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. 1997 Dec;71(12):9713–9721. doi: 10.1128/jvi.71.12.9713-9721.1997

Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1.

S M Smith 1, F Maldarelli 1, K T Jeang 1
PMCID: PMC230281  PMID: 9371637

Abstract

Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.

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Selected References

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  1. Berthold E., Maldarelli F. cis-acting elements in human immunodeficiency virus type 1 RNAs direct viral transcripts to distinct intranuclear locations. J Virol. 1996 Jul;70(7):4667–4682. doi: 10.1128/jvi.70.7.4667-4682.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cech T. R. The efficiency and versatility of catalytic RNA: implications for an RNA world. Gene. 1993 Dec 15;135(1-2):33–36. doi: 10.1016/0378-1119(93)90046-6. [DOI] [PubMed] [Google Scholar]
  3. Cotten M., Birnstiel M. L. Ribozyme mediated destruction of RNA in vivo. EMBO J. 1989 Dec 1;8(12):3861–3866. doi: 10.1002/j.1460-2075.1989.tb08564.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dropulic B., Smith S. M., Jeang K. T. Activation and inactivation of gene expression using RNA sequences. Adv Pharmacol. 1994;30:247–270. doi: 10.1016/s1054-3589(08)60176-3. [DOI] [PubMed] [Google Scholar]
  5. Dropulić B., Lin N. H., Jeang K. T. A method to increase the cumulative cleavage efficiency of ribozymes: thermal cycling. Nucleic Acids Res. 1993 May 11;21(9):2273–2274. doi: 10.1093/nar/21.9.2273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dropulić B., Lin N. H., Martin M. A., Jeang K. T. Functional characterization of a U5 ribozyme: intracellular suppression of human immunodeficiency virus type 1 expression. J Virol. 1992 Mar;66(3):1432–1441. doi: 10.1128/jvi.66.3.1432-1441.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frolov I., Hoffman T. A., Prágai B. M., Dryga S. A., Huang H. V., Schlesinger S., Rice C. M. Alphavirus-based expression vectors: strategies and applications. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11371–11377. doi: 10.1073/pnas.93.21.11371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Homann M., Tzortzakaki S., Rittner K., Sczakiel G., Tabler M. Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1. Nucleic Acids Res. 1993 Jun 25;21(12):2809–2814. doi: 10.1093/nar/21.12.2809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Israel M. A. Molecular approaches to cancer therapy. Adv Cancer Res. 1993;61:57–85. doi: 10.1016/s0065-230x(08)60955-0. [DOI] [PubMed] [Google Scholar]
  10. Kashani-Sabet M., Funato T., Florenes V. A., Fodstad O., Scanlon K. J. Suppression of the neoplastic phenotype in vivo by an anti-ras ribozyme. Cancer Res. 1994 Feb 15;54(4):900–902. [PubMed] [Google Scholar]
  11. Kashani-Sabet M., Funato T., Tone T., Jiao L., Wang W., Yoshida E., Kashfinn B. I., Shitara T., Wu A. M., Moreno J. G. Reversal of the malignant phenotype by an anti-ras ribozyme. Antisense Res Dev. 1992 Spring;2(1):3–15. doi: 10.1089/ard.1992.2.3. [DOI] [PubMed] [Google Scholar]
  12. Liljeström P., Garoff H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 1991 Dec;9(12):1356–1361. doi: 10.1038/nbt1291-1356. [DOI] [PubMed] [Google Scholar]
  13. Littman D. R., Thomas Y., Maddon P. J., Chess L., Axel R. The isolation and sequence of the gene encoding T8: a molecule defining functional classes of T lymphocytes. Cell. 1985 Feb;40(2):237–246. doi: 10.1016/0092-8674(85)90138-2. [DOI] [PubMed] [Google Scholar]
  14. Lustig B., Lin N. H., Smith S. M., Jernigan R. L., Jeang K. T. A small modified hammerhead ribozyme and its conformational characteristics determined by mutagenesis and lattice calculation. Nucleic Acids Res. 1995 Sep 11;23(17):3531–3538. doi: 10.1093/nar/23.17.3531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mebatsion T., Schnell M. J., Cox J. H., Finke S., Conzelmann K. K. Highly stable expression of a foreign gene from rabies virus vectors. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7310–7314. doi: 10.1073/pnas.93.14.7310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Paulus W., Baur I., Boyce F. M., Breakefield X. O., Reeves S. A. Self-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells. J Virol. 1996 Jan;70(1):62–67. doi: 10.1128/jvi.70.1.62-67.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rossi J. J., Elkins D., Taylor N., Zaia J., Sullivan S., Deshler J. O. Exploring the use of antisense, enzymatic RNA molecules (ribozymes) as therapeutic agents. Antisense Res Dev. 1991 Fall;1(3):285–288. [PubMed] [Google Scholar]
  18. 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]
  19. Schlesinger S. Alphaviruses--vectors for the expression of heterologous genes. Trends Biotechnol. 1993 Jan;11(1):18–22. doi: 10.1016/0167-7799(93)90070-P. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schlesinger S. RNA viruses as vectors for the expression of heterologous proteins. Mol Biotechnol. 1995 Apr;3(2):155–165. doi: 10.1007/BF02789111. [DOI] [PubMed] [Google Scholar]
  21. Steinecke P., Herget T., Schreier P. H. Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo. EMBO J. 1992 Apr;11(4):1525–1530. doi: 10.1002/j.1460-2075.1992.tb05197.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Strauss J. H., Strauss E. G. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994 Sep;58(3):491–562. doi: 10.1128/mr.58.3.491-562.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang S., Dolnick B. J. Quantitative evaluation of intracellular sense: antisense RNA hybrid duplexes. Nucleic Acids Res. 1993 Sep 11;21(18):4383–4391. doi: 10.1093/nar/21.18.4383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Xiong C., Levis R., Shen P., Schlesinger S., Rice C. M., Huang H. V. Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science. 1989 Mar 3;243(4895):1188–1191. doi: 10.1126/science.2922607. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. el Kharroubi A., Martin M. A. cis-acting sequences located downstream of the human immunodeficiency virus type 1 promoter affect its chromatin structure and transcriptional activity. Mol Cell Biol. 1996 Jun;16(6):2958–2966. doi: 10.1128/mcb.16.6.2958. [DOI] [PMC free article] [PubMed] [Google Scholar]

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