., Luban J., Goff S. P. Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. J Virol. 1993 Dec;67(12):7190–7200. doi: 10.1128/jvi.67.12.7190-7200.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Bryant F. R., Lehman I. R. On the mechanism of renaturation of complementary DNA strands by the recA protein of Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jan;82(2):297–301. doi: 10.1073/pnas.82.2.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chase J. W., Williams K. R. Single-stranded DNA binding proteins required for DNA replication. Annu Rev Biochem. 1986;55:103–136. doi: 10.1146/annurev.bi.55.070186.000535. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Dupraz P., Spahr P. F. Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging. J Virol. 1992 Aug;66(8):4662–4670. doi: 10.1128/jvi.66.8.4662-4670.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fodor S. P., Rava R. P., Huang X. C., Pease A. C., Holmes C. P., Adams C. L. Multiplexed biochemical assays with biological chips. Nature. 1993 Aug 5;364(6437):555–556. doi: 10.1038/364555a0. [DOI] [PubMed] [Google Scholar]
  16. Fu W., Rein A. Maturation of dimeric viral RNA of Moloney murine leukemia virus. J Virol. 1993 Sep;67(9):5443–5449. doi: 10.1128/jvi.67.9.5443-5449.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Gorelick R. J., Chabot D. J., Rein A., Henderson L. E., Arthur L. O. The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent. J Virol. 1993 Jul;67(7):4027–4036. doi: 10.1128/jvi.67.7.4027-4036.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Herschlag D. Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn't always better. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6921–6925. doi: 10.1073/pnas.88.16.6921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  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. Kumar A., Wilson S. H. Studies of the strand-annealing activity of mammalian hnRNP complex protein A1. Biochemistry. 1990 Dec 4;29(48):10717–10722. doi: 10.1021/bi00500a001. [DOI] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Leis J., Jentoft J. Characteristics and regulation of interaction of avian retrovirus pp12 protein with viral RNA. J Virol. 1983 Nov;48(2):361–369. doi: 10.1128/jvi.48.2.361-369.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Luban J., Goff S. P. Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein. J Virol. 1991 Jun;65(6):3203–3212. doi: 10.1128/jvi.65.6.3203-3212.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Munroe S. H., Dong X. F. Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):895–899. doi: 10.1073/pnas.89.3.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Méric C., Gouilloud E., Spahr P. F. Mutations in Rous sarcoma virus nucleocapsid protein p12 (NC): deletions of Cys-His boxes. J Virol. 1988 Sep;62(9):3328–3333. doi: 10.1128/jvi.62.9.3328-3333.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Nelson S. F., McCusker J. H., Sander M. A., Kee Y., Modrich P., Brown P. O. Genomic mismatch scanning: a new approach to genetic linkage mapping. Nat Genet. 1993 May;4(1):11–18. doi: 10.1038/ng0593-11. [DOI] [PubMed] [Google Scholar]
  35. Nissen-Meyer J., Abraham A. K. Specificity of RNA binding by the structural protein (p10) of Friend murine leukemia virus. J Mol Biol. 1980 Sep 5;142(1):19–28. doi: 10.1016/0022-2836(80)90203-x. [DOI] [PubMed] [Google Scholar]
  36. Oertle S., Spahr P. F. Role of the gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA. J Virol. 1990 Dec;64(12):5757–5763. doi: 10.1128/jvi.64.12.5757-5763.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pontius B. W., Berg P. Rapid assembly and disassembly of complementary DNA strands through an equilibrium intermediate state mediated by A1 hnRNP protein. J Biol Chem. 1992 Jul 15;267(20):13815–13818. [PubMed] [Google Scholar]
  38. 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]
  39. Portman D. S., Dreyfuss G. RNA annealing activities in HeLa nuclei. EMBO J. 1994 Jan 1;13(1):213–221. doi: 10.1002/j.1460-2075.1994.tb06251.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Sikorav J. L., Church G. M. Complementary recognition in condensed DNA: accelerated DNA renaturation. J Mol Biol. 1991 Dec 20;222(4):1085–1108. doi: 10.1016/0022-2836(91)90595-w. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Surovoy A., Dannull J., Moelling K., Jung G. Conformational and nucleic acid binding studies on the synthetic nucleocapsid protein of HIV-1. J Mol Biol. 1993 Jan 5;229(1):94–104. doi: 10.1006/jmbi.1993.1011. [DOI] [PubMed] [Google Scholar]
  46. Tabor S., Richardson C. C. Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis. J Biol Chem. 1989 Apr 15;264(11):6447–6458. [PubMed] [Google Scholar]
  47. 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]
  48. You J. C., McHenry C. S. HIV nucleocapsid protein. Expression in Escherichia coli, purification, and characterization. J Biol Chem. 1993 Aug 5;268(22):16519–16527. [PubMed] [Google Scholar]

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