Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1994 Nov;68(11):6924–6932. doi: 10.1128/jvi.68.11.6924-6932.1994

An internal deletion enhances the transcriptional activity of a recombinant retrovirus in hematopoietic cells in vivo.

K L MacKenzie 1, L Bonham 1, G Symonds 1
PMCID: PMC237128  PMID: 7933073

Abstract

Lv-myc is a recombinant retrovirus that spontaneously arose during experiments designed to express the provirus LNAv-myc in the hematopoietic system of bone marrow-reconstituted mice (L. Bonham, K. MacKenzie, S. Wood, P. B. Rowe, and G. Symonds, Oncogene 7:2219-2229, 1992). The recombinant provirus is of interest because it is able to promote long terminal repeat-initiated transcription in hematopoietic cells in vivo, whereas the parental provirus, LNAv-myc, is transcriptionally repressed in the same cells. Here we report that Lv-myc was generated by precise deletion of the neomycin resistance gene (neo) and the human gamma-actin promoter from LNAv-myc. In comparison with LNAv-myc, no sequence alterations in the viral regulatory regions of Lv-myc were detected. Thus, it appears that neo and/or the gamma-actin promoter exerted a cis-acting repressor effect on the long terminal repeat of LNAv-myc in vivo. The origin of Lv-myc was also investigated, and it was shown that Lv-myc was harbored as a productive provirus in a G418-resistant subpopulation of the LNAv-myc producer cell line, psi 2AV. It appears that Lv-myc arose during propagation of the psi 2AV cell line. Repeated sequence detected at the sites of the deletion suggest that Lv-myc was generated by a template misalignment during reverse transcription of LNAv-myc.

Full text

PDF
6924

Images in this article

6926Image
on p.6926
6927Image
on p.6927
6929Image
on p.6929

Selected References

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

  1. Akgün E., Ziegler M., Grez M. Determinants of retrovirus gene expression in embryonal carcinoma cells. J Virol. 1991 Jan;65(1):382–388. doi: 10.1128/jvi.65.1.382-388.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Apperley J. F., Luskey B. D., Williams D. A. Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression. Blood. 1991 Jul 15;78(2):310–317. [PubMed] [Google Scholar]
  3. Artelt P., Grannemann R., Stocking C., Friel J., Bartsch J., Hauser H. The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells. Gene. 1991 Mar 15;99(2):249–254. doi: 10.1016/0378-1119(91)90134-w. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Beck-Engeser G., Stocking C., Just U., Albritton L., Dexter M., Spooncer E., Ostertag W. Retroviral vectors related to the myeloproliferative sarcoma virus allow efficient expression in hematopoietic stem and precursor cell lines, but retroviral infection is reduced in more primitive cells. Hum Gene Ther. 1991 Spring;2(1):61–70. doi: 10.1089/hum.1991.2.1-61. [DOI] [PubMed] [Google Scholar]
  6. Bender M. A., Gelinas R. E., Miller A. D. A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells. Mol Cell Biol. 1989 Apr;9(4):1426–1434. doi: 10.1128/mcb.9.4.1426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bonham L., MacKenzie K., Wood S., Rowe P. B., Symonds G. Both myeloproliferative disease and leukemia are induced by transplantation of bone marrow cells expressing v-myc. Oncogene. 1992 Nov;7(11):2219–2229. [PubMed] [Google Scholar]
  9. Bowtell D. D., Cory S., Johnson G. R., Gonda T. J. Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes. J Virol. 1988 Jul;62(7):2464–2473. doi: 10.1128/jvi.62.7.2464-2473.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bowtell D. D., Johnson G. R., Kelso A., Cory S. Expression of genes transferred to haemopoietic stem cells by recombinant retroviruses. Mol Biol Med. 1987 Aug;4(4):229–250. [PubMed] [Google Scholar]
  11. Casanova J. L., Pannetier C., Jaulin C., Kourilsky P. Optimal conditions for directly sequencing double-stranded PCR products with sequenase. Nucleic Acids Res. 1990 Jul 11;18(13):4028–4028. doi: 10.1093/nar/18.13.4028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Correll P. H., Fink J. K., Brady R. O., Perry L. K., Karlsson S. Production of human glucocerebrosidase in mice after retroviral gene transfer into multipotential hematopoietic progenitor cells. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8912–8916. doi: 10.1073/pnas.86.22.8912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Demarquoy J., Herman G. E., Lorenzo I., Trentin J., Beaudet A. L., O'Brien W. E. Long-term expression of human argininosuccinate synthetase in mice following bone marrow transplantation with retrovirus-transduced hematopoietic stem cells. Hum Gene Ther. 1992 Feb;3(1):3–10. doi: 10.1089/hum.1992.3.1-3. [DOI] [PubMed] [Google Scholar]
  14. Dick J. E., Magli M. C., Huszar D., Phillips R. A., Bernstein A. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell. 1985 Aug;42(1):71–79. doi: 10.1016/s0092-8674(85)80102-1. [DOI] [PubMed] [Google Scholar]
  15. Drews R. E., Chan V. T., Schnipper L. E. Oncogenes result in genomic alterations that activate a transcriptionally silent, dominantly selectable reporter gene (neo). Mol Cell Biol. 1992 Jan;12(1):198–206. doi: 10.1128/mcb.12.1.198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dunbar C. E., Crosier P. S., Nienhuis A. W. Introduction of an activated RAS oncogene into murine bone marrow lymphoid progenitors via retroviral gene transfer results in thymic lymphomas. Oncogene Res. 1991;6(1):39–51. [PubMed] [Google Scholar]
  17. Eglitis M. A., Kantoff P., Gilboa E., Anderson W. F. Gene expression in mice after high efficiency retroviral-mediated gene transfer. Science. 1985 Dec 20;230(4732):1395–1398. doi: 10.1126/science.2999985. [DOI] [PubMed] [Google Scholar]
  18. Emerman M., Temin H. M. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell. 1984 Dec;39(3 Pt 2):449–467. [PubMed] [Google Scholar]
  19. Emerman M., Temin H. M. High-frequency deletion in recovered retrovirus vectors containing exogenous DNA with promoters. J Virol. 1984 Apr;50(1):42–49. doi: 10.1128/jvi.50.1.42-49.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gautsch J. W., Meier H. A short-term quantitative XC assay for murine leukemia virus. Virology. 1976 Jul 15;72(2):509–513. doi: 10.1016/0042-6822(76)90179-3. [DOI] [PubMed] [Google Scholar]
  21. Gorman C. M., Rigby P. W., Lane D. P. Negative regulation of viral enhancers in undifferentiated embryonic stem cells. Cell. 1985 Sep;42(2):519–526. doi: 10.1016/0092-8674(85)90109-6. [DOI] [PubMed] [Google Scholar]
  22. Grez M., Zörnig M., Nowock J., Ziegler M. A single point mutation activates the Moloney murine leukemia virus long terminal repeat in embryonal stem cells. J Virol. 1991 Sep;65(9):4691–4698. doi: 10.1128/jvi.65.9.4691-4698.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hilberg F., Stocking C., Ostertag W., Grez M. Functional analysis of a retroviral host-range mutant: altered long terminal repeat sequences allow expression in embryonal carcinoma cells. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5232–5236. doi: 10.1073/pnas.84.15.5232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hirsh J., Levine M. N. Low molecular weight heparin. Blood. 1992 Jan 1;79(1):1–17. [PubMed] [Google Scholar]
  25. Hoeben R. C., Einerhand M. P., Briët E., van Ormondt H., Valerio D., van der Eb A. J. Toward gene therapy in haemophilia A: retrovirus-mediated transfer of a factor VIII gene into murine haematopoietic progenitor cells. Thromb Haemost. 1992 Mar 2;67(3):341–345. [PubMed] [Google Scholar]
  26. Jolly D. J., Willis R. C., Friedmann T. Variable stability of a selectable provirus after retroviral vector gene transfer into human cells. Mol Cell Biol. 1986 Apr;6(4):1141–1147. doi: 10.1128/mcb.6.4.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Just U., Stocking C., Spooncer E., Dexter T. M., Ostertag W. Expression of the GM-CSF gene after retroviral transfer in hematopoietic stem cell lines induces synchronous granulocyte-macrophage differentiation. Cell. 1991 Mar 22;64(6):1163–1173. doi: 10.1016/0092-8674(91)90271-y. [DOI] [PubMed] [Google Scholar]
  28. Keith W. N., Brown R., Pragnell I. B. Retrovirus mediated transfer and expression of GM-CSF in haematopoietic cells. Br J Cancer. 1990 Sep;62(3):388–394. doi: 10.1038/bjc.1990.304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Keller G., Wagner E. F. Expression of v-src induces a myeloproliferative disease in bone-marrow-reconstituted mice. Genes Dev. 1989 Jun;3(6):827–837. doi: 10.1101/gad.3.6.827. [DOI] [PubMed] [Google Scholar]
  30. Lang R. A., Metcalf D., Gough N. M., Dunn A. R., Gonda T. J. Expression of a hemopoietic growth factor cDNA in a factor-dependent cell line results in autonomous growth and tumorigenicity. Cell. 1985 Dec;43(2 Pt 1):531–542. doi: 10.1016/0092-8674(85)90182-5. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. McIvor R. S. Deletion in a recombinant retroviral vector resulting from a cryptic splice donor signal in the Moloney leukemia virus envelope gene. Virology. 1990 Jun;176(2):652–655. doi: 10.1016/0042-6822(90)90039-t. [DOI] [PubMed] [Google Scholar]
  34. McIvor R. S., Johnson M. J., Miller A. D., Pitts S., Williams S. R., Valerio D., Martin D. W., Jr, Verma I. M. Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses. Mol Cell Biol. 1987 Feb;7(2):838–846. doi: 10.1128/mcb.7.2.838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miller A. D., Trauber D. R., Buttimore C. Factors involved in production of helper virus-free retrovirus vectors. Somat Cell Mol Genet. 1986 Mar;12(2):175–183. doi: 10.1007/BF01560664. [DOI] [PubMed] [Google Scholar]
  36. Miller M., Symonds G. Induction of pre-B lymphoid leukemia following reconstitution of lethally irradiated mice with v-erb-B virus-infected bone marrow progenitor cells. Cell Growth Differ. 1993 Feb;4(2):125–135. [PubMed] [Google Scholar]
  37. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Olson P., Temin H. M., Dornburg R. Unusually high frequency of reconstitution of long terminal repeats in U3-minus retrovirus vectors by DNA recombination or gene conversion. J Virol. 1992 Mar;66(3):1336–1343. doi: 10.1128/jvi.66.3.1336-1343.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ostertag W., Vehmeyer K., Fagg B., Pragnell I. B., Paetz W., Le Bousse M. C., Smadja-Joffe F., Klein B., Jasmin C., Eisen H. Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice. J Virol. 1980 Feb;33(2):573–582. doi: 10.1128/jvi.33.2.573-582.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pathak V. K., Temin H. M. Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: deletions and deletions with insertions. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6024–6028. doi: 10.1073/pnas.87.16.6024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Prince V. E., Rigby P. W. Derivatives of Moloney murine sarcoma virus capable of being transcribed in embryonal carcinoma stem cells have gained a functional Sp1 binding site. J Virol. 1991 Apr;65(4):1803–1811. doi: 10.1128/jvi.65.4.1803-1811.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pulsinelli G. A., Temin H. M. Characterization of large deletions occurring during a single round of retrovirus vector replication: novel deletion mechanism involving errors in strand transfer. J Virol. 1991 Sep;65(9):4786–4797. doi: 10.1128/jvi.65.9.4786-4797.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ralph P., Prichard J., Cohn M. Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity. J Immunol. 1975 Feb;114(2 Pt 2):898–905. [PubMed] [Google Scholar]
  45. Rhode B. W., Emerman M., Temin H. M. Instability of large direct repeats in retrovirus vectors. J Virol. 1987 Mar;61(3):925–927. doi: 10.1128/jvi.61.3.925-927.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stocking C., Kollek R., Bergholz U., Ostertag W. Long terminal repeat sequences impart hematopoietic transformation properties to the myeloproliferative sarcoma virus. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5746–5750. doi: 10.1073/pnas.82.17.5746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Taketo M., Shaffer D. J. Deletions in a recombinant retrovirus genome associated with its expression in embryonal carcinoma cells. J Virol. 1989 Oct;63(10):4431–4433. doi: 10.1128/jvi.63.10.4431-4433.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Temin H. M. Retrovirus variation and reverse transcription: abnormal strand transfers result in retrovirus genetic variation. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6900–6903. doi: 10.1073/pnas.90.15.6900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tikhonenko A. T., Linial M. L. Transforming variants of the avian myc-containing retrovirus FH3 arise prior to phenotypic selection. J Virol. 1993 Jun;67(6):3635–3638. doi: 10.1128/jvi.67.6.3635-3638.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tsukiyama T., Niwa O., Yokoro K. Mechanism of suppression of the long terminal repeat of Moloney leukemia virus in mouse embryonal carcinoma cells. Mol Cell Biol. 1989 Nov;9(11):4670–4676. doi: 10.1128/mcb.9.11.4670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Varela-Echavarría A., Prorock C. M., Ron Y., Dougherty J. P. High rate of genetic rearrangement during replication of a Moloney murine leukemia virus-based vector. J Virol. 1993 Nov;67(11):6357–6364. doi: 10.1128/jvi.67.11.6357-6364.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Williams D. A., Orkin S. H., Mulligan R. C. Retrovirus-mediated transfer of human adenosine deaminase gene sequences into cells in culture and into murine hematopoietic cells in vivo. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2566–2570. doi: 10.1073/pnas.83.8.2566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wilson J. M., Danos O., Grossman M., Raulet D. H., Mulligan R. C. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells. Proc Natl Acad Sci U S A. 1990 Jan;87(1):439–443. doi: 10.1073/pnas.87.1.439. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES