A chimeric human immunodeficiency virus type 1 (HIV-1) minimal Rev response element-ribozyme molecule exhibits dual antiviral function and inhibits cell-cell transmission of HIV-1 (original) (raw)

Abstract

We have previously shown that hairpin ribozymes targeting the human immunodeficiency virus (HIV) genome can effectively inhibit virus replication in a variety of primary and cultured hematopoietic cells. To further increase antiviral potency and minimize the chance of viral resistance, we have now cloned the stem-loop II sequences of the HIV type 1 Rev response element into ribozyme transcription cassettes. Fusion RNA molecules were shown to function both as RNA decoys and ribozymes. Stable Molt-4/8 cell lines expressing fusion RNA of stem-loop II and a ribozyme directed at the HIV-type 1 U5 sequence (MSLMJT) or its disabled counterpart (MSLdMJT) were generated. The expression of fusion RNA was persistent for at least 6 months without apparent cytotoxicity. When virus inhibition was examined after the cocultivation of transduced cells with chronically infected Jurkat cells, much greater protection was observed in MSLMJT cells than in MSLdMJT or MMJT (expressing only the ribozyme) cells. Furthermore, to specifically compare the ribozyme activities in various transduced cells, we determined the quantitative levels of proviral DNA in the first round of virus replication (7 h after infection with HXB2). By competitive PCR, the proviral DNA levels in MSLMJT and MMJT cells were found to be reduced to 1/7 and 1/3, respectively, compared with those in MSLdMJT and MdMJT cells. These results suggest not only that the greater inhibition afforded by this fusion RNA was due to its function both as decoy and ribozyme but also that the ribozyme activity may be facilitated.

Full Text

The Full Text of this article is available as a PDF (311.9 KB).

Selected References

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

  1. Bertrand E. L., Rossi J. J. Facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of HIV-1 and the heterogeneous nuclear ribonucleoprotein A1. EMBO J. 1994 Jun 15;13(12):2904–2912. doi: 10.1002/j.1460-2075.1994.tb06585.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blaese R. M., Culver K. W., Miller A. D., Carter C. S., Fleisher T., Clerici M., Shearer G., Chang L., Chiang Y., Tolstoshev P. T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science. 1995 Oct 20;270(5235):475–480. doi: 10.1126/science.270.5235.475. [DOI] [PubMed] [Google Scholar]
  3. Bordignon C., Notarangelo L. D., Nobili N., Ferrari G., Casorati G., Panina P., Mazzolari E., Maggioni D., Rossi C., Servida P. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients. Science. 1995 Oct 20;270(5235):470–475. doi: 10.1126/science.270.5235.470. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Chowrira B. M., Berzal-Herranz A., Burke J. M. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature. 1991 Nov 28;354(6351):320–322. doi: 10.1038/354320a0. [DOI] [PubMed] [Google Scholar]
  6. Cochrane A. W., Chen C. H., Rosen C. A. Specific interaction of the human immunodeficiency virus Rev protein with a structured region in the env mRNA. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1198–1202. doi: 10.1073/pnas.87.3.1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coffin J. M. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science. 1995 Jan 27;267(5197):483–489. doi: 10.1126/science.7824947. [DOI] [PubMed] [Google Scholar]
  8. Daefler S., Klotman M. E., Wong-Staal F. Trans-activating rev protein of the human immunodeficiency virus 1 interacts directly and specifically with its target RNA. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4571–4575. doi: 10.1073/pnas.87.12.4571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feinberg M. B., Jarrett R. F., Aldovini A., Gallo R. C., Wong-Staal F. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell. 1986 Sep 12;46(6):807–817. doi: 10.1016/0092-8674(86)90062-0. [DOI] [PubMed] [Google Scholar]
  10. Felber B. K., Hadzopoulou-Cladaras M., Cladaras C., Copeland T., Pavlakis G. N. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1495–1499. doi: 10.1073/pnas.86.5.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heaphy S., Dingwall C., Ernberg I., Gait M. J., Green S. M., Karn J., Lowe A. D., Singh M., Skinner M. A. HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region. Cell. 1990 Feb 23;60(4):685–693. doi: 10.1016/0092-8674(90)90671-z. [DOI] [PubMed] [Google Scholar]
  12. Ho D. D., Neumann A. U., Perelson A. S., Chen W., Leonard J. M., Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature. 1995 Jan 12;373(6510):123–126. doi: 10.1038/373123a0. [DOI] [PubMed] [Google Scholar]
  13. Joseph S., Burke J. M. Optimization of an anti-HIV hairpin ribozyme by in vitro selection. J Biol Chem. 1993 Nov 25;268(33):24515–24518. [PubMed] [Google Scholar]
  14. Kjems J., Frankel A. D., Sharp P. A. Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev. Cell. 1991 Oct 4;67(1):169–178. doi: 10.1016/0092-8674(91)90580-r. [DOI] [PubMed] [Google Scholar]
  15. Leavitt M. C., Yu M., Yamada O., Kraus G., Looney D., Poeschla E., Wong-Staal F. Transfer of an anti-HIV-1 ribozyme gene into primary human lymphocytes. Hum Gene Ther. 1994 Sep;5(9):1115–1120. doi: 10.1089/hum.1994.5.9-1115. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Lee T. C., Sullenger B. A., Gallardo H. F., Ungers G. E., Gilboa E. Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells. New Biol. 1992 Jan;4(1):66–74. [PubMed] [Google Scholar]
  18. Luznik L., Martone M. E., Kraus G., Zhang Y., Xu Y., Ellisman M. H., Wong-Staal F. Localization of human immunodeficiency virus Rev in transfected and virus-infected cells. AIDS Res Hum Retroviruses. 1995 Jul;11(7):795–804. doi: 10.1089/aid.1995.11.795. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Malim M. H., Tiley L. S., McCarn D. F., Rusche J. R., Hauber J., Cullen B. R. HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence. Cell. 1990 Feb 23;60(4):675–683. doi: 10.1016/0092-8674(90)90670-a. [DOI] [PubMed] [Google Scholar]
  21. Meyer B. E., Malim M. H. The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm. Genes Dev. 1994 Jul 1;8(13):1538–1547. doi: 10.1101/gad.8.13.1538. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Ou C. Y., Kwok S., Mitchell S. W., Mack D. H., Sninsky J. J., Krebs J. W., Feorino P., Warfield D., Schochetman G. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science. 1988 Jan 15;239(4837):295–297. doi: 10.1126/science.3336784. [DOI] [PubMed] [Google Scholar]
  24. Rossi J. J., Sarver N. RNA enzymes (ribozymes) as antiviral therapeutic agents. Trends Biotechnol. 1990 Jul;8(7):179–183. doi: 10.1016/0167-7799(90)90169-x. [DOI] [PubMed] [Google Scholar]
  25. Sarver N., Cantin E. M., Chang P. S., Zaia J. A., Ladne P. A., Stephens D. A., Rossi J. J. Ribozymes as potential anti-HIV-1 therapeutic agents. Science. 1990 Mar 9;247(4947):1222–1225. doi: 10.1126/science.2107573. [DOI] [PubMed] [Google Scholar]
  26. Sullenger B. A., Cech T. R. Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA. Science. 1993 Dec 3;262(5139):1566–1569. doi: 10.1126/science.8248806. [DOI] [PubMed] [Google Scholar]
  27. Vaishnav Y. N., Vaishnav M., Wong-Staal F. Identification and characterization of a nuclear factor that specifically binds to the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV-1). New Biol. 1991 Feb;3(2):142–150. [PubMed] [Google Scholar]
  28. Wei X., Ghosh S. K., Taylor M. E., Johnson V. A., Emini E. A., Deutsch P., Lifson J. D., Bonhoeffer S., Nowak M. A., Hahn B. H. Viral dynamics in human immunodeficiency virus type 1 infection. Nature. 1995 Jan 12;373(6510):117–122. doi: 10.1038/373117a0. [DOI] [PubMed] [Google Scholar]
  29. Yamada O., Kraus G., Leavitt M. C., Yu M., Wong-Staal F. Activity and cleavage site specificity of an anti-HIV-1 hairpin ribozyme in human T cells. Virology. 1994 Nov 15;205(1):121–126. doi: 10.1006/viro.1994.1626. [DOI] [PubMed] [Google Scholar]
  30. Yamada O., Yu M., Yee J. K., Kraus G., Looney D., Wong-Staal F. Intracellular immunization of human T cells with a hairpin ribozyme against human immunodeficiency virus type 1. Gene Ther. 1994 Jan;1(1):38–45. [PubMed] [Google Scholar]
  31. Yu M., Leavitt M. C., Maruyama M., Yamada O., Young D., Ho A. D., Wong-Staal F. Intracellular immunization of human fetal cord blood stem/progenitor cells with a ribozyme against human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):699–703. doi: 10.1073/pnas.92.3.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yu M., Ojwang J., Yamada O., Hampel A., Rapapport J., Looney D., Wong-Staal F. A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6340–6344. doi: 10.1073/pnas.90.13.6340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yu M., Poeschla E., Wong-Staal F. Progress towards gene therapy for HIV infection. Gene Ther. 1994 Jan;1(1):13–26. [PubMed] [Google Scholar]
  34. Yu M., Poeschla E., Yamada O., Degrandis P., Leavitt M. C., Heusch M., Yees J. K., Wong-Staal F., Hampel A. In vitro and in vivo characterization of a second functional hairpin ribozyme against HIV-1. Virology. 1995 Jan 10;206(1):381–386. doi: 10.1016/s0042-6822(95)80053-0. [DOI] [PubMed] [Google Scholar]
  35. Zapp M. L., Green M. R. Sequence-specific RNA binding by the HIV-1 Rev protein. Nature. 1989 Dec 7;342(6250):714–716. doi: 10.1038/342714a0. [DOI] [PubMed] [Google Scholar]