1887

Abstract

While RNA interference (RNAi) has been widely used to study rotavirus gene function , the potential therapeutic role for RNAi has not been explored. To this end, we constructed two recombinant lentiviral vectors containing short hairpin RNA (shRNA) against non-structural protein-4 (NSP4) of bovine rotavirus (BRV), RNAi-351 and RNAi-492. RNAi-351 and RNAi-492 strongly suppressed the transient expression of a FLAG-tagged NSP4 fusion protein in 293T cells. In BRV-susceptible MA104 cells, RNAi-492 more potently silenced NSP4 mRNA than RNAi-351 and combination of the two shRNAs almost completely silenced viral NSP4 gene expression. While 100 % of suckling mice exposed to BRV and control shRNA developed severe diarrhoea, no suckling mice exposed to BRV in the presence of RNAi-492 or a combination of RNAi-492/RNAi-351 developed severe diarrhoea, and only 20 and 3.3 % developed mild diarrhoea, respectively. In addition, RNAi-492 and RNAi-351 markedly abrogated rotaviral replication in MA104 cells and significantly inhibited BRV replication in mouse pups. These results indicated that shRNAs silencing NSP4 gene had substantial antiviral properties and inhibited replication of BRV in a sequence-specific manner that may have clinical application.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.027680-0
2011-04-01
2019-11-17
Loading full text...

Full text loading...

/deliver/fulltext/jgv/92/4/945.html?itemId=/content/journal/jgv/10.1099/vir.0.027680-0&mimeType=html&fmt=ahah

References

  1. Alkhalil, A., Strand, S., Mucker, E., Huggins, J. W., Jahrling, P. B. & Ibrahim, S. M. ( 2009; ). Inhibition of monkeypox virus replication by RNA interference. Virol J 6, 188.[CrossRef]
    [Google Scholar]
  2. Ayala-Breton, C., Arias, M., Espinosa, R., Romero, P., Arias, C. F. & López, S. ( 2009; ). Analysis of the kinetics of transcription and replication of the rotavirus genome by RNA interference. J Virol 83, 8819–8831.[CrossRef]
    [Google Scholar]
  3. 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, 34–44.[CrossRef]
    [Google Scholar]
  4. Campagna, M., Eichwald, C., Vascotto, F. & Burrone, O. R. ( 2005; ). RNA interference of rotavirus segment 11 mRNA reveals the essential role of NSP5 in the virus replicative cycle. J Gen Virol 86, 1481–1487.[CrossRef]
    [Google Scholar]
  5. Chauhan, R. S. & Singh, N. P. ( 1996; ). Epidemiology of rotavirus infection in calves in India. International Journal of Animal Sciences 11, 221–223.
    [Google Scholar]
  6. Cuadras, M. A., Bordier, B. B., Zambrano, J. L., Ludert, J. E. & Greenberg, H. B. ( 2006; ). Dissecting rotavirus particle-raft interaction with small interfering RNAs: insights into rotavirus transit through the secretory pathway. J Virol 80, 3935–3946.[CrossRef]
    [Google Scholar]
  7. Déctor, M. A., Romero, P., López, S. & Arias, C. F. ( 2002; ). Rotavirus gene silencing by small interfering RNAs. EMBO Rep 3, 1175–1180.[CrossRef]
    [Google Scholar]
  8. Dhama, K., Chauhan, R. S., Mahendran, M. & Malik, S. V. ( 2009; ). Rotavirus diarrhea in bovines and other domestic animals. Vet Res Commun 33, 1–23.
    [Google Scholar]
  9. Díaz, Y., Chemello, M. E., Peña, F., Aristimuño, O. C., Zambrano, J. L., Rojas, H., Bartoli, F., Salazar, L., Chwetzoff, S. & other authors ( 2008; ). Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells. J Virol 82, 11331–11343.[CrossRef]
    [Google Scholar]
  10. Gitlin, L., Karelsky, S. & Andino, R. ( 2002; ). Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 418, 430–434.[CrossRef]
    [Google Scholar]
  11. He, H. & Sun, Y. ( 2007; ). Ribosomal protein S27L is a direct p53 target that regulates apoptosis. Oncogene 26, 2707–2716.[CrossRef]
    [Google Scholar]
  12. He, H., Tan, M., Pamarthy, D., Wang, G., Ahmed, K. & Sun, Y. ( 2007; ). CK2 phosphorylation of SAG at Thr10 regulates SAG stability, but not its E3 ligase activity. Mol Cell Biochem 295, 179–188.[CrossRef]
    [Google Scholar]
  13. Hummler, E., Cole, T. J., Blendy, J. A., Ganss, R., Aguzzi, A., Schmid, W., Beermann, F. & Schütz, G. ( 1994; ). Targeted mutation of the CREB gene: compensation within the CREB/ATF family of transcription factors. Proc Natl Acad Sci U S A 91, 5647–5651.[CrossRef]
    [Google Scholar]
  14. Kobinger, G. P., Weiner, D. J., Yu, Q. C. & Wilson, J. M. ( 2001; ). Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nat Biotechnol 19, 225–230.[CrossRef]
    [Google Scholar]
  15. Leonard, J. N. & Schaffer, D. V. ( 2006; ). Antiviral RNAi therapy: emerging approaches for hitting a moving target. Gene Ther 13, 532–540.[CrossRef]
    [Google Scholar]
  16. Li, M. J., Kim, J., Li, S., Zaia, J., Yee, J. K., Anderson, J., Akkina, R. & Rossi, J. J. ( 2005; ). Long-term inhibition of HIV-1 infection in primary hematopoietic cells by lentiviral vector delivery of a triple combination of anti-HIV shRNA, anti-CCR5 ribozyme, and a nucleolar-localizing TAR decoy. Mol Ther 12, 900–909.[CrossRef]
    [Google Scholar]
  17. López, T., Rojas, M., Ayala-Bretón, C., López, S. & Arias, C. F. ( 2005a; ). Reduced expression of the rotavirus NSP5 gene has a pleiotropic effect on virus replication. J Gen Virol 86, 1609–1617.[CrossRef]
    [Google Scholar]
  18. López, T., Camacho, M., Zayas, M., Nájera, R., Sánchez, R., Arias, C. F. & López, S. ( 2005b; ). Silencing the morphogenesis of rotavirus. J Virol 79, 184–192.[CrossRef]
    [Google Scholar]
  19. Montero, H., Arias, C. F. & Lopez, S. ( 2006; ). Rotavirus nonstructural protein NSP3 is not required for viral protein synthesis. J Virol 80, 9031–9038.[CrossRef]
    [Google Scholar]
  20. Parez, N., Fourgeux, C., Mohamed, A., Dubuquoy, C., Pillot, M., Dehee, A., Charpilienne, A., Poncet, D., Schwartz-Cornil, I. & Garbarg-Chenon, A. ( 2006; ). Rectal immunization with rotavirus virus-like particles induces systemic and mucosal humoral immune responses and protects mice against rotavirus infection. J Virol 80, 1752–1761.[CrossRef]
    [Google Scholar]
  21. Perrimon, N., Ni, J. Q. & Perkins, L. ( 2010; ). In vivo RNAi: today and tomorrow. Cold Spring Harb Perspect Biol 2, a003640.
    [Google Scholar]
  22. Poeschla, E., Corbeau, P. & Wong-Staal, F. ( 1996; ). Development of HIV vectors for anti-HIV gene therapy. Proc Natl Acad Sci U S A 93, 11395–11399.[CrossRef]
    [Google Scholar]
  23. Reed, L. J. & Muench, H. ( 1938; ). A simple method of estimating fifty per cent endpoints. Am J Hyg 27, 493–497.
    [Google Scholar]
  24. Sandrin, V., Russell, S. J. & Cosset, F. L. ( 2003; ). Targeting retroviral and lentiviral vectors. Curr Top Microbiol Immunol 281, 137–178.
    [Google Scholar]
  25. Sebat, J., Lakshmi, B., Troge, J., Alexander, J., Young, J., Lundin, P., Månér, S., Massa, H., Walker, M. & other authors ( 2004; ). Large-scale copy number polymorphism in the human genome. Science 305, 525–528.[CrossRef]
    [Google Scholar]
  26. Silvestri, L. S., Taraporewala, Z. F. & Patton, J. T. ( 2004; ). Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms. J Virol 78, 7763–7774.[CrossRef]
    [Google Scholar]
  27. Silvestri, L. S., Tortorici, M. A., Vasquez-Del Carpio, R. & Patton, J. T. ( 2005; ). Rotavirus glycoprotein NSP4 is a modulator of viral transcription in the infected cell. J Virol 79, 15165–15174.[CrossRef]
    [Google Scholar]
  28. Waterhouse, P. M., Wang, M. B. & Lough, T. ( 2001; ). Gene silencing as an adaptive defence against viruses. Nature 411, 834–842.[CrossRef]
    [Google Scholar]
  29. Westerhout, E. M., Ooms, M., Vink, M., Das, A. T. & Berkhout, B. ( 2005; ). HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res 33, 796–804.[CrossRef]
    [Google Scholar]
  30. Wolber, F. M., Broomfield, A. M., Fray, L., Cross, M. L. & Dey, D. ( 2005; ). Supplemental dietary whey protein concentrate reduces rotavirus-induced disease symptoms in suckling mice. J Nutr 135, 1470–1474.
    [Google Scholar]
  31. Wu, X., Li, Y., Crise, B. & Burgess, S. M. ( 2003; ). Transcription start regions in the human genome are favored targets for MLV integration. Science 300, 1749–1751.[CrossRef]
    [Google Scholar]
  32. Zambrano, J. L., Díaz, Y., Peña, F., Vizzi, E., Ruiz, M. C., Michelangeli, F., Liprandi, F. & Ludert, J. E. ( 2008; ). Silencing of rotavirus NSP4 or VP7 expression reduces alterations in Ca2+ homeostasis induced by infection of cultured cells. J Virol 82, 5815–5824.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.027680-0
Loading
/content/journal/jgv/10.1099/vir.0.027680-0
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error