Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Journal of Virology logoLink to Journal of Virology
. 1995 Jun;69(6):3282–3289. doi: 10.1128/jvi.69.6.3282-3289.1995

The bovine leukemia virus encapsidation signal is discontinuous and extends into the 5' end of the gag gene.

L M Mansky 1, A E Krueger 1, H M Temin 1
PMCID: PMC189039  PMID: 7745675

Abstract

In order to define bovine leukemia virus (BLV) sequences required for efficient vector replication, a series of mutations were made in a BLV vector. Testing the replication efficiency of the vectors with a helper virus and helper plasmids allowed for separation of the mutant vectors into three groups. The replication efficiency of the first group was reduced by a factor of 7; these mutants contained deletions in the 5' end of the gag gene. The second group of mutants had replication reduced by a factor of 50 and had deletions including the 5' untranslated leader region. The third group of mutants replicated at levels comparable to those of the parental vector and contained deletions of the 3' end of the gag gene, the pol gene, and the env gene. Analysis of cytoplasmic and virion RNA levels indicated that vector RNA expression was not affected but that the vector RNA encapsidation was less efficient for group 1 and group 2 mutants. Additional mutations revealed two regions important for RNA encapsidation. The first region is a 132-nucleotide-base sequence within the gag gene (nucleotides 1015 to 1147 of the proviral DNA) and facilitates efficient RNA encapsidation in the presence of the second region. The second region includes a 147-nucleotide-base sequence downstream of the primer binding site (nucleotide 551) and near the gag gene start codon (nucleotide 698; gag begins at nucleotide 628) and is essential for RNA encapsidation. We conclude that the encapsidation signal is discontinuous; a primary signal, essential for RNA encapsidation, is largely in the untranslated leader region between the primer binding site and near the gag start codon. A secondary signal, which facilitates efficient RNA encapsidation, is in a 132-nucleotide-base region within the 5' end of the gag gene.

Full Text

The Full Text of this article is available as a PDF (693.6 KB).

Selected References

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

  1. Abrahams J. P., van den Berg M., van Batenburg E., Pleij C. Prediction of RNA secondary structure, including pseudoknotting, by computer simulation. Nucleic Acids Res. 1990 May 25;18(10):3035–3044. doi: 10.1093/nar/18.10.3035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adam M. A., Miller A. D. Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions. J Virol. 1988 Oct;62(10):3802–3806. doi: 10.1128/jvi.62.10.3802-3806.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aldovini A., Young R. A. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990 May;64(5):1920–1926. doi: 10.1128/jvi.64.5.1920-1926.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Aronoff R., Hajjar A. M., Linial M. L. Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs. J Virol. 1993 Jan;67(1):178–188. doi: 10.1128/jvi.67.1.178-188.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Aronoff R., Linial M. Specificity of retroviral RNA packaging. J Virol. 1991 Jan;65(1):71–80. doi: 10.1128/jvi.65.1.71-80.1991. [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. Bieth E., Gabus C., Darlix J. L. A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro. Nucleic Acids Res. 1990 Jan 11;18(1):119–127. doi: 10.1093/nar/18.1.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Boris-Lawrie K., Temin H. M. Genetically simpler bovine leukemia virus derivatives can replicate independently of Tax and Rex. J Virol. 1995 Mar;69(3):1920–1924. doi: 10.1128/jvi.69.3.1920-1924.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Buchschacher G. L., Jr, Panganiban A. T. Human immunodeficiency virus vectors for inducible expression of foreign genes. J Virol. 1992 May;66(5):2731–2739. doi: 10.1128/jvi.66.5.2731-2739.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clavel F., Orenstein J. M. A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology. J Virol. 1990 Oct;64(10):5230–5234. doi: 10.1128/jvi.64.10.5230-5234.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Darlix J. L., Gabus C., Nugeyre M. T., Clavel F., Barré-Sinoussi F. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1. J Mol Biol. 1990 Dec 5;216(3):689–699. doi: 10.1016/0022-2836(90)90392-Y. [DOI] [PubMed] [Google Scholar]
  13. Derse D., Martarano L. Construction of a recombinant bovine leukemia virus vector for analysis of virus infectivity. J Virol. 1990 Jan;64(1):401–405. doi: 10.1128/jvi.64.1.401-405.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dougherty J. P., Temin H. M. High mutation rate of a spleen necrosis virus-based retrovirus vector. Mol Cell Biol. 1986 Dec;6(12):4387–4395. doi: 10.1128/mcb.6.12.4387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dougherty J. P., Wisniewski R., Yang S. L., Rhode B. W., Temin H. M. New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors. J Virol. 1989 Jul;63(7):3209–3212. doi: 10.1128/jvi.63.7.3209-3212.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Embretson J. E., Temin H. M. Lack of competition results in efficient packaging of heterologous murine retroviral RNAs and reticuloendotheliosis virus encapsidation-minus RNAs by the reticuloendotheliosis virus helper cell line. J Virol. 1987 Sep;61(9):2675–2683. doi: 10.1128/jvi.61.9.2675-2683.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gallis B., Linial M., Eisenman R. An avian oncovirus mutant deficient in genomic RNA: characterization of the packaged RNA as cellular messenger RNA. Virology. 1979 Apr 15;94(1):146–161. doi: 10.1016/0042-6822(79)90445-8. [DOI] [PubMed] [Google Scholar]
  19. Giordano T. J., McAllister W. T. Optimization of the hygromycin B resistance-conferring gene as a dominant selectable marker in mammalian cells. Gene. 1990 Apr 16;88(2):285–288. doi: 10.1016/0378-1119(90)90045-s. [DOI] [PubMed] [Google Scholar]
  20. Harrison G. P., Lever A. M. The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure. J Virol. 1992 Jul;66(7):4144–4153. doi: 10.1128/jvi.66.7.4144-4153.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hayashi T., Shioda T., Iwakura Y., Shibuta H. RNA packaging signal of human immunodeficiency virus type 1. Virology. 1992 Jun;188(2):590–599. doi: 10.1016/0042-6822(92)90513-o. [DOI] [PubMed] [Google Scholar]
  22. Jaeger J. A., Turner D. H., Zuker M. Improved predictions of secondary structures for RNA. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7706–7710. doi: 10.1073/pnas.86.20.7706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Katoh I., Kyushiki H., Sakamoto Y., Ikawa Y., Yoshinaka Y. Bovine leukemia virus matrix-associated protein MA(p15): further processing and formation of a specific complex with the dimer of the 5'-terminal genomic RNA fragment. J Virol. 1991 Dec;65(12):6845–6855. doi: 10.1128/jvi.65.12.6845-6855.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Katoh I., Yasunaga T., Yoshinaka Y. Bovine leukemia virus RNA sequences involved in dimerization and specific gag protein binding: close relation to the packaging sites of avian, murine, and human retroviruses. J Virol. 1993 Apr;67(4):1830–1839. doi: 10.1128/jvi.67.4.1830-1839.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Landau N. R., Littman D. R. Packaging system for rapid production of murine leukemia virus vectors with variable tropism. J Virol. 1992 Aug;66(8):5110–5113. doi: 10.1128/jvi.66.8.5110-5113.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lever A., Gottlinger H., Haseltine W., Sodroski J. Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J Virol. 1989 Sep;63(9):4085–4087. doi: 10.1128/jvi.63.9.4085-4087.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Luban J., Goff S. P. Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. J Virol. 1994 Jun;68(6):3784–3793. doi: 10.1128/jvi.68.6.3784-3793.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  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. Mansky L. M., Temin H. M. Lower mutation rate of bovine leukemia virus relative to that of spleen necrosis virus. J Virol. 1994 Jan;68(1):494–499. doi: 10.1128/jvi.68.1.494-499.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Marquet R., Baudin F., Gabus C., Darlix J. L., Mougel M., Ehresmann C., Ehresmann B. Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism. Nucleic Acids Res. 1991 May 11;19(9):2349–2357. doi: 10.1093/nar/19.9.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Milan D., Nicolas J. F. Activator-dependent and activator-independent defective recombinant retroviruses from bovine leukemia virus. J Virol. 1991 Apr;65(4):1938–1945. doi: 10.1128/jvi.65.4.1938-1945.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Osborne W. R., Miller A. D. Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6851–6855. doi: 10.1073/pnas.85.18.6851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Parolin C., Dorfman T., Palú G., Göttlinger H., Sodroski J. Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes. J Virol. 1994 Jun;68(6):3888–3895. doi: 10.1128/jvi.68.6.3888-3895.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Prats A. C., Roy C., Wang P. A., Erard M., Housset V., Gabus C., Paoletti C., Darlix J. L. cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA. J Virol. 1990 Feb;64(2):774–783. doi: 10.1128/jvi.64.2.774-783.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Rein A. Retroviral RNA packaging: a review. Arch Virol Suppl. 1994;9:513–522. doi: 10.1007/978-3-7091-9326-6_49. [DOI] [PubMed] [Google Scholar]
  41. Rizvi T. A., Panganiban A. T. Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles. J Virol. 1993 May;67(5):2681–2688. doi: 10.1128/jvi.67.5.2681-2688.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sagata N., Yasunaga T., Tsuzuku-Kawamura J., Ohishi K., Ogawa Y., Ikawa Y. Complete nucleotide sequence of the genome of bovine leukemia virus: its evolutionary relationship to other retroviruses. Proc Natl Acad Sci U S A. 1985 Feb;82(3):677–681. doi: 10.1073/pnas.82.3.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sorge J., Ricci W., Hughes S. H. cis-Acting RNA packaging locus in the 115-nucleotide direct repeat of Rous sarcoma virus. J Virol. 1983 Dec;48(3):667–675. doi: 10.1128/jvi.48.3.667-675.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sundquist W. I., Heaphy S. Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3393–3397. doi: 10.1073/pnas.90.8.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Temin H. M. Studies on carcinogenesis by avian sarcoma viruses. 8. Glycolysis and cell multiplication. Int J Cancer. 1968 Mar 15;3(2):273–282. doi: 10.1002/ijc.2910030213. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Yang S., Temin H. M. A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA. EMBO J. 1994 Feb 1;13(3):713–726. doi: 10.1002/j.1460-2075.1994.tb06311.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]
  49. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES