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. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987

Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region.

M A Bender, T D Palmer, R E Gelinas, A D Miller
PMCID: PMC254146  PMID: 3502707

Abstract

Replication-competent retroviruses can be modified to carry nonviral genes. Such gene transfer vectors help define regions of the retroviral genome that are required in cis for retroviral replication. Moloney murine leukemia virus has been used extensively in vector construction, and all of the internal protein-encoding regions can be removed and replaced with other genes while still allowing production of virions containing and transmitting the altered retroviral genome. However, inclusion of a portion of the gag region from Moloney murine leukemia virus markedly increases the titer of virus derived from these vectors. We determined that this effect was due to more efficient packaging of the vector RNA into particles and did not depend on protein synthesis from the gag region. We conclude that the retrovirus packaging signal extends into the gag region. We have found that retroviral vectors containing the complete packaging signal allow more efficient gene transfer into a variety of cell types. In addition, these results may help explain why many oncogenic retroviruses have retained gag sequences and often express transforming proteins that are gag-onc hybrids.

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Selected References

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  1. Armentano D., Yu S. F., Kantoff P. W., von Ruden T., Anderson W. F., Gilboa E. Effect of internal viral sequences on the utility of retroviral vectors. J Virol. 1987 May;61(5):1647–1650. doi: 10.1128/jvi.61.5.1647-1650.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Broome S., Gilbert W. Rous sarcoma virus encodes a transcriptional activator. Cell. 1985 Mar;40(3):537–546. doi: 10.1016/0092-8674(85)90202-8. [DOI] [PubMed] [Google Scholar]
  3. Colbère-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. C. A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol. 1981 Jul 25;150(1):1–14. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  4. Edwards S. A., Fan H. Sequence relationship of glycosylated and unglycosylated gag polyproteins of Moloney murine leukemia virus. J Virol. 1980 Jul;35(1):41–51. doi: 10.1128/jvi.35.1.41-51.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Edwards S. A., Fan H. gag-Related polyproteins of Moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms. J Virol. 1979 May;30(2):551–563. doi: 10.1128/jvi.30.2.551-563.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Gritz L., Davies J. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene. 1983 Nov;25(2-3):179–188. doi: 10.1016/0378-1119(83)90223-8. [DOI] [PubMed] [Google Scholar]
  8. Hock R. A., Miller A. D. Retrovirus-mediated transfer and expression of drug resistance genes in human haematopoietic progenitor cells. Nature. 1986 Mar 20;320(6059):275–277. doi: 10.1038/320275a0. [DOI] [PubMed] [Google Scholar]
  9. Keller G., Paige C., Gilboa E., Wagner E. F. Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors. Nature. 1985 Nov 14;318(6042):149–154. doi: 10.1038/318149a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Kwok W. W., Schuening F., Stead R. B., Miller A. D. Retroviral transfer of genes into canine hemopoietic progenitor cells in culture: a model for human gene therapy. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4552–4555. doi: 10.1073/pnas.83.12.4552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mann R., Baltimore D. Varying the position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAs. J Virol. 1985 May;54(2):401–407. doi: 10.1128/jvi.54.2.401-407.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Miller A. D., Jolly D. J., Friedmann T., Verma I. M. A transmissible retrovirus expressing human hypoxanthine phosphoribosyltransferase (HPRT): gene transfer into cells obtained from humans deficient in HPRT. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4709–4713. doi: 10.1073/pnas.80.15.4709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Palmer T. D., Hock R. A., Osborne W. R., Miller A. D. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human. Proc Natl Acad Sci U S A. 1987 Feb;84(4):1055–1059. doi: 10.1073/pnas.84.4.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schultz A. M., Oroszlan S. Murine leukemia virus gag polyproteins: the peptide chain unique to Pr80 is located at the amino terminus. Virology. 1978 Dec;91(2):481–486. doi: 10.1016/0042-6822(78)90395-1. [DOI] [PubMed] [Google Scholar]
  23. Schwartzberg P., Colicelli J., Goff S. P. Deletion mutants of Moloney murine leukemia virus which lack glycosylated gag protein are replication competent. J Virol. 1983 May;46(2):538–546. doi: 10.1128/jvi.46.2.538-546.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Van Beveren C., Verma I. M. Homology among oncogenes. Curr Top Microbiol Immunol. 1986;123:73–98. doi: 10.1007/978-3-642-70810-7_4. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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