1887

Abstract

A retroviral vector-rescue system in which co-packaging of the two co-expressed vectors is required for transduction of one of the vectors has been established previously. By using this rescue system, two distinct packaging-cell populations have been generated. One cell population expressed retroviral RNA from co-localized transcription sites, resulting in local and overlapping accumulation of both RNA transcripts. In the other cell population, the two transcription cassettes were introduced separately, leading to distinct transcription sites of the two RNAs and no significant co-localization of the RNAs. Titre measurements from the two distinct cell populations showed large differences in rescue titre, which is an indirect measure of co-packaging efficiency. Thus, the cell populations with overlapping RNA accumulation gave rise to 15–80-fold-higher rescue titres than cell populations with non-overlapping RNA accumulation. These data show that the spatial position of proviral transcription sites affects the level of retroviral RNA co-packaging and suggest that there is already a linkage of RNAs for co-packaging at the transcription site. It is hypothesized that this linkage is due to RNA dimerization taking place at the transcription site.

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2006-08-01
2024-04-24
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References

  1. Aagaard L., Rasmussen S. V., Mikkelsen J. G., Pedersen F. S. 2004; Efficient replication of full-length murine leukemia viruses modified at the dimer initiation site regions. Virology 318:360–370 [CrossRef]
    [Google Scholar]
  2. Basyuk E., Boulon S., Skou Pedersen F., Bertrand E., Vestergaard Rasmussen S. 2005; The packaging signal of MLV is an integrated module that mediates intracellular transport of genomic RNAs. J Mol Biol 354:330–339 [CrossRef]
    [Google Scholar]
  3. Boris-Lawrie K., Roberts T. M., Hull S. 2001; Retroviral RNA elements integrate components of post-transcriptional gene expression. Life Sci 69:2697–2709 [CrossRef]
    [Google Scholar]
  4. Chartrand P., Bertrand E., Singer R. H., Long R. M. 2000; Sensitive and high-resolution detection of RNA in situ. Methods Enzymol 318:493–506
    [Google Scholar]
  5. Chen C., Chasin L. A. 1998; Cointegration of DNA molecules introduced into mammalian cells by electroporation. Somat Cell Mol Genet 24:249–256 [CrossRef]
    [Google Scholar]
  6. De Tapia M., Metzler V., Mougel M., Ehresmann B., Ehresmann C. 1998; Dimerization of MoMuLV genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278-303) and new roles for stem-loops H2 (310-352) and H3 (355-374). Biochemistry 37:6077–6085 [CrossRef]
    [Google Scholar]
  7. D'Souza V., Summers M. F. 2004; Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus. Nature 431:586–590 [CrossRef]
    [Google Scholar]
  8. Fisher J., Goff S. P. 1998; Mutational analysis of stem-loops in the RNA packaging signal of the Moloney murine leukemia virus. Virology 244:133–145 [CrossRef]
    [Google Scholar]
  9. Flynn J. A., Telesnitsky A. 2006; Two distinct Moloney murine leukemia virus RNAs produced from a single locus dimerize at random. Virology 344:391–400 [CrossRef]
    [Google Scholar]
  10. Fu W., Rein A. 1993; Maturation of dimeric viral RNA of Moloney murine leukemia virus. J Virol 67:5443–5449
    [Google Scholar]
  11. Girard P.-M., Bonnet-Mathonière B., Muriaux D., Paoletti J. 1995; A short autocomplementary sequence in the 5′ leader region is responsible for dimerization of MoMuLV genomic RNA. Biochemistry 34:9785–9794 [CrossRef]
    [Google Scholar]
  12. Girard P.-M., de Rocquigny H., Roques B.-P., Paoletti J. 1996; A model of PSI dimerization: destabilization of the C278-G303 stem-loop by the nucleocapsid protein (NCp10) of MoMuLV. Biochemistry 35:8705–8714 [CrossRef]
    [Google Scholar]
  13. Hamada T., Sasaki H., Seki R., Sakaki Y. 1993; Mechanism of chromosomal integration of transgenes in microinjected mouse eggs: sequence analysis of genome-transgene and transgene-transgene junctions at two loci. Gene 128:197–202 [CrossRef]
    [Google Scholar]
  14. Hu W.-S., Temin H. M. 1990; Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination. Proc Natl Acad Sci U S A 87:1556–1560 [CrossRef]
    [Google Scholar]
  15. Janicki S. M., Tsukamoto T., Salghetti S. E. & 8 other authors 2004; From silencing to gene expression: real-time analysis in single cells. Cell 116:683–698 [CrossRef]
    [Google Scholar]
  16. Kharytonchyk S. A., Kireyeva A. I., Osipovich A. B., Fomin I. K. 2005; Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site. Retrovirology 2:3 [CrossRef]
    [Google Scholar]
  17. Levin J. G., Rosenak M. J. 1976; Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA. Proc Natl Acad Sci U S A 73:1154–1158 [CrossRef]
    [Google Scholar]
  18. Mann R., Mulligan R. C., Baltimore D. 1983; Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33:153–159 [CrossRef]
    [Google Scholar]
  19. Méric C., Goff S. P. 1989; Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein. J Virol 63:1558–1568
    [Google Scholar]
  20. Mikkelsen J. G., Lund A. H., Kristensen K. D., Duch M., Sørensen M. S., Jørgensen P., Pedersen F. S. 1996; A preferred region for recombinational patch repair in the 5′ untranslated region of primer binding site-impaired murine leukemia virus vectors. J Virol 70:1439–1447
    [Google Scholar]
  21. Mikkelsen J. G., Lund A. H., Duch M., Pedersen F. S. 1998; Recombination in the 5′ leader of murine leukemia virus is accurate and influenced by sequence identity with a strong bias toward the kissing-loop dimerization region. J Virol 72:6967–6978
    [Google Scholar]
  22. Mougel M., Zhang Y., Barklis E. 1996; cis -Active structural motifs involved in specific encapsidation of Moloney murine leukemia virus RNA. J Virol 70:5043–5050
    [Google Scholar]
  23. Nicolás A. L., Young C. S. H. 1994; Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific. Mol Cell Biol 14:170–180
    [Google Scholar]
  24. Oroudjev E. M., Kang P. C. E., Kohlstaedt L. A. 1999; An additional dimer linkage structure in Moloney murine leukemia virus RNA. J Mol Biol 291:603–613 [CrossRef]
    [Google Scholar]
  25. Perucho M., Hanahan D., Wigler M. 1980; Genetic and physical linkage of exogenous sequences in transformed cells. Cell 22:309–317 [CrossRef]
    [Google Scholar]
  26. Rasmussen S. V., Pedersen F. S. 2004; Complementarity between RNA dimerization elements favors formation of functional heterozygous murine leukemia viruses. Virology 329:440–453 [CrossRef]
    [Google Scholar]
  27. Shav-Tal Y., Darzacq X., Shenoy S. M., Fusco D., Janicki S. M., Spector D. L., Singer R. H. 2004; Dynamics of single mRNPs in nuclei of living cells. Science 304:1797–1800 [CrossRef]
    [Google Scholar]
  28. Stuhlmann H., Berg P. 1992; Homologous recombination of copackaged retrovirus RNAs during reverse transcription. J Virol 66:2378–2388
    [Google Scholar]
  29. Tange T. Ø., Nott A., Moore M. J. 2004; The ever-increasing complexities of the exon junction complex. Curr Opin Cell Biol 16:279–284 [CrossRef]
    [Google Scholar]
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