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. 1991 Mar;65(3):1160–1167. doi: 10.1128/jvi.65.3.1160-1167.1991

Defining nucleic acid-binding properties of avian retrovirus integrase by deletion analysis.

S R Mumm 1, D P Grandgenett 1
PMCID: PMC239882  PMID: 1847445

Abstract

Integration of retroviral DNA into the host genome requires the activity of retrovirus-encoded integration protein IN. We expressed Rous sarcoma virus (RSV) IN, 286 amino acid residues in length, by using in vitro transcription, followed by in vitro translation in rabbit reticulocyte lysate. The nucleic acid-binding activity of in vitro-translated IN was assessed by using DNA-cellulose affinity chromatography and poly(U)-Sepharose affinity chromatography and by sedimentation analysis in the presence or absence of DNA. In vitro-translated RSV IN exhibited nucleic acid-binding activity similar to that of IN purified from avian myeloblastosis virus. To identify regions of IN which bind to nucleic acids, several deletions of RSV IN were generated. The NH2-terminal 26 amino acids, including the two His residues of a His-Cys box, were not necessary for IN nucleic acid binding with any of the substrates tested. The substrates included native calf thymus DNA, poly(U), and a double-stranded linear DNA molecule with RSV long terminal repeat sequences at its termini. The COOH-terminal region (residues 178 to 286) of IN bound quantitatively (greater than 90%) to poly(U) and to single-stranded circular phi X174 DNA but did not exhibit the double-stranded linear DNA-binding ability of the entire IN molecule.

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Selected References

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

  1. Alexander F., Leis J., Soltis D. A., Crowl R. M., Danho W., Poonian M. S., Pan Y. C., Skalka A. M. Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain. J Virol. 1987 Feb;61(2):534–542. doi: 10.1128/jvi.61.2.534-542.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2525–2529. doi: 10.1073/pnas.86.8.2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Craigie R., Fujiwara T., Bushman F. The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro. Cell. 1990 Aug 24;62(4):829–837. doi: 10.1016/0092-8674(90)90126-y. [DOI] [PubMed] [Google Scholar]
  5. Donehower L. A., Varmus H. E. A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6461–6465. doi: 10.1073/pnas.81.20.6461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Draper D. E., von Hippel P. H. Measurement of macromolecular equilibrium binding constants by a sucrose gradient band sedimentation method. Application to protein-nucleic acid interactions. Biochemistry. 1979 Mar 6;18(5):753–760. doi: 10.1021/bi00572a003. [DOI] [PubMed] [Google Scholar]
  7. Eisenman R. N., Mason W. S., Linial M. Synthesis and processing of polymerase proteins of wild-type and mutant avian retroviruses. J Virol. 1980 Oct;36(1):62–78. doi: 10.1128/jvi.36.1.62-78.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fujiwara T., Craigie R. Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration. Proc Natl Acad Sci U S A. 1989 May;86(9):3065–3069. doi: 10.1073/pnas.86.9.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fujiwara T., Mizuuchi K. Retroviral DNA integration: structure of an integration intermediate. Cell. 1988 Aug 12;54(4):497–504. doi: 10.1016/0092-8674(88)90071-2. [DOI] [PubMed] [Google Scholar]
  10. Godowski P. J., Rusconi S., Miesfeld R., Yamamoto K. R. Glucocorticoid receptor mutants that are constitutive activators of transcriptional enhancement. Nature. 1987 Jan 22;325(6102):365–368. doi: 10.1038/325365a0. [DOI] [PubMed] [Google Scholar]
  11. Grandgenett D. P., Mumm S. R. Unraveling retrovirus integration. Cell. 1990 Jan 12;60(1):3–4. doi: 10.1016/0092-8674(90)90707-l. [DOI] [PubMed] [Google Scholar]
  12. Grandgenett D. P., Vora A. C., Schiff R. D. A 32,000-dalton nucleic acid-binding protein from avian retravirus cores possesses DNA endonuclease activity. Virology. 1978 Aug;89(1):119–132. doi: 10.1016/0042-6822(78)90046-6. [DOI] [PubMed] [Google Scholar]
  13. Grandgenett D. P., Vora A. C., Swanstrom R., Olsen J. C. Nuclease mechanism of the avian retrovirus pp32 endonuclease. J Virol. 1986 Jun;58(3):970–974. doi: 10.1128/jvi.58.3.970-974.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grandgenett D., Quinn T., Hippenmeyer P. J., Oroszlan S. Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains. J Biol Chem. 1985 Jul 15;260(14):8243–8249. [PubMed] [Google Scholar]
  15. Halazonetis T. D., Georgopoulos K., Greenberg M. E., Leder P. c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell. 1988 Dec 2;55(5):917–924. doi: 10.1016/0092-8674(88)90147-x. [DOI] [PubMed] [Google Scholar]
  16. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  17. Hippenmeyer P. J., Grandgenett D. P. Requirement of the avian retrovirus pp32 DNA binding protein domain for replication. Virology. 1984 Sep;137(2):358–370. doi: 10.1016/0042-6822(84)90228-9. [DOI] [PubMed] [Google Scholar]
  18. Hollenberg S. M., Giguere V., Segui P., Evans R. M. Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor. Cell. 1987 Apr 10;49(1):39–46. doi: 10.1016/0092-8674(87)90753-7. [DOI] [PubMed] [Google Scholar]
  19. Hope I. A., Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell. 1986 Sep 12;46(6):885–894. doi: 10.1016/0092-8674(86)90070-x. [DOI] [PubMed] [Google Scholar]
  20. Horton R., Mumm S., Grandgenett D. P. Avian retrovirus pp32 DNA endonuclease is phosphorylated on Ser in the carboxyl-terminal region. J Virol. 1988 Jun;62(6):2067–2075. doi: 10.1128/jvi.62.6.2067-2075.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Johnson A. D., Herskowitz I. A repressor (MAT alpha 2 Product) and its operator control expression of a set of cell type specific genes in yeast. Cell. 1985 Aug;42(1):237–247. doi: 10.1016/s0092-8674(85)80119-7. [DOI] [PubMed] [Google Scholar]
  22. Katz R. A., Skalka A. M. A C-terminal domain in the avian sarcoma-leukosis virus pol gene product is not essential for viral replication. J Virol. 1988 Feb;62(2):528–533. doi: 10.1128/jvi.62.2.528-533.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Katzman M., Katz R. A., Skalka A. M., Leis J. The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration. J Virol. 1989 Dec;63(12):5319–5327. doi: 10.1128/jvi.63.12.5319-5327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Knaus R. J., Hippenmeyer P. J., Misra T. K., Grandgenett D. P., Müller U. R., Fitch W. M. Avian retrovirus pp32 DNA binding protein. Preferential binding to the promoter region of long terminal repeat DNA. Biochemistry. 1984 Jan 17;23(2):350–359. doi: 10.1021/bi00297a026. [DOI] [PubMed] [Google Scholar]
  25. Krogstad P. A., Champoux J. J. Sequence-specific binding of DNA by the Moloney murine leukemia virus integrase protein. J Virol. 1990 Jun;64(6):2796–2801. doi: 10.1128/jvi.64.6.2796-2801.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Lightfoote M. M., Coligan J. E., Folks T. M., Fauci A. S., Martin M. A., Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol. 1986 Nov;60(2):771–775. doi: 10.1128/jvi.60.2.771-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miller J., McLachlan A. D., Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun;4(6):1609–1614. doi: 10.1002/j.1460-2075.1985.tb03825.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nakabeppu Y., Ryder K., Nathans D. DNA binding activities of three murine Jun proteins: stimulation by Fos. Cell. 1988 Dec 2;55(5):907–915. doi: 10.1016/0092-8674(88)90146-8. [DOI] [PubMed] [Google Scholar]
  30. Palmiter R. D. Prevention of NH2-terminal acetylation of proteins synthesized in cell-free systems. J Biol Chem. 1977 Dec 25;252(24):8781–8783. [PubMed] [Google Scholar]
  31. Panganiban A. T., Temin H. M. The retrovirus pol gene encodes a product required for DNA integration: identification of a retrovirus int locus. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7885–7889. doi: 10.1073/pnas.81.24.7885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Quinn T. P., Grandgenett D. P. Genetic evidence that the avian retrovirus DNA endonuclease domain of pol is necessary for viral integration. J Virol. 1988 Jul;62(7):2307–2312. doi: 10.1128/jvi.62.7.2307-2312.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roth M. J., Schwartzberg P. L., Goff S. P. Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence. Cell. 1989 Jul 14;58(1):47–54. doi: 10.1016/0092-8674(89)90401-7. [DOI] [PubMed] [Google Scholar]
  34. Roth M. J., Tanese N., Goff S. P. Gene product of Moloney murine leukemia virus required for proviral integration is a DNA-binding protein. J Mol Biol. 1988 Sep 5;203(1):131–139. doi: 10.1016/0022-2836(88)90097-6. [DOI] [PubMed] [Google Scholar]
  35. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  36. Schwartzberg P., Colicelli J., Goff S. P. Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for productive infection. Cell. 1984 Jul;37(3):1043–1052. doi: 10.1016/0092-8674(84)90439-2. [DOI] [PubMed] [Google Scholar]
  37. Steeg C. M., Vogt V. M. RNA-binding properties of the matrix protein (p19gag) of avian sarcoma and leukemia viruses. J Virol. 1990 Feb;64(2):847–855. doi: 10.1128/jvi.64.2.847-855.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tanese N., Roth M. J., Goff S. P. Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli. J Virol. 1986 Aug;59(2):328–340. doi: 10.1128/jvi.59.2.328-340.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Terry R., Soltis D. A., Katzman M., Cobrinik D., Leis J., Skalka A. M. Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli. J Virol. 1988 Jul;62(7):2358–2365. doi: 10.1128/jvi.62.7.2358-2365.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vora A. C., Fitzgerald M. L., Grandgenett D. P. Removal of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat termini by the avian retrovirus integration protein. J Virol. 1990 Nov;64(11):5656–5659. doi: 10.1128/jvi.64.11.5656-5659.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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