Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Jul 11;23(13):2434–2441. doi: 10.1093/nar/23.13.2434

Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1.

M Lapadat-Tapolsky 1, C Pernelle 1, C Borie 1, J L Darlix 1
PMCID: PMC307048  PMID: 7543198

Abstract

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex.

Full text

PDF
2441

Images in this article

2436Image
on p.2436
2436Image
on p.2436
2438Image
on p.2438
2440Image
on p.2440

Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. Bakalkin G., Yakovleva T., Selivanova G., Magnusson K. P., Szekely L., Kiseleva E., Klein G., Terenius L., Wiman K. G. p53 binds single-stranded DNA ends and catalyzes DNA renaturation and strand transfer. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):413–417. doi: 10.1073/pnas.91.1.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barat C., Le Grice S. F., Darlix J. L. Interaction of HIV-1 reverse transcriptase with a synthetic form of its replication primer, tRNA(Lys,3). Nucleic Acids Res. 1991 Feb 25;19(4):751–757. doi: 10.1093/nar/19.4.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Baudin F., Bach C., Cusack S., Ruigrok R. W. Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent. EMBO J. 1994 Jul 1;13(13):3158–3165. doi: 10.1002/j.1460-2075.1994.tb06614.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bertrand E. L., Rossi J. J. Facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of HIV-1 and the heterogeneous nuclear ribonucleoprotein A1. EMBO J. 1994 Jun 15;13(12):2904–2912. doi: 10.1002/j.1460-2075.1994.tb06585.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Boehmer P. E., Lehman I. R. Physical interaction between the herpes simplex virus 1 origin-binding protein and single-stranded DNA-binding protein ICP8. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8444–8448. doi: 10.1073/pnas.90.18.8444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cesareni G., Muesing M. A., Polisky B. Control of ColE1 DNA replication: the rop gene product negatively affects transcription from the replication primer promoter. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6313–6317. doi: 10.1073/pnas.79.20.6313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Coffin J. M. Structure, replication, and recombination of retrovirus genomes: some unifying hypotheses. J Gen Virol. 1979 Jan;42(1):1–26. doi: 10.1099/0022-1317-42-1-1. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Darlix J. L., Vincent A., Gabus C., de Rocquigny H., Roques B. Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA. C R Acad Sci III. 1993 Aug;316(8):763–771. [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Echols H. Nucleoprotein structures initiating DNA replication, transcription, and site-specific recombination. J Biol Chem. 1990 Sep 5;265(25):14697–14700. [PubMed] [Google Scholar]
  19. Göttlinger H. G., Sodroski J. G., Haseltine W. A. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5781–5785. doi: 10.1073/pnas.86.15.5781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Herschlag D., Khosla M., Tsuchihashi Z., Karpel R. L. An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis. EMBO J. 1994 Jun 15;13(12):2913–2924. doi: 10.1002/j.1460-2075.1994.tb06586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jentoft J. E., Smith L. M., Fu X. D., Johnson M., Leis J. Conserved cysteine and histidine residues of the avian myeloblastosis virus nucleocapsid protein are essential for viral replication but are not "zinc-binding fingers". Proc Natl Acad Sci U S A. 1988 Oct;85(19):7094–7098. doi: 10.1073/pnas.85.19.7094. [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. 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]
  26. 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]
  27. 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]
  28. Oberosler P., Hloch P., Ramsperger U., Stahl H. p53-catalyzed annealing of complementary single-stranded nucleic acids. EMBO J. 1993 Jun;12(6):2389–2396. doi: 10.1002/j.1460-2075.1993.tb05893.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. Zijderveld D. C., van der Vliet P. C. Helix-destabilizing properties of the adenovirus DNA-binding protein. J Virol. 1994 Feb;68(2):1158–1164. doi: 10.1128/jvi.68.2.1158-1164.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]

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

RESOURCES