Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1993 Jul;67(7):3997–4005. doi: 10.1128/jvi.67.7.3997-4005.1993

Packaging of human immunodeficiency virus type 1 RNA requires cis-acting sequences outside the 5' leader region.

J H Richardson 1, L A Child 1, A M Lever 1
PMCID: PMC237767  PMID: 8510213

Abstract

cis elements required for the encapsidation of human immunodeficiency virus type 1 (HIV-1) RNA have been investigated by using a replication-competent helper virus to package a series of HIV-1-based vectors which had been stably transfected into human CD4 T-cell lines. A previously identified packaging signal in the 5' leader region was not sufficient for the encapsidation of small vectors containing heterologous genes. In contrast, vectors containing additional gag and env sequences were packaged with high efficiency and transduced into CD4-expressing target cells with titers exceeding 10(4) CFU/ml. The presence of gag sequences did not enhance vector packaging efficiency. A 1.1-kb env gene fragment encompassing the Rev-responsive element was absolutely required for the expression and encapsidation of vectors containing cis-acting repressive sequences and appeared also to contain an important packaging signal. Vectors as small as 2.6 kb were successfully packaged in this system. The presence of abundant, packageable vector RNA did not appear to interfere with encapsidation of the wild-type HIV-1 genome, suggesting that HIV-1 RNA packaging capacity is not saturated during acute infection.

Full text

PDF
3997

Images in this article

4000Image
on p.4000
4001Image
on p.4001
4001Image
on p.4001
4002Image
on p.4002

Selected References

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

  1. 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]
  2. 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]
  3. Alford R. L., Honda S., Lawrence C. B., Belmont J. W. RNA secondary structure analysis of the packaging signal for Moloney murine leukemia virus. Virology. 1991 Aug;183(2):611–619. doi: 10.1016/0042-6822(91)90990-s. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Danos O., Mulligan R. C. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6460–6464. doi: 10.1073/pnas.85.17.6460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Fisher A. G., Collalti E., Ratner L., Gallo R. C., Wong-Staal F. A molecular clone of HTLV-III with biological activity. Nature. 1985 Jul 18;316(6025):262–265. doi: 10.1038/316262a0. [DOI] [PubMed] [Google Scholar]
  13. Gorelick R. J., Nigida S. M., Jr, Bess J. W., Jr, Arthur L. O., Henderson L. E., Rein A. Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA. J Virol. 1990 Jul;64(7):3207–3211. doi: 10.1128/jvi.64.7.3207-3211.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  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. Kowalski M., Potz J., Basiripour L., Dorfman T., Goh W. C., Terwilliger E., Dayton A., Rosen C., Haseltine W., Sodroski J. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science. 1987 Sep 11;237(4820):1351–1355. doi: 10.1126/science.3629244. [DOI] [PubMed] [Google Scholar]
  18. Lever A. M., Richardson J. H., Harrison G. P. Retroviral RNA packaging. Biochem Soc Trans. 1991 Nov;19(4):963–966. doi: 10.1042/bst0190963. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Levin J. G., Rosenak M. J. Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1154–1158. doi: 10.1073/pnas.73.4.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Linial M. L., Miller A. D. Retroviral RNA packaging: sequence requirements and implications. Curr Top Microbiol Immunol. 1990;157:125–152. doi: 10.1007/978-3-642-75218-6_5. [DOI] [PubMed] [Google Scholar]
  22. LoSardo J. E., Boral A. L., Lenz J. Relative importance of elements within the SL3-3 virus enhancer for T-cell specificity. J Virol. 1990 Apr;64(4):1756–1763. doi: 10.1128/jvi.64.4.1756-1763.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maddon P. J., Dalgleish A. G., McDougal J. S., Clapham P. R., Weiss R. A., Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell. 1986 Nov 7;47(3):333–348. doi: 10.1016/0092-8674(86)90590-8. [DOI] [PubMed] [Google Scholar]
  24. Malim M. H., Hauber J., Le S. Y., Maizel J. V., Cullen B. R. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989 Mar 16;338(6212):254–257. doi: 10.1038/338254a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Mazarin V., Gourdou I., Querat G., Sauze N., Audoly G., Vitu C., Russo P., Rousselot C., Filippi P., Vigne R. Subcellular localization of rev-gene product in visna virus-infected cells. Virology. 1990 Sep;178(1):305–310. doi: 10.1016/0042-6822(90)90410-s. [DOI] [PubMed] [Google Scholar]
  28. Morgenstern J. P., Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 1990 Jun 25;18(12):3587–3596. doi: 10.1093/nar/18.12.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Méric C., Goff S. P. Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein. J Virol. 1989 Apr;63(4):1558–1568. doi: 10.1128/jvi.63.4.1558-1568.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nishizawa M., Koyama T., Kawai S. Frequent segregation of more-defective variants from a Rous sarcoma virus packaging mutant, TK15. J Virol. 1987 Oct;61(10):3208–3213. doi: 10.1128/jvi.61.10.3208-3213.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Poznansky M., Lever A., Bergeron L., Haseltine W., Sodroski J. Gene transfer into human lymphocytes by a defective human immunodeficiency virus type 1 vector. J Virol. 1991 Jan;65(1):532–536. doi: 10.1128/jvi.65.1.532-536.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Rosen C. A., Sodroski J. G., Campbell K., Haseltine W. A. Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans activator genes. J Virol. 1986 Jan;57(1):379–384. doi: 10.1128/jvi.57.1.379-384.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rosen C. A., Terwilliger E., Dayton A., Sodroski J. G., Haseltine W. A. Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2071–2075. doi: 10.1073/pnas.85.7.2071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schwartz S., Felber B. K., Pavlakis G. N. Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein. J Virol. 1992 Jan;66(1):150–159. doi: 10.1128/jvi.66.1.150-159.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Shimada T., Fujii H., Mitsuya H., Nienhuis A. W. Targeted and highly efficient gene transfer into CD4+ cells by a recombinant human immunodeficiency virus retroviral vector. J Clin Invest. 1991 Sep;88(3):1043–1047. doi: 10.1172/JCI115365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Terwilliger E. F., Godin B., Sodroski J. G., Haseltine W. A. Construction and use of a replication-competent human immunodeficiency virus (HIV-1) that expresses the chloramphenicol acetyltransferase enzyme. Proc Natl Acad Sci U S A. 1989 May;86(10):3857–3861. doi: 10.1073/pnas.86.10.3857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]

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

RESOURCES