Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1983 Dec;3(12):2180–2190. doi: 10.1128/mcb.3.12.2180

Retrovirus transduction: generation of infectious retroviruses expressing dominant and selectable genes is associated with in vivo recombination and deletion events.

A L Joyner, A Bernstein
PMCID: PMC370089  PMID: 6318087

Abstract

We describe the generation of infectious retroviruses containing foreign genes by an in vivo recombination-deletion mechanism. Cotransfection into mouse cells of chimeric plasmids carrying a murine retrovirus 5' long terminal repeat and either the thymidine kinase (tk) gene of herpesvirus or the dominant selectable bacterial gene for neomycin resistance (neo), along with a clone of Moloney murine leukemia virus, results in the generation of infectious thymidine kinase or neomycin-resistant viruses. Expression of the selectable marker in these viruses can be regulated by the homologous transcriptional promoter of the gene, by the promoter contained within the Friend spleen focus-forming virus long terminal repeat, or by the simian virus 40 early region promoter. In all cases, the rescued viruses appeared to arise by recombination in vivo with Moloney murine leukemia virus sequences, resulting in the acquisition of the Moloney 3' long terminal repeat and variable amounts of the 3' adjacent Moloney genome. In two of the thymidine kinase constructs where tk was inserted 200 base pairs downstream from the long terminal repeat, the rescued viruses acquired a large part of the murine leukemia virus genome, including the region involved in packaging genomic RNA into virions. The generation of infectious neomycin-resistant virus is associated with deletions of simian virus 40 splicing and polyadenylation sequences. These results demonstrate that nonhomologous recombination and deletion events can take place in animal cells, resulting in the acquisition or removal of cis-acting sequences required for, or inhibitory to, retrovirus infectivity.

Full text

PDF
2180

Images in this article

2183Image
on p.2183
2184Image
on p.2184
2187Image
on p.2187

Selected References

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

  1. Alitalo K., Bishop J. M., Smith D. H., Chen E. Y., Colby W. W., Levinson A. D. Nucleotide sequence to the v-myc oncogene of avian retrovirus MC29. Proc Natl Acad Sci U S A. 1983 Jan;80(1):100–104. doi: 10.1073/pnas.80.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. A., Krakauer T., Camerini-Otero R. D. DNA-mediated gene transfer: recombination between cotransferred DNA sequences and recovery of recombinants in a plasmid. Proc Natl Acad Sci U S A. 1982 May;79(9):2748–2752. doi: 10.1073/pnas.79.9.2748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Clark S. P., Mak T. W. Complete nucleotide sequence of an infectious clone of Friend spleen focus-forming provirus: gp55 is an envelope fusion glycoprotein. Proc Natl Acad Sci U S A. 1983 Aug;80(16):5037–5041. doi: 10.1073/pnas.80.16.5037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Edgell M. H., Polsky F. I. Use of preparative gel electrophoresis for DNA fragment isolation. Methods Enzymol. 1980;65(1):319–327. doi: 10.1016/s0076-6879(80)65042-3. [DOI] [PubMed] [Google Scholar]
  6. Enquist L. W., Vande Woude G. F., Wagner M., Smiley J. R., Summers W. C. Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus. Gene. 1979 Nov;7(3-4):335–342. doi: 10.1016/0378-1119(79)90052-0. [DOI] [PubMed] [Google Scholar]
  7. Goff S. P., Tabin C. J., Wang J. Y., Weinberg R., Baltimore D. Transfection of fibroblasts by cloned Abelson murine leukemia virus DNA and recovery of transmissible virus by recombination with helper virus. J Virol. 1982 Jan;41(1):271–285. doi: 10.1128/jvi.41.1.271-285.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldfarb M. P., Weinberg R. A. Generation of novel, biologically active Harvey sarcoma viruses via apparent illegitimate recombination. J Virol. 1981 Apr;38(1):136–150. doi: 10.1128/jvi.38.1.136-150.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  10. Harbers K., Schnieke A., Stuhlmann H., Jähner D., Jaenisch R. DNA methylation and gene expression: endogenous retroviral genome becomes infectious after molecular cloning. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7609–7613. doi: 10.1073/pnas.78.12.7609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  12. Jainchill J. L., Aaronson S. A., Todaro G. J. Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J Virol. 1969 Nov;4(5):549–553. doi: 10.1128/jvi.4.5.549-553.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Josephs S. F., Dalla-Favera R., Gelmann E. P., Gallo R. C., Wong-Staal F. 5' viral and human cellular sequences corresponding to the transforming gene of simian sarcoma virus. Science. 1983 Feb 4;219(4584):503–505. doi: 10.1126/science.6297002. [DOI] [PubMed] [Google Scholar]
  14. Joyner A. L., Bernstein A. Retrovirus transduction: segregation of the viral transforming function and the herpes simplex virus tk gene in infectious Friend spleen focus-forming virus thymidine kinase vectors. Mol Cell Biol. 1983 Dec;3(12):2191–2202. doi: 10.1128/mcb.3.12.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Joyner A., Yamamoto Y., Bernstein A. Retrovirus long terminal repeats activate expression of coding sequences for the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1573–1577. doi: 10.1073/pnas.79.5.1573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. KIT S., DUBBS D. R., PIEKARSKI L. J., HSU T. C. DELETION OF THYMIDINE KINASE ACTIVITY FROM L CELLS RESISTANT TO BROMODEOXYURIDINE. Exp Cell Res. 1963 Aug;31:297–312. doi: 10.1016/0014-4827(63)90007-7. [DOI] [PubMed] [Google Scholar]
  17. Klempnauer K. H., Gonda T. J., Bishop J. M. Nucleotide sequence of the retroviral leukemia gene v-myb and its cellular progenitor c-myb: the architecture of a transduced oncogene. Cell. 1982 Dec;31(2 Pt 1):453–463. doi: 10.1016/0092-8674(82)90138-6. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Perkins A. S., Kirschmeier P. T., Gattoni-Celli S., Weinstein I. B. Design of a retrovirus-derived vector for expression and transduction of exogenous genes in mammalian cells. Mol Cell Biol. 1983 Jun;3(6):1123–1132. doi: 10.1128/mcb.3.6.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Reddy V. B., Ghosh P. K., Lebowitz P., Piatak M., Weissman S. M. Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells. J Virol. 1979 Apr;30(1):279–296. doi: 10.1128/jvi.30.1.279-296.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Shank P. R., Linial M. Avian oncovirus mutant (SE21Q1b) deficient in genomic RNA: characterization of a deletion in the provirus. J Virol. 1980 Nov;36(2):450–456. doi: 10.1128/jvi.36.2.450-456.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shimotohno K., Temin H. M. Formation of infectious progeny virus after insertion of herpes simplex thymidine kinase gene into DNA of an avian retrovirus. Cell. 1981 Oct;26(1 Pt 1):67–77. doi: 10.1016/0092-8674(81)90034-9. [DOI] [PubMed] [Google Scholar]
  23. Shimotohno K., Temin H. M. Spontaneous variation and synthesis in the U3 region of the long terminal repeat of an avian retrovirus. J Virol. 1982 Jan;41(1):163–171. doi: 10.1128/jvi.41.1.163-171.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Small J., Scangos G. Recombination during gene transfer into mouse cells can restore the function of deleted genes. Science. 1983 Jan 14;219(4581):174–176. doi: 10.1126/science.6294829. [DOI] [PubMed] [Google Scholar]
  25. Sorge J., Hughes S. H. Splicing of intervening sequences introduced into an infectious retroviral vector. J Mol Appl Genet. 1982;1(6):547–559. [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. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  28. Stanners C. P., Eliceiri G. L., Green H. Two types of ribosome in mouse-hamster hybrid cells. Nat New Biol. 1971 Mar 10;230(10):52–54. doi: 10.1038/newbio230052a0. [DOI] [PubMed] [Google Scholar]
  29. Tabin C. J., Hoffmann J. W., Goff S. P., Weinberg R. A. Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene. Mol Cell Biol. 1982 Apr;2(4):426–436. doi: 10.1128/mcb.2.4.426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Topp W. C. Normal rat cell lines deficient in nuclear thymidine kinase. Virology. 1981 Aug;113(1):408–411. doi: 10.1016/0042-6822(81)90168-9. [DOI] [PubMed] [Google Scholar]
  31. Varmus H. E. Form and function of retroviral proviruses. Science. 1982 May 21;216(4548):812–820. doi: 10.1126/science.6177038. [DOI] [PubMed] [Google Scholar]
  32. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Watanabe S., Temin H. M. Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5' long terminal repeat and the start of the gag gene. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5986–5990. doi: 10.1073/pnas.79.19.5986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wei C. M., Gibson M., Spear P. G., Scolnick E. M. Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1. J Virol. 1981 Sep;39(3):935–944. doi: 10.1128/jvi.39.3.935-944.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yamamoto Y., Gamble C. L., Clark S. P., Joyner A., Shibuya T., MacDonald M. E., Mager D., Bernstein A., Mak T. W. Clonal analysis of early and late stages of erythroleukemia induced by molecular clones of integrated spleen focus-forming virus. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6893–6897. doi: 10.1073/pnas.78.11.6893. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES