1887

Abstract

High mortality rates and lack of an available vaccine against Marburg haemorrhagic fever (MHF) highlight the need for a defensive therapy against MHF and greater knowledge of the causative agent, the Marburg virus (MARV). Here, RNA interference (RNAi) is employed to destroy MARV transcripts, disrupting replication and allowing analysis of various roles of MARV proteins. Small interfering RNAs (siRNAs) homologous to three MARV transcripts (NP, VP35 and VP30) were co-transfected into cells with plasmids encoding the corresponding nucleocapsid proteins. The resulting decrease in MARV nucleocapsid-protein levels was shown to be specific, as siRNA that was not homologous to the MARV genome did not decrease the levels of viral nucleocapsid proteins. Additionally, transcript levels of double-stranded RNA (dsRNA)-sensor proteins, the dsRNA-activated protein kinase and 2′,5′-oligoadenylate synthetase 1 remained unchanged, suggesting that the decrease in viral proteins was not a result of activation of the antiviral properties of the interferon system. Subsequently, siRNAs were shown to reduce intracellular viral proteins in MARV-infected cells and viral material released into the medium. Targeted reduction of VP30 downregulated the intracellular levels of all other viral proteins, suggesting that VP30 plays an essential role for transcription/replication. The efficient reduction of MARV replication also suggests that RNAi may provide an agent against MHF.

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2005-04-01
2019-11-12
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References

  1. Baize, S., Leroy, E. M., Georges-Courbot, M.-C., Capron, M., Lansoud-Soukate, J., Debré, P., Fisher-Hoch, S. P., McCormick, J. B. & Georges, A. J. ( 1999; ). Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients. Nat Med 5, 423–426.[CrossRef]
    [Google Scholar]
  2. Becker, S., Rinne, C., Hofsäß, U., Klenk, H.-D. & Mühlberger, E. ( 1998; ). Interactions of Marburg virus nucleocapsid proteins. Virology 249, 406–417.[CrossRef]
    [Google Scholar]
  3. Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. ( 2001; ). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366.[CrossRef]
    [Google Scholar]
  4. Bitko, V. & Barik, S. ( 2001; ). Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses. BMC Microbiol 1, 34.[CrossRef]
    [Google Scholar]
  5. Bridge, A. J., Pebernard, S., Ducraux, A., Nicoulaz, A.-L. & Iggo, R. ( 2003; ). Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 34, 263–264.[CrossRef]
    [Google Scholar]
  6. Der, S. D., Zhou, A., Williams, B. R. G. & Silverman, R. H. ( 1998; ). Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays. Proc Natl Acad Sci U S A 95, 15623–15628.[CrossRef]
    [Google Scholar]
  7. Elbashir, S. M., Lendeckel, W. & Tuschl, T. ( 2001a; ). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15, 188–200.[CrossRef]
    [Google Scholar]
  8. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. ( 2001b; ). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.[CrossRef]
    [Google Scholar]
  9. Feldmann, H. & Kiley, M. P. ( 1999; ). Classification, structure, and replication of filoviruses. Curr Top Microbiol Immunol 235, 1–21.
    [Google Scholar]
  10. Funke, C., Becker, S., Dartsch, H., Klenk, H.-D. & Mühlberger, E. ( 1995; ). Acylation of the Marburg virus glycoprotein. Virology 208, 289–297.[CrossRef]
    [Google Scholar]
  11. Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. ( 2000; ). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296.[CrossRef]
    [Google Scholar]
  12. Hutvágner, G., McLachlan, J., Pasquinelli, A. E., Bálint, E., Tuschl, T. & Zamore, P. D. ( 2001; ). A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838.[CrossRef]
    [Google Scholar]
  13. Kubota, K., Tsuda, S., Tamai, A. & Meshi, T. ( 2003; ). Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing. J Virol 77, 11016–11026.[CrossRef]
    [Google Scholar]
  14. Manche, L., Green, S. R., Schmedt, C. & Mathews, M. B. ( 1992; ). Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Biol 12, 5238–5248.
    [Google Scholar]
  15. McCaffrey, A. P., Nakai, H., Pandey, K., Huang, Z., Salazar, F. H., Xu, H., Wieland, S. F., Marion, P. L. & Kay, M. A. ( 2003; ). Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol 21, 639–644.[CrossRef]
    [Google Scholar]
  16. Modrof, J., Mühlberger, E., Klenk, H.-D. & Becker, S. ( 2002; ). Phosphorylation of VP30 impairs Ebola virus transcription. J Biol Chem 277, 33099–33104.[CrossRef]
    [Google Scholar]
  17. Modrof, J., Becker, S. & Mühlberger, E. ( 2003; ). Ebola virus transcription activator VP30 is a zinc-binding protein. J Virol 77, 3334–3338.[CrossRef]
    [Google Scholar]
  18. Mühlberger, E., Lötfering, B., Klenk, H.-D. & Becker, S. ( 1998; ). Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to mediate replication and transcription of Marburg virus-specific monocistronic minigenomes. J Virol 72, 8756–8764.
    [Google Scholar]
  19. Mühlberger, E., Weik, M., Volchkov, V. E., Klenk, H.-D. & Becker, S. ( 1999; ). Comparison of the transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems. J Virol 73, 2333–2342.
    [Google Scholar]
  20. Niwa, H., Yamamura, K. & Miyazaki, J. ( 1991; ). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199.[CrossRef]
    [Google Scholar]
  21. Reed, J. C., Kasschau, K. D., Prokhnevsky, A. I., Gopinath, K., Pogue, G. P., Carrington, J. C. & Dolja, V. V. ( 2003; ). Suppressor of RNA silencing encoded by Beet yellows virus. Virology 306, 203–209.[CrossRef]
    [Google Scholar]
  22. Saksela, K. ( 2003; ). Human viruses under attack by small inhibitory RNA. Trends Microbiol 11, 345–347.[CrossRef]
    [Google Scholar]
  23. Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H. & Williams, B. R. G. ( 2003; ). Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5, 834–839.[CrossRef]
    [Google Scholar]
  24. Stark, G. R., Kerr, I. M., Williams, B. R. G., Silverman, R. H. & Schreiber, R. D. ( 1998; ). How cells respond to interferons. Annu Rev Biochem 67, 227–264.[CrossRef]
    [Google Scholar]
  25. Thomas, C. L., Leh, V., Lederer, C. & Maule, A. J. ( 2003; ). Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306, 33–41.[CrossRef]
    [Google Scholar]
  26. Weik, M., Modrof, J., Klenk, H.-D., Becker, S. & Mühlberger, E. ( 2002; ). Ebola virus VP30-mediated transcription is regulated by RNA secondary structure formation. J Virol 76, 8532–8539.[CrossRef]
    [Google Scholar]
  27. Will, C., Mühlberger, E., Linder, D., Slenczka, W., Klenk, H.-D. & Feldmann, H. ( 1993; ). Marburg virus gene 4 encodes the virion membrane protein, a type I transmembrane glycoprotein. J Virol 67, 1203–1210.
    [Google Scholar]
  28. Zamore, P. D., Tuschl, T., Sharp, P. A. & Bartel, D. P. ( 2000; ). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33.[CrossRef]
    [Google Scholar]
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