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. 1989 Feb;63(2):513–522. doi: 10.1128/jvi.63.2.513-522.1989

Generation of a helper cell line for packaging avian leukosis virus-based vectors.

P Savatier 1, C Bagnis 1, P Thoraval 1, D Poncet 1, M Belakebi 1, F Mallet 1, C Legras 1, F L Cosset 1, J L Thomas 1, Y Chebloune 1, et al.
PMCID: PMC247719  PMID: 2536089

Abstract

We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produced replication-defective virus at high titer but they did not release any replication-competent particles. Proviral DNA was shown to be correctly integrated as well as correctly expressed. Viral RNAs were shown to be correctly translated into gag-related polypeptides.

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Selected References

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  1. Bosselman R. A., Hsu R. Y., Bruszewski J., Hu S., Martin F., Nicolson M. Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter. Mol Cell Biol. 1987 May;7(5):1797–1806. doi: 10.1128/mcb.7.5.1797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Clark D. P., Dougherty R. M. Detection of avian oncovirus group-specific antigens by the enzyme-linked immunosorbent assay. J Gen Virol. 1980 Apr;47(2):283–291. doi: 10.1099/0022-1317-47-2-283. [DOI] [PubMed] [Google Scholar]
  4. Cone R. D., Mulligan R. C. High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6349–6353. doi: 10.1073/pnas.81.20.6349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Darlix J. L. Circularization of retroviral genomic RNA and the control of RNA translation, packaging and reverse transcription. Biochimie. 1986 Jul-Aug;68(7-8):941–949. doi: 10.1016/s0300-9084(86)81057-4. [DOI] [PubMed] [Google Scholar]
  6. Darlix J. L. Control of Rous sarcoma virus RNA translation and packaging by the 5' and 3' untranslated sequences. J Mol Biol. 1986 Jun 5;189(3):421–434. doi: 10.1016/0022-2836(86)90314-1. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Gandrillon O., Jurdic P., Benchaibi M., Xiao J. H., Ghysdael J., Samarut J. Expression of the v-erbA oncogene in chicken embryo fibroblasts stimulates their proliferation in vitro and enhances tumor growth in vivo. Cell. 1987 Jun 5;49(5):687–697. doi: 10.1016/0092-8674(87)90545-9. [DOI] [PubMed] [Google Scholar]
  9. Ghysdael J., Gegonne A., Pognonec P., Boulukos K., Leprince D., Dernis D., Lagrou C., Stehelin D. Identification in chicken macrophages of a set of proteins related to, but distinct from, the chicken cellular c-ets-encoded protein p54c-ets. EMBO J. 1986 Sep;5(9):2251–2256. doi: 10.1002/j.1460-2075.1986.tb04492.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  11. Hanafusa H., Hanafusa T., Kawai S. Genetic control of expression of endogenous virus genes in chicken cells. Virology. 1974 Apr;58(2):439–448. doi: 10.1016/0042-6822(74)90078-6. [DOI] [PubMed] [Google Scholar]
  12. Hanafusa H. Rapid transformation of cells by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1969 Jun;63(2):318–325. doi: 10.1073/pnas.63.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hughes S. H., Kosik E., Fadly A. M., Salter D. W., Crittenden L. B. Design of retroviral vectors for the insertion of foreign deoxyribonucleic acid sequences into the avian germ line. Poult Sci. 1986 Aug;65(8):1459–1467. doi: 10.3382/ps.0651459. [DOI] [PubMed] [Google Scholar]
  14. Hwang L. H., Gilboa E. Expression of genes introduced into cells by retroviral infection is more efficient than that of genes introduced into cells by DNA transfection. J Virol. 1984 May;50(2):417–424. doi: 10.1128/jvi.50.2.417-424.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Katz R. A., Cullen B. R., Malavarca R., Skalka A. M. Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control. Mol Cell Biol. 1986 Feb;6(2):372–379. doi: 10.1128/mcb.6.2.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Katz R. A., Terry R. W., Skalka A. M. A conserved cis-acting sequence in the 5' leader of avian sarcoma virus RNA is required for packaging. J Virol. 1986 Jul;59(1):163–167. doi: 10.1128/jvi.59.1.163-167.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kawai S., Koyama T. Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biological properties of mutant TK15 and mutant-induced transformants. J Virol. 1984 Jul;51(1):147–153. doi: 10.1128/jvi.51.1.147-153.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kawai S., Nishizawa M. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol Cell Biol. 1984 Jun;4(6):1172–1174. doi: 10.1128/mcb.4.6.1172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Koyama T., Harada F., Kawai S. Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biochemical properties of mutant TK15 and mutant-induced transformants. J Virol. 1984 Jul;51(1):154–162. doi: 10.1128/jvi.51.1.154-162.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Markowitz D., Goff S., Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988 Apr;62(4):1120–1124. doi: 10.1128/jvi.62.4.1120-1124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Miller A. D., Law M. F., Verma I. M. Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene. Mol Cell Biol. 1985 Mar;5(3):431–437. doi: 10.1128/mcb.5.3.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moscovici C., Moscovici M. G., Jimenez H., Lai M. M., Hayman M. J., Vogt P. K. Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail. Cell. 1977 May;11(1):95–103. doi: 10.1016/0092-8674(77)90320-8. [DOI] [PubMed] [Google Scholar]
  26. Norton P. A., Coffin J. M. Characterization of Rous sarcoma virus sequences essential for viral gene expression. J Virol. 1987 Apr;61(4):1171–1179. doi: 10.1128/jvi.61.4.1171-1179.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Panganiban A. T. Retroviral DNA integration. Cell. 1985 Aug;42(1):5–6. doi: 10.1016/s0092-8674(85)80092-1. [DOI] [PubMed] [Google Scholar]
  28. Panganiban A. T., Temin H. M. The retrovirus pol gene encodes a product required for DNA integration: identification of a retrovirus int locus. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7885–7889. doi: 10.1073/pnas.81.24.7885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pugatsch T., Stacey D. W. Identification of a sequence likely to be required for avian retroviral packaging. Virology. 1983 Jul 30;128(2):505–511. doi: 10.1016/0042-6822(83)90279-9. [DOI] [PubMed] [Google Scholar]
  30. Robinson H. L., Astrin S. M., Senior A. M., Salazar F. H. Host Susceptibility to endogenous viruses: defective, glycoprotein-expressing proviruses interfere with infections. J Virol. 1981 Dec;40(3):745–751. doi: 10.1128/jvi.40.3.745-751.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Robinson H. L., Lamoreux W. F. Expression of endogenous ALV antigens and susceptibility to subgroup E ALV in three strains of chickens (endogenous avian C-type virus). Virology. 1976 Jan;69(1):50–62. doi: 10.1016/0042-6822(76)90193-8. [DOI] [PubMed] [Google Scholar]
  32. Rovigatti U. G., Astrin S. M. Avian endogenous viral genes. Curr Top Microbiol Immunol. 1983;103:1–21. doi: 10.1007/978-3-642-68943-7_1. [DOI] [PubMed] [Google Scholar]
  33. Salter D. W., Smith E. J., Hughes S. H., Wright S. E., Crittenden L. B. Transgenic chickens: insertion of retroviral genes into the chicken germ line. Virology. 1987 Mar;157(1):236–240. doi: 10.1016/0042-6822(87)90334-5. [DOI] [PubMed] [Google Scholar]
  34. Salter D. W., Smith E. J., Hughes S. H., Wright S. E., Fadly A. M., Witter R. L., Crittenden L. B. Gene insertion into the chicken germ line by retroviruses. Poult Sci. 1986 Aug;65(8):1445–1458. doi: 10.3382/ps.0651445. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Sorge J., Wright D., Erdman V. D., Cutting A. E. Amphotropic retrovirus vector system for human cell gene transfer. Mol Cell Biol. 1984 Sep;4(9):1730–1737. doi: 10.1128/mcb.4.9.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Stoker A. W., Bissell M. J. Development of avian sarcoma and leukosis virus-based vector-packaging cell lines. J Virol. 1988 Mar;62(3):1008–1015. doi: 10.1128/jvi.62.3.1008-1015.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Swanstrom R., Varmus H. E., Bishop J. M. The terminal redundancy of the retrovirus genome facilitates chain elongation by reverse transcriptase. J Biol Chem. 1981 Feb 10;256(3):1115–1121. [PubMed] [Google Scholar]
  40. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vogt V. M., Eisenman R., Diggelmann H. Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide. J Mol Biol. 1975 Aug 15;96(3):471–493. doi: 10.1016/0022-2836(75)90174-6. [DOI] [PubMed] [Google Scholar]
  42. Vogt V. M., Eisenman R. Identification of a large polypeptide precursor of avian oncornavirus proteins. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1734–1738. doi: 10.1073/pnas.70.6.1734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Watanabe S., Temin H. M. Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. Mol Cell Biol. 1983 Dec;3(12):2241–2249. doi: 10.1128/mcb.3.12.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]

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