Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1996 Dec;70(12):8792–8800. doi: 10.1128/jvi.70.12.8792-8800.1996

Intracellular expression of RNA transcripts complementary to the human immunodeficiency virus type 1 gag gene inhibits viral replication in human CD4+ lymphocytes.

G Veres 1, S Escaich 1, J Baker 1, C Barske 1, C Kalfoglou 1, H Ilves 1, H Kaneshima 1, E Böhnlein 1
PMCID: PMC190976  PMID: 8971008

Abstract

Intracellular expression of antisense transcripts was evaluated for its potential to interfere with human immunodeficiency virus type 1 (HIV-1) replication. Retroviral vectors encoding HIV-1 psi-gag complementary sequences downstream of a selectable gene (neo, puromycin gene, or Lyt2 gene) were stable and yielded high titers. Human CEMSS T cells were transduced with amphotropic retroviral vectors to express RNA complementary to the psi-gag sequence of HIV-1. Replication of laboratory-adapted HIV-1 strains was inhibited by more than 1 order of magnitude (log10) in these transduced cells even at high inoculation doses (4 x 10(4) 50% tissue culture infective doses). Antisense-mediated anti-HIV efficacy was further demonstrated by survival of CD4+ cells in these cultures relative to controls. The level of anti-HIV-1 activity of the psi-gag antisense sequence correlated with the length of the antisense transcript. Maximal anti-HIV efficacy was observed with complementary sequence more than 1,000 nucleotides long, whereas transcripts less than 400 nucleotides long failed to inhibit HIV-1 replication. Expression of psi-gag antisense RNA also reduced HIV-1 JR-CSF replication 10-fold in primary CD4+ lymphocytes. These results obtained with a T-cell line and primary peripheral blood lymphocytes indicate the potential of long antisense RNAs as an efficient anti-HIV-1 therapeutic agent for gene therapy.

Full Text

The Full Text of this article is available as a PDF (501.6 KB).

Selected References

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

  1. Anazodo M. I., Wainberg M. A., Friesen A. D., Wright J. A. Sequence-specific inhibition of gene expression by a novel antisense oligodeoxynucleotide phosphorothioate directed against a nonregulatory region of the human immunodeficiency virus type 1 genome. J Virol. 1995 Mar;69(3):1794–1801. doi: 10.1128/jvi.69.3.1794-1801.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bahner I., Zhou C., Yu X. J., Hao Q. L., Guatelli J. C., Kohn D. B. Comparison of trans-dominant inhibitory mutant human immunodeficiency virus type 1 genes expressed by retroviral vectors in human T lymphocytes. J Virol. 1993 Jun;67(6):3199–3207. doi: 10.1128/jvi.67.6.3199-3207.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baltimore D. Gene therapy. Intracellular immunization. Nature. 1988 Sep 29;335(6189):395–396. doi: 10.1038/335395a0. [DOI] [PubMed] [Google Scholar]
  4. Bevec D., Dobrovnik M., Hauber J., Böhnlein E. Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative Rev trans-activator. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9870–9874. doi: 10.1073/pnas.89.20.9870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bevec D., Volc-Platzer B., Zimmermann K., Dobrovnik M., Hauber J., Veres G., Böhnlein E. Constitutive expression of chimeric neo-Rev response element transcripts suppresses HIV-1 replication in human CD4+ T lymphocytes. Hum Gene Ther. 1994 Feb;5(2):193–201. doi: 10.1089/hum.1994.5.2-193. [DOI] [PubMed] [Google Scholar]
  6. Cann A. J. Stable and safe HIV provirus clones. Nucleic Acids Res. 1990 Oct 25;18(20):6153–6154. doi: 10.1093/nar/18.20.6153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohli H., Fan B., Joshi R. L., Ramezani A., Li X., Joshi S. Inhibition of HIV-1 multiplication in a human CD4+ lymphocytic cell line expressing antisense and sense RNA molecules containing HIV-1 packaging signal and Rev response element(s). Antisense Res Dev. 1994 Spring;4(1):19–26. doi: 10.1089/ard.1994.4.19. [DOI] [PubMed] [Google Scholar]
  8. Escaich S., Kalfoglou C., Plavec I., Kaushal S., Mosca J. D., Böhnlein E. RevM10-mediated inhibition of HIV-1 replication in chronically infected T cells. Hum Gene Ther. 1995 May;6(5):625–634. doi: 10.1089/hum.1995.6.5-625. [DOI] [PubMed] [Google Scholar]
  9. Gilboa E., Smith C. Gene therapy for infectious diseases: the AIDS model. Trends Genet. 1994 Apr;10(4):139–144. doi: 10.1016/0168-9525(94)90216-x. [DOI] [PubMed] [Google Scholar]
  10. Jacks T., Power M. D., Masiarz F. R., Luciw P. A., Barr P. J., Varmus H. E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988 Jan 21;331(6153):280–283. doi: 10.1038/331280a0. [DOI] [PubMed] [Google Scholar]
  11. Joshi S., Van Brunschot A., Asad S., van der Elst I., Read S. E., Bernstein A. Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression. J Virol. 1991 Oct;65(10):5524–5530. doi: 10.1128/jvi.65.10.5524-5530.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kotani H., Newton P. B., 3rd, Zhang S., Chiang Y. L., Otto E., Weaver L., Blaese R. M., Anderson W. F., McGarrity G. J. Improved methods of retroviral vector transduction and production for gene therapy. Hum Gene Ther. 1994 Jan;5(1):19–28. doi: 10.1089/hum.1994.5.1-19. [DOI] [PubMed] [Google Scholar]
  13. Lee S. W., Gallardo H. F., Gilboa E., Smith C. Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev response element decoy consisting of the 13-nucleotide minimal Rev-binding domain. J Virol. 1994 Dec;68(12):8254–8264. doi: 10.1128/jvi.68.12.8254-8264.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Malim M. H., Böhnlein S., Hauber J., Cullen B. R. Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function. Cell. 1989 Jul 14;58(1):205–214. doi: 10.1016/0092-8674(89)90416-9. [DOI] [PubMed] [Google Scholar]
  15. Malim M. H., Freimuth W. W., Liu J., Boyle T. J., Lyerly H. K., Cullen B. R., Nabel G. J. Stable expression of transdominant Rev protein in human T cells inhibits human immunodeficiency virus replication. J Exp Med. 1992 Oct 1;176(4):1197–1201. doi: 10.1084/jem.176.4.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Meyer J., Nick S., Stamminger T., Grummt F., Jahn G., Lipps H. J. Inhibition of HIV-1 replication by a high-copy-number vector expressing antisense RNA for reverse transcriptase. Gene. 1993 Jul 30;129(2):263–268. doi: 10.1016/0378-1119(93)90277-a. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  19. Nabel G. J., Fox B. A., Post L., Thompson C. B., Woffendin C. A molecular genetic intervention for AIDS--effects of a transdominant negative form of Rev. Hum Gene Ther. 1994 Jan;5(1):79–92. doi: 10.1089/hum.1994.5.1-79. [DOI] [PubMed] [Google Scholar]
  20. Ojwang J. O., Hampel A., Looney D. J., Wong-Staal F., Rappaport J. Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10802–10806. doi: 10.1073/pnas.89.22.10802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pear W. S., Nolan G. P., Scott M. L., Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8392–8396. doi: 10.1073/pnas.90.18.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rhodes A., James W. Inhibition of human immunodeficiency virus replication in cell culture by endogenously synthesized antisense RNA. J Gen Virol. 1990 Sep;71(Pt 9):1965–1974. doi: 10.1099/0022-1317-71-9-1965. [DOI] [PubMed] [Google Scholar]
  23. Riddell S. R., Elliott M., Lewinsohn D. A., Gilbert M. J., Wilson L., Manley S. A., Lupton S. D., Overell R. W., Reynolds T. C., Corey L. T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nat Med. 1996 Feb;2(2):216–223. doi: 10.1038/nm0296-216. [DOI] [PubMed] [Google Scholar]
  24. Rigg R. J., Dando J. S., Escaich S., Plavec I., Böhnlein E. Detection of intracellular HIV-1 Rev protein by flow cytometry. J Immunol Methods. 1995 Dec 27;188(2):187–195. doi: 10.1016/0022-1759(95)00237-5. [DOI] [PubMed] [Google Scholar]
  25. Sczakiel G., Homann M., Rittner K. Computer-aided search for effective antisense RNA target sequences of the human immunodeficiency virus type 1. Antisense Res Dev. 1993 Spring;3(1):45–52. doi: 10.1089/ard.1993.3.45. [DOI] [PubMed] [Google Scholar]
  26. Sczakiel G., Oppenländer M., Rittner K., Pawlita M. Tat- and Rev-directed antisense RNA expression inhibits and abolishes replication of human immunodeficiency virus type 1: a temporal analysis. J Virol. 1992 Sep;66(9):5576–5581. doi: 10.1128/jvi.66.9.5576-5581.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sczakiel G., Pawlita M. Inhibition of human immunodeficiency virus type 1 replication in human T cells stably expressing antisense RNA. J Virol. 1991 Jan;65(1):468–472. doi: 10.1128/jvi.65.1.468-472.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sczakiel G., Pawlita M., Kleinheinz A. Specific inhibition of human immunodeficiency virus type 1 replication by RNA transcribed in sense and antisense orientation from the 5'-leader/gag region. Biochem Biophys Res Commun. 1990 Jun 15;169(2):643–651. doi: 10.1016/0006-291x(90)90379-2. [DOI] [PubMed] [Google Scholar]
  29. Stout J. T., Caskey C. T. Antisense RNA inhibition of endogenous genes. Methods Enzymol. 1987;151:519–530. doi: 10.1016/s0076-6879(87)51041-2. [DOI] [PubMed] [Google Scholar]
  30. Sullenger B. A., Gallardo H. F., Ungers G. E., Gilboa E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell. 1990 Nov 2;63(3):601–608. doi: 10.1016/0092-8674(90)90455-n. [DOI] [PubMed] [Google Scholar]
  31. Tagawa M., Nakauchi H., Herzenberg L. A., Nolan G. P. Formal proof that different-size Lyt-2 polypeptides arise from differential splicing and post-transcriptional regulation. Proc Natl Acad Sci U S A. 1986 May;83(10):3422–3426. doi: 10.1073/pnas.83.10.3422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Trono D., Feinberg M. B., Baltimore D. HIV-1 Gag mutants can dominantly interfere with the replication of the wild-type virus. Cell. 1989 Oct 6;59(1):113–120. doi: 10.1016/0092-8674(89)90874-x. [DOI] [PubMed] [Google Scholar]
  33. Tung F. Y. Suppression of simian immunodeficiency virus replication in primary peripheral mononuclear cells by antisense RNA. J Med Virol. 1994 Mar;42(3):255–258. doi: 10.1002/jmv.1890420310. [DOI] [PubMed] [Google Scholar]
  34. Woffendin C., Ranga U., Yang Z., Xu L., Nabel G. J. Expression of a protective gene-prolongs survival of T cells in human immunodeficiency virus-infected patients. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2889–2894. doi: 10.1073/pnas.93.7.2889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zhou C., Bahner I. C., Larson G. P., Zaia J. A., Rossi J. J., Kohn E. B. Inhibition of HIV-1 in human T-lymphocytes by retrovirally transduced anti-tat and rev hammerhead ribozymes. Gene. 1994 Nov 4;149(1):33–39. doi: 10.1016/0378-1119(94)90409-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES