Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 May 25;21(10):2399–2407. doi: 10.1093/nar/21.10.2399

Retrovirus-mediated insertion of expressed and non-expressed genes at identical chromosomal locations.

B Berwin 1, E Barklis 1
PMCID: PMC309539  PMID: 8506135

Abstract

During retrovirus replication, a cellularly derived tRNA is annealed to the viral RNA at the primer binding site (PBS) to prime reverse transcription, and both the tRNA and the PBS become copied and matched together on complementary proviral DNA strands prior to integration. Using a viral PBS single base pair mutant which affects provirus expression in undifferentiated cells, we show that reversion to wild type (wt) occurs at a frequency of approximately 50%. Daughter cell lines containing wt or mutant proviruses at identical chromosomal sites have been isolated, supporting a model where an integrated PBS-mismatched provirus was copied before mismatch correction could occur. Virus expression in daughter cells containing the mutant provirus was 100-fold higher than in cells bearing the wt counterpart. Additionally, proviral 5' DNA and cellular 5' flanking DNA became methylated in daughter cells containing wt but not mutant integrants. These results strongly support the current model of retrovirus reverse transcription, and indicate that the wt PBS region contains an element which suppresses virus expression and directs the methylation of viral and neighboring cellular DNA.

Full text

PDF
2399

Images in this article

2402Image
on p.2402
2404Image
on p.2404
2404Image
on p.2404
2404Image
on p.2404
2405Image
on p.2405
2405Image
on p.2405

Selected References

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

  1. Antequera F., Boyes J., Bird A. High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell. 1990 Aug 10;62(3):503–514. doi: 10.1016/0092-8674(90)90015-7. [DOI] [PubMed] [Google Scholar]
  2. Barklis E., Mulligan R. C., Jaenisch R. Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells. Cell. 1986 Nov 7;47(3):391–399. doi: 10.1016/0092-8674(86)90596-9. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  5. Colicelli J., Goff S. P. Structure of a cloned circular retroviral DNA containing a tRNA sequence between the terminal repeats. J Virol. 1986 Feb;57(2):674–677. doi: 10.1128/jvi.57.2.674-677.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  7. Gilboa E., Mitra S. W., Goff S., Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. doi: 10.1016/0092-8674(79)90357-x. [DOI] [PubMed] [Google Scholar]
  8. Hasty P., Ramírez-Solis R., Krumlauf R., Bradley A. Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells. Nature. 1991 Mar 21;350(6315):243–246. doi: 10.1038/350243a0. [DOI] [PubMed] [Google Scholar]
  9. Hoeben R. C., Migchielsen A. A., van der Jagt R. C., van Ormondt H., van der Eb A. J. Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position. J Virol. 1991 Feb;65(2):904–912. doi: 10.1128/jvi.65.2.904-912.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jaenisch R., Harbers K., Schnieke A., Löhler J., Chumakov I., Jähner D., Grotkopp D., Hoffmann E. Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death. Cell. 1983 Jan;32(1):209–216. doi: 10.1016/0092-8674(83)90511-1. [DOI] [PubMed] [Google Scholar]
  11. Johnson T. R., Ilan J. Proteins associated with poly(A)+RNA of cockerel liver: effects of estradiol stimulation. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4088–4092. doi: 10.1073/pnas.79.13.4088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jones T. A., Blaug G., Hansen M., Barklis E. Assembly of gag-beta-galactosidase proteins into retrovirus particles. J Virol. 1990 May;64(5):2265–2279. doi: 10.1128/jvi.64.5.2265-2279.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Loh T. P., Sievert L. L., Scott R. W. Evidence for a stem cell-specific repressor of Moloney murine leukemia virus expression in embryonal carcinoma cells. Mol Cell Biol. 1990 Aug;10(8):4045–4057. doi: 10.1128/mcb.10.8.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Loh T. P., Sievert L. L., Scott R. W. Proviral sequences that restrict retroviral expression in mouse embryonal carcinoma cells. Mol Cell Biol. 1987 Oct;7(10):3775–3784. doi: 10.1128/mcb.7.10.3775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  16. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Niwa O., Yokota Y., Ishida H., Sugahara T. Independent mechanisms involved in suppression of the Moloney leukemia virus genome during differentiation of murine teratocarcinoma cells. Cell. 1983 Apr;32(4):1105–1113. doi: 10.1016/0092-8674(83)90294-5. [DOI] [PubMed] [Google Scholar]
  18. Nolan G. P., Fiering S., Nicolas J. F., Herzenberg L. A. Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2603–2607. doi: 10.1073/pnas.85.8.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Norton P. A., Coffin J. M. Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication. Mol Cell Biol. 1985 Feb;5(2):281–290. doi: 10.1128/mcb.5.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Peckham I., Sobel S., Comer J., Jaenisch R., Barklis E. Retrovirus activation in embryonal carcinoma cells by cellular promoters. Genes Dev. 1989 Dec;3(12B):2062–2071. doi: 10.1101/gad.3.12b.2062. [DOI] [PubMed] [Google Scholar]
  22. Petersen R., Kempler G., Barklis E. A stem cell-specific silencer in the primer-binding site of a retrovirus. Mol Cell Biol. 1991 Mar;11(3):1214–1221. doi: 10.1128/mcb.11.3.1214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rhim H., Park J., Morrow C. D. Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription. J Virol. 1991 Sep;65(9):4555–4564. doi: 10.1128/jvi.65.9.4555-4564.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  25. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  26. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  27. Teich N. M., Weiss R. A., Martin G. R., Lowy D. R. Virus infection of murine teratocarcinoma stem cell lines. Cell. 1977 Dec;12(4):973–982. doi: 10.1016/0092-8674(77)90162-3. [DOI] [PubMed] [Google Scholar]
  28. Valancius V., Smithies O. Testing an "in-out" targeting procedure for making subtle genomic modifications in mouse embryonic stem cells. Mol Cell Biol. 1991 Mar;11(3):1402–1408. doi: 10.1128/mcb.11.3.1402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Walsh C., Cepko C. L. Clonally related cortical cells show several migration patterns. Science. 1988 Sep 9;241(4871):1342–1345. doi: 10.1126/science.3137660. [DOI] [PubMed] [Google Scholar]
  30. Weiher H., Barklis E., Ostertag W., Jaenisch R. Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells. J Virol. 1987 Sep;61(9):2742–2746. doi: 10.1128/jvi.61.9.2742-2746.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES