1887

Abstract

DNAzyme (Dz) molecules have been shown to be highly efficient inhibitors of virus replication. Hepatitis C virus RNA translation is mediated by an internal ribosome entry site (IRES) element located mostly in the 5′ untranslated region (UTR), the mechanism of which is fundamentally different from cap-dependent translation of cellular mRNAs, and thus an attractive target for designing antiviral drugs. Inhibition of HCV IRES-mediated translation has drastic consequences for the replication of viral RNA as well. We have designed several Dzs, targeting different regions of HCV IRES specific for 1b and also sequences conserved across genotypes. The RNA cleavage and translation inhibitory activities of these molecules were tested in a cell-free system and in cell culture using transient transfections. The majority of Dzs efficiently inhibited HCV IRES-mediated translation. However, these Dz molecules did not show significant inhibition of coxsackievirus B3 IRES-mediated translation or cap-dependent translation of reporter gene, showing high level of specificity towards target RNA. Also, Northern blot hybridization analysis showed significant cleavage of HCV IRES by the Dz molecules in Huh7 cells transiently transfected with the HCV–FLuc monocistronic construct. Interestingly, one of the Dzs was more effective against genotype1b, whereas the other showed significant inhibition of viral RNA replication in Huh7 cells harbouring a HCV 2a monocistronic replicon. As expected, mutant-Dz failed to cleave RNA and inhibit HCV RNA translation, showing the specificity of inhibition. Taken together, these findings suggest that the Dz molecule can be used as selective and effective inhibitor of HCV RNA replication, which can be explored further for development of a potent therapeutic agent against HCV infection.

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2008-07-01
2019-11-22
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References

  1. Ackermann, J. M., Kanugula, S. & Pegg, A. E. ( 2005; ). DNAzyme-mediated silencing of ornithine decarboxylase. Biochemistry 44, 2143–2152.[CrossRef]
    [Google Scholar]
  2. Asahina, Y., Ito, Y., Wu, C. H. & Wu, G. Y. ( 1998; ). DNA ribonucleases that are active against intracellular hepatitis B viral RNA targets. Hepatology 28, 547–554.[CrossRef]
    [Google Scholar]
  3. Banerjea, A. C., Chakraborti, S., Unwalla, H., Goila, R., Shrabani, B., Dash, B. C. Sriram, B., Parasivam, M. & Viswanathan, S. ( 2004; ). Potential therapeutic application of DNA enzymes and siRNAs against viral and cellular genes. In Synthetic Nucleic Acids as Inhibitors of Gene Expression: Mechanisms, Applications, and Therapeutic Implications, pp. 115–134. Edited by L. M. Khachigian. Boca Raton, FL: CRC Press.
  4. Bartenschlager, R., Frese, M. & Pietschmann, T. ( 2004; ). Novel insights into hepatitis C virus replication and persistence. Adv Virus Res 63, 71–180.
    [Google Scholar]
  5. Blight, K. J., McKeating, J. A. & Rice, C. M. ( 2002; ). Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 76, 13001–13014.[CrossRef]
    [Google Scholar]
  6. Brown, E. A., Zhang, H., Ping, L. H. & Lemon, S. M. ( 1992; ). Secondary structure of the 5′ nontranslated regions of hepatitis C virus and pestivirus. Nucleic Acids Res 20, 5041–5045.[CrossRef]
    [Google Scholar]
  7. Dasgupta, A., Das, S., Izumi, R., Venkatesan, A. & Barat, B. ( 2004; ). Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses. FEMS Microbiol Lett 15, 189–199.
    [Google Scholar]
  8. Dash, B. C. & Banerjea, A. C. ( 2004; ). Sequence-specific cleavage activities of DNA enzymes targeted against HIV-1 Gag and Nef regions. Oligonucleotides 14, 41–47.[CrossRef]
    [Google Scholar]
  9. Dhar, D., Roy, S. & Das, S. ( 2007; ). Translational control of the interferon regulatory factor 2 mRNA by IRES element. Nucleic Acids Res 35, 5409–5421.[CrossRef]
    [Google Scholar]
  10. Gil, J. & Esteban, M. ( 2000; ). Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 5, 107–114.[CrossRef]
    [Google Scholar]
  11. Goila, R. & Banerjea, A. C. ( 1998; ). Sequence specific cleavage of the HIV-1 coreceptor CCR5 gene by a hammer-head ribozyme and a DNA-enzyme: Inhibitions of the coreceptor function by DNA-enzyme. FEBS Lett 436, 233–238.[CrossRef]
    [Google Scholar]
  12. Goila, R. & Banerjea, A. C. ( 2001; ). Inhibition of hepatitis B virus X gene expression by novel DNA enzymes. Biochem J 353, 701–708.[CrossRef]
    [Google Scholar]
  13. Goila, R. & Banerjea, A. C. ( 2004; ). Sequence-specific cleavage of hepatitis X RNA in cis and trans by novel monotarget and multitarget hammerhead motif-containing ribozymes. Oligonucleotides 14, 249–262.[CrossRef]
    [Google Scholar]
  14. Gupta, R., Subramani, M., Khaja, M. N., Madhavi, C., Roy, S., Habibullah, M. C. & Das, S. ( 2006; ). Analysis of mutations within the 5′ untranslated region, interferon sensitivity region, and PePHD region as a function of response to interferon therapy in hepatitis C virus-infected patients in India. J Clin Microbiol 44, 709–715.[CrossRef]
    [Google Scholar]
  15. Hellen, C. U. T. & Sarnow, P. ( 2001; ). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev 15, 1593–1612.[CrossRef]
    [Google Scholar]
  16. Jarczak, D., Kofr, M., Beger, C., Manns, M. P. & Kruger, M. ( 2005; ). Hairpin ribozymes in combination with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons. FEBS J 272, 5910–5922.[CrossRef]
    [Google Scholar]
  17. Joyce, G. F. ( 2004; ). Directed evolution of nucleic acid enzymes. Annu Rev Biochem 73, 791–836.[CrossRef]
    [Google Scholar]
  18. Kusunoki, A., Miyano-Kurosaki, N. & Takaku, H. ( 2003; ). A novel single-stranded DNA enzyme expression system using HIV-1 reverse transcriptase. Biochem Biophys Res Commun 301, 535–539.[CrossRef]
    [Google Scholar]
  19. Lohmann, V., Körner, F., Koch, J., Herian, U., Theilmann, L. & Bartenschlager, R. ( 1999; ). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113.[CrossRef]
    [Google Scholar]
  20. Oketani, M., Asahina, Y., Wu, C. H. & Wu, G. Y. ( 1999; ). Inhibition of hepatitis C virus-directed gene expression by a DNA ribonuclease. J Hepatol 31, 628–634.[CrossRef]
    [Google Scholar]
  21. Pudi, R., Abhiman, S., Srinivasan, N. & Das, S. ( 2003; ). Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by specific interaction of independent regions of human La autoantigen. J Biol Chem 278, 12231–12240.[CrossRef]
    [Google Scholar]
  22. Ramani, K., Bora, R. S., Kumar, M., Tyagi, S. K. & Sarkar, D. P. ( 1997; ). Novel gene delivery to liver cells using engineered virosomes. FEBS Lett 404, 164–168.[CrossRef]
    [Google Scholar]
  23. Santoro, S. W. & Joyce, G. F. ( 1997; ). A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci U S A 94, 4262–4266.[CrossRef]
    [Google Scholar]
  24. Santoro, S. W. & Joyce, G. F. ( 1998; ). Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry 37, 13330–13342.[CrossRef]
    [Google Scholar]
  25. Silverman, S. K. ( 2005; ). In vitro selection, characterization, and application of deoxyribozymes that cleave RNA. Nucleic Acids Res 33, 6151–6163.[CrossRef]
    [Google Scholar]
  26. Sood, V., Unwalla, H., Gupta, N., Chakraborti, S. & Banerjea, A. C. ( 2007; ). Potent knock down of HIV-1 replication by targeting HIV-1 Tat/Rev RNA sequences synergistically with catalytic RNA and DNA. AIDS 21, 31–40.[CrossRef]
    [Google Scholar]
  27. Sun, L. Q., Cairns, M. J., Gerlach, W. L., Witherington, C., Wang, L. & King, A. ( 1999; ). Suppression of smooth muscle cell proliferation by a c-myc RNA-cleaving deoxyribozyme. J Biol Chem 274, 17236–17241.[CrossRef]
    [Google Scholar]
  28. Trepanier, J., Tanner, J. E., Momparler, R. L., Le, O. N. L., Alvarez, F. & Alfieri, C. ( 2006; ). Cleavage of intracellular hepatitis C RNA i the virus core protein coding region by deoxyribozymes. J Viral Hepat 13, 131–138.[CrossRef]
    [Google Scholar]
  29. Unwalla, H. & Banerjea, A. C. ( 2001; ). Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA. Biochem J 357, 147–155.[CrossRef]
    [Google Scholar]
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