1887

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Volume 86, Issue 9

Yeast system as a model to study Moloney murine leukemia virus integrase: expression, mutagenesis and search for eukaryotic partners Free

Abstract

Moloney murine leukemia virus (M-MuLV) integrase (IN) catalyses the insertion of the viral genome into the host chromosomal DNA. The limited solubility of the recombinant protein produced in led the authors to explore the use of for expression of M-MuLV IN. IN was expressed in yeast and purified by chromatography on nickel–NTA agarose. IN migrated as a single band in SDS-PAGE and did not contain IN degradation products. The enzyme was about twofold more active than the enzyme purified from and was free of nucleases. Using the yeast system, the substitution of the putative catalytic amino acid Asp184 by alanine was also analysed. The mutated enzyme was inactive in the assays. This is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN. Finally, was used as a model to assess the ability of M-MuLV IN to interact with eukaryotic protein partners. The expression of an active M-MuLV IN in yeast strains deficient in RAD52 induced a lethal effect. This phenotype could be attributed to cellular damage, as suggested by the viability of cells expressing inactive D184A IN. Furthermore, when active IN was expressed in a yeast strain lacking the ySNF5 transcription factor, the lethal effect was abolished, suggesting the involvement of ySNF5 in the cellular damage induced by IN. These results indicate that could be a useful model to study the interaction of IN with cellular components in order to identify potential counterparts of the natural host.

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2005-09-01
2024-04-16
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References

  1. Bailis A. M., Rothstein R. 1990; A defect in mismatch repair in Saccharomyces cerevisiae stimulates ectopic recombination between homologous genes by an excision repair-dependent process. Genetics 126:535–547
     [Google Scholar]
  2. Bao K. K., Wang H., Miller J. K., Erie D. A., Skalka A. M., Wong I. 2003; Functional oligomeric state of avian sarcoma virus integrase. J Biol Chem 278:1323–1327 [CrossRef]
     [Google Scholar]
  3. Barnes G., Rine J. 1985; Regulated expression of endonuclease Eco RI in Saccharomyces cerevisiae : nuclear entry and biological consequences. Proc Natl Acad Sci U S A 82:1354–1358 [CrossRef]
     [Google Scholar]
  4. Caumont A. B., Jamieson G. A., Pichuantes S., Nguyen A. T., Litvak S., Dupont C. H. 1996; Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening. Curr Genet 29:503–510 [CrossRef]
     [Google Scholar]
  5. Chalker D. L., Sandmeyer S. B. 1992; Ty3 integrates within the region of RNA polymerase III transcription initiation. Genes Dev 6:117–128 [CrossRef]
     [Google Scholar]
  6. Chen H., Engelman A. 1998; The barrier-to-autointegration protein is a host factor for HIV-1 integration. Proc Natl Acad Sci U S A 95:15270–15274 [CrossRef]
     [Google Scholar]
  7. Chen D. C., Yang B. C., Kuo T. T. 1992; One-step transformation of yeast in stationary phase. Curr Genet 21:83–84 [CrossRef]
     [Google Scholar]
  8. Chiu T. K., Davies D. R. 2004; Structure and function of HIV-1 integrase. Curr Top Med Chem 4:965–977 [CrossRef]
     [Google Scholar]
  9. Chow S. A. 1992; Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus. Science 255:723–726 [CrossRef]
     [Google Scholar]
  10. Cousens L. S., Shuster J. R., Gallegos C., Ku I., Stempien M. M., Urdea M. S., Sanchez-Pescador R., Taylor A., Tekamp-Olson P. 1987; High level of expression of proinsulin in the yeast Saccharomyces cerevisiae . Gene 61:265–275 [CrossRef]
     [Google Scholar]
  11. Craigie R. 2001; HIV-1 integrase: a brief overview from chemistry to therapeutics. J Biol Chem 276:23213–23216 [CrossRef]
     [Google Scholar]
  12. Craigie R., Fujiwara T., Bushman F. 1990; The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro. Cell 62:829–837 [CrossRef]
     [Google Scholar]
  13. Donzella G. D., Leon O., Roth M. J. 1998; Implication of a central cysteine residue and the HHCC domain of Moloney murine leukemia virus integrase protein in functional multimerization. J Virol 72:1691–1698
     [Google Scholar]
  14. Drelich M., Wilhelm R., Mous J. 1992; Identification of amino acids residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro . Virology 188:459–468 [CrossRef]
     [Google Scholar]
  15. Engelman A. 2003; The roles of cellular factors in retroviral integration. Curr Top Microbiol Immunol 281:209–237
     [Google Scholar]
  16. Engelman A., Craigie R. 1992; Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function. J Virol 66:6361–6369
     [Google Scholar]
  17. Engelman A., Mizuuchi K., Craigie R. 1991; HIV-1 integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell 67:1211–1221 [CrossRef]
     [Google Scholar]
  18. Engelman A., Hickman A. B., Craigie R. 1994; The core and carboxyl-terminal domains of the integrase protein of HIV-1 each contribute to nonspecific DNA binding. J Virol 9:5911–5917
     [Google Scholar]
  19. Erhart E., Hollenberg C. P. 1983; The presence of defective LEU2 gene on a 2 μ DNA recombinant plasmid of Saccharomyces cerevisiae is responsible for curing and high copy number. J Bacteriol 156:625–635
     [Google Scholar]
  20. Farnet C. M., Bushman F. D. 1997; HIV-1 cDNA integration: requirement of HMGI(Y) protein for function of preintegration complexes in vitro. Cell 88:483–492 [CrossRef]
     [Google Scholar]
  21. Fassati A., Goff S. P. 1999; Characterization of intracellular reverse transcription complexes of Moloney murine leukemia virus. J Virol 73:8919–8925
     [Google Scholar]
  22. Hanahan D. 1983; Studies on transformation of E. coli with plasmids. J Mol Biol 166:557–580 [CrossRef]
     [Google Scholar]
  23. Jonsson C. B., Donzella G. A., Roth M. J. 1993; Characterization of the forward and reverse integration reactions of the Moloney murine leukemia virus integrase protein purified from Escherichia coli . J Biol Chem 268:1462–1469
     [Google Scholar]
  24. Jonsson C. B., Donzella G. A., Gaucan E., Smith C. M., Roth M. J. 1996; Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis. J Virol 70:4585–4597
     [Google Scholar]
  25. Kalpana G. V., Marmon S., Wang W., Crabtree G. R., Goff S. P. 1994; Binding and stimulation of HIV-1 integrase by human homolog of yeast transcription factor SNF5. Science 266:2002–2006 [CrossRef]
     [Google Scholar]
  26. Kulkosky J., Jones K. S., Katz R. A., Leis J., Skalka A. M. 1992; Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol 12:2331–2338
     [Google Scholar]
  27. Leavitt A. D., Shiue L., Varmus H. E. 1993; Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro. J Biol Chem 268:2113–2119
     [Google Scholar]
  28. Leon O., Roth M. J. 2000; Zinc fingers: DNA binding and protein–protein interactions. Biol Res 33:21–30
     [Google Scholar]
  29. Maertens G., Cherepanow P., Debyser Z., Engelborghs Y., Engelman A. 2004; Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75. J Biol Chem 279:33421–33429 [CrossRef]
     [Google Scholar]
  30. Parissi V., Caumont A., Richard de Soultrait V., Dupont C. H., Pichuantes S., Litvak S. 2000a; Inactivation of the SNF5 transcription factor gene abolishes the lethal phenotype induced by the expression of HIV-1 integrase in yeast. Gene 247:129–136 [CrossRef]
     [Google Scholar]
  31. Parissi V., Caumont A. B., de Soultrait V. R., Calmels C., Pichuantes S., Litvak S., Dupont C. H. 2000b; Selection of amino acid substitutions restoring activity of HIV-1 integrase mutated in its catalytic site using the yeast Saccharomyces cerevisiae . J Mol Biol 295:755–765 [CrossRef]
     [Google Scholar]
  32. Parissi V., Caumont A., de Soultrait V. R. 7 other authors 2003; The lethal phenotype observed after HIV-1 integrase expression in yeast cells is related to DNA repair and recombination events. Gene 322:157–168 [CrossRef]
     [Google Scholar]
  33. Pichuantes S., Babe L. M., Barr P. J., DeCamp D. L., Craik C. S. 1990; Recombinant HIV-2 protease processes HIV-1 pr53gag and analogous gene peptides in vitro. J Biol Chem 265:13890–13898
     [Google Scholar]
  34. Roe T., Reynolds T. C., Yu G., Brown P. O. 1993; Integration of murine leukemia virus depends on mitosis. EMBO J 12:2099–2108
     [Google Scholar]
  35. Suzuki Y., Yang H., Craigie R. 2004; LAP2 alpha and BAF collaborate to organize the Moloney murine leukemia virus preintegration complex. EMBO J 23:4670–4678 [CrossRef]
     [Google Scholar]
  36. Turlure F., Devroe E., Silver P. A., Engelman A. 2004; Human cell proteins and human immunodeficiency virus DNA integration. Front Biosci 9:3187–3208 [CrossRef]
     [Google Scholar]
  37. Villanueva R. A., Jonsson C. B., Jones J., Georgiadis M. M., Roth M. J. 2003; Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions. Virology 316:146–160 [CrossRef]
     [Google Scholar]
  38. Wilhelm M., Wilhelm F. X. 2001; Reverse transcription of retroviruses and LTR retrotransposons. Cell Mol Life Sci 58:1246–1262 [CrossRef]
     [Google Scholar]
  39. Yang F., Roth M. J. 2001; Assembly and catalysis of concerted two-end integration events by Moloney murine leukemia virus integrase. J Virol 75:9561–9570 [CrossRef]
     [Google Scholar]
  40. Yang F., Seamon J. A., Roth M. J. 2001; Mutational analysis of the N-terminus of Moloney murine leukemia virus integrase. Virology 291:32–45 [CrossRef]
     [Google Scholar]
  41. Yang F., Leon O., Greenfield N. J., Roth M. J. 1999; Functional interactions of the HHCC domain of moloney murine leukemia virus integrase revealed by nonoverlapping complementation and zinc-dependent dimerization. J Virol 73:1809–1817
     [Google Scholar]
  42. Zou S., Voytas D. F. 1997; Silent chromatin determines target preferences of the Saccharomyces retrotransposons Ty5. Proc Natl Acad Sci U S A 94:7412–7416 [CrossRef]
     [Google Scholar]
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Yeast system as a model to study Moloney murine leukemia virus integrase: expression, mutagenesis and search for eukaryotic partners
J. Gen. Virol. 86, 2481 (2005); https://doi.org/10.1099/vir.0.81006-0
/content/journal/jgv/10.1099/vir.0.81006-0
/content/journal/jgv/10.1099/vir.0.81006-0
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