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Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

Gene Therapy volume 10, pages 1258–1267 (2003)Cite this article

Abstract

The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5′/3′ ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems – exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5′/3′ ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.

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References

  1. Guerrier-Takada C et al. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 1983; 35: 849–857.

    Article  CAS  PubMed  Google Scholar 

  2. Macnaughton TB, Wang YJ, Lai MM . Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites. J Virol 1993; 67: 2228–2234.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Pyle AM, Cech TR . Ribozyme recognition of RNA by tertiary interactions with specific ribose 2′-OH groups. Nature 1991; 350: 628–631.

    Article  CAS  PubMed  Google Scholar 

  4. Kruger K et al. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 1982; 31: 147–157.

    Article  CAS  PubMed  Google Scholar 

  5. Burgin AB, Pace NR . Mapping the active site of ribonuclease P RNA using a substrate containing a photoaffinity agent. EMBO J 1990; 9: 4111–4118.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Forster AC, Symons RH . Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell 1987; 49: 211–220.

    Article  CAS  PubMed  Google Scholar 

  7. Luo G et al. A specific base transition occurs on replicating hepatitis delta virus RNA. J Virol 1990; 64: 1021–1027.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Chowrira BM, Berzal-Herranz A, Burke JM . Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature 1991; 354: 320–322.

    Article  CAS  PubMed  Google Scholar 

  9. Cotten M, Birnstiel M . Ribozyme mediated destruction of RNA in vivo. The EMBO J 1989; 8: 3861–3866.

    Article  CAS  PubMed  Google Scholar 

  10. Sarver NA et al. Ribozymes as potential anti-HIV-1 therapeutic agents. Science 1990; 247: 1222–1225.

    Article  CAS  PubMed  Google Scholar 

  11. He YK, Lu CD, Qi GR . In vitro cleavage of HPV16 E6 and E7 RNA fragments by synthetic ribozymes and transcribed ribozymes from RNA-trimming plasmids. FEBS Lett 1993; 322: 21–24.

    Article  CAS  PubMed  Google Scholar 

  12. Huang Y et al. Stable expression of anti-HPV 16 E7-ribozyme in CV-1 cell lines. Chin J Biotechnol 1996; 12: 215–220.

    CAS  PubMed  Google Scholar 

  13. Liu B, Tabler M, Tsagris M . Episomal expression of a hammerhead ribozyme directed against plum pox virus. Virus Res 2000; 68: 15–23.

    Article  CAS  PubMed  Google Scholar 

  14. Han S et al. Ribozyme-mediated resistance to rice dwarf virus and the transgene silencing in the progeny of transgenic rice plants. Transgenic Res 2000; 9: 195–203.

    Article  CAS  PubMed  Google Scholar 

  15. Lee NS et al. Functional colocalization of ribozymes and target mRNAs in Drosophila oocytes. FASEB J 2001; 15: 2390–2400.

    Article  CAS  PubMed  Google Scholar 

  16. Kashani-Sabet M et al. Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice. Proc Natl Acad Sci USA 2002; 99: 3878–3883.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Merlo AO et al. Ribozymes targeted to stearoyl-ACP delta9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves. Plant Cell 1998; 10: 1603–1622.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Perriman H, Bruening G, Dennis E, Peacock W . Effective ribozyme delivery in plant cells. Proc Natl Acad Sci USA 1995; 92: 6175–6179.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bertrand E et al. The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. RNA 1997; 3: 75–88.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Vaish NK, Kore AR, Eckstein F . Recent developments in the hammerhead ribozyme field. Nucleic Acids Res 1998; 26: 5237–5242.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Guo P, Erickson S, Anderson D . A small viral RNA is required for in vitro packaging of bacteriophage phi29 DNA. Science 1987; 236: 690–694.

    Article  CAS  PubMed  Google Scholar 

  22. Hoeprich S, Guo P . Computer modeling of three-dimensional structure of DNA-packaging RNA(pRNA) monomer, dimer, and hexamer of Phi29 DNA packaging motor. J Biol Chem 2002; 277: 20794–20803.

    Article  CAS  PubMed  Google Scholar 

  23. Garver K, Guo P . Boundary of pRNA functional domains and minimum pRNA sequence requirement for specific connector binding and DNA packaging of phage phi29. RNA 1997; 3: 1068–1079.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Chen C, Guo P . Magnesium-induced conformational change of packaging RNA for procapsid recognition and binding during phage phi29 DNA encapsidation. J Virol 1997; 71: 495–500.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Mat-Arip Y et al. Three-dimensional interaction of Phi29 pRNA dimer probed by chemical modification interference, Cryo-AFM, and cross-linking. J Biol Chem 2001; 276: 32575–32584.

    Article  CAS  PubMed  Google Scholar 

  26. Zhang C, Trottier M, Chen C, Guo P . Chemical modification patterns of active and inactive as well as procapsid-bound and unbound DNA-packaging RNA of bacterial virus Phi29. Virology 2001; 281: 281–293.

    Article  CAS  PubMed  Google Scholar 

  27. Trottier M et al. Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification. RNA 2000; 6: 1257–1266.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhang CL, Tellinghuisen T, Guo P . Conformation of the helical structure of the 5′/3′ termini of the essential DNA packaging pRNA of phage phi29. RNA 1995; 1: 1041–1050.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Zhang CL, Lee C-S, Guo P . The proximate 5′ and 3′ ends of the 120-base viral RNA (pRNA) are crucial for the packaging of bacteriophage phi29 DNA. Virology 1994; 201: 77–85.

    Article  CAS  PubMed  Google Scholar 

  30. Zhang CL, Tellinghuisen T, Guo P . Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29. RNA 1997; 3: 315–322.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Reid RJD, Zhang F, Benson S, Anderson D . Probing the structure of bacteriophage phi29 prohead RNA with specific mutations. J Biol Chem 1994; 269: 18656–18661.

    CAS  PubMed  Google Scholar 

  32. Wichitwechkarn J, Johnson D, Anderson D . Mutant Prohead RNAs in vitro packaging of bacteriophage phi29 DNA-gp3. Mol Biol 1992; 223: 991–998.

    Article  CAS  Google Scholar 

  33. Reid RJD, Bodley JW, Anderson D . Characterization of the prohead-pRNA interaction of bacteriophage phi29. J Biol Chem 1994; 269: 5157–5162.

    CAS  PubMed  Google Scholar 

  34. Zhang CL, Garver K, Guo P . Inhibition of phage phi29 assembly by antisense oligonucleotides targeting viral pRNA essential for DNA packaging. Virology 1995; 211: 568–576.

    Article  CAS  PubMed  Google Scholar 

  35. Trottier M, Guo P . Approaches to determine stoichiometry of viral assembly components. J Virol 1997; 71: 487–494.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Trottier M, Zhang CL, Guo P . Complete inhibition of virion assembly in vivo with mutant pRNA essential for phage phi29 DNA packaging. J Virol 1996; 70: 55–61.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Chen C, Sheng S, Shao Z, Guo P . A dimer as a building block in assembling RNA. A hexamer that gears bacterial virus phi29 DNA-translocating machinery. J Biol Chem 2000; 275: 17510–17516.

    Article  CAS  PubMed  Google Scholar 

  38. Zhang CL, Trottier M, Guo PX . Circularly permuted viral pRNA active and specific in the packaging of bacteriophage phi29 DNA. Virology 1995; 207: 442–451.

    Article  CAS  PubMed  Google Scholar 

  39. Guo P . Structure and function of phi29 hexameric RNA that drive viral DNA packaging motor: review. Prog Nucl Acid Res Mol Biol 2002; 72: 415–472.

    Article  CAS  Google Scholar 

  40. Feng Y, Kong Y, Wang Y, Qi G . Antiviral activity of a hammerhead ribozyme against HBV in HepG2.2.15 cells. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002; 34: 204–208.

    CAS  Google Scholar 

  41. Feng Y, Kong YY, Wang Y, Qi GR . Inhibition of hepatitis B virus by hammerhead ribozyme targeted to the poly(A) signal sequence in cultured cells. Biol Chem 2001; 382: 655–660.

    Article  CAS  PubMed  Google Scholar 

  42. Chen C, Zhang C, Guo P . Sequence requirement for hand-in-hand interaction in formation of pRNA dimers and hexamers to gear phi29 DNA translocation motor. RNA 1999; 5: 805–818.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Guo P et al. Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation. Mol Cell 1998; 2: 149–155.

    Article  CAS  PubMed  Google Scholar 

  44. Zhang F et al. Function of hexameric RNA in packaging of bacteriophage phi29 DNA in vitro. Mol Cell 1998; 2: 141–147.

    Article  CAS  PubMed  Google Scholar 

  45. Hendrix RW . Bacteriophage DNA packaging: RNA gears in a DNA transport machine (Minireview). Cell 1998; 94: 147–150.

    Article  CAS  PubMed  Google Scholar 

  46. Tuerk G, Gold L . Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA ploymerase. Science 1990; 249: 505–510.

    Article  CAS  PubMed  Google Scholar 

  47. Ellington AD, Szostak JW . In vitro selection of RNA molecules that bind specific ligands. Nature 1990; 346: 818–822.

    Article  CAS  PubMed  Google Scholar 

  48. Ciesiolka J, Gorskl J, Yarus M . Selection of an RNA domain that binds Zn++. RNA 1995; 1: 538–550.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Kraus E, James W, Barclay AN . Cutting edge: novel RNA ligands able to bind CD4 antigen and inhibit CD4+ T lymphocyte function. J Immunol 1998; 160(11): 5209–5212.

    CAS  PubMed  Google Scholar 

  50. Wang C, Jin YX, Wang DB . Selection with SELEX method of small RNA molecules specifically binding to starch. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1998; 30: 402–404.

    CAS  Google Scholar 

  51. Sayer N, Ibrahim J, Turner K, Tahiri-Alaoui A, James W . Structural characterization of a 2′F-RNA aptamer that binds a HIV-I SU glycoprotein, gp120. Biochem Biophys Res Commun 2002; 293(3): 924–931.

    Article  CAS  PubMed  Google Scholar 

  52. Bailey S et al. Phylogenetic analysis and secondary structure of the Bacillus subtilis bacteriophage RNA required for DNA packaging. J Biol Chem 1990; 265: 22365–22370.

    CAS  PubMed  Google Scholar 

  53. Pecenkova T et al. Bacteriophage B103: complete DNA sequence of its genome and relationship to other Bacillus phages. Gene 1997; 199: 157–163.

    Article  CAS  PubMed  Google Scholar 

  54. Wichitwechkarn J, Bailey S, Bodley JW, Anderson D . Prohead RNA of bacteriophage phi29: size, stoichiometry and biological activity. Nucleic Acids Res 1989; 17: 3459–3468.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Shu D, Guo P . A Viral RNA that binds ATP and contains a motif similar to an ATP-binding aptamer from SELEX. J Biol Chem 2003; 278(9): 7119–7125.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by NIH Grants GM59944, GM48159, and NSF Grant MCB-9723923 to Guo, a Grant G1999054105 from The Basic Research Program of the Ministry of Science and Technology of China to Wang. Hoeprich was supported by a Purdue University Presidential Scholarship and Showalter Foundation grant. We thank Dr Elikplimi Asem and Dr Harm Hogenesch for their comments, Jane Kovach, and Jeremy Hall for their assistance in the preparation of this manuscript.

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Authors and Affiliations

  1. Department of Pathobiology and Cancer Research Center, Purdue University, USA

    S Hoeprich, S Guo, D Shu & P Guo

  2. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, Shanghai, China

    Q Zhou, G Qi & Y Wang

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Hoeprich, S., Zhou, Q., Guo, S. et al. Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus. Gene Ther 10, 1258–1267 (2003). https://doi.org/10.1038/sj.gt.3302002

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