Skip to main content

Suppression of RNA interference pathway in vitro by Grass carp reovirus

Virologica Sinica volume 27, pages 109–119 (2012)Cite this article

Abstract

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

39,95 €

Price includes VAT (Finland)

References

  1. Aliyari R, Ding S W. 2009. RNA-based viral immunity initiated by the Dicer family of host immune receptors. Immunol Rev, 227(1): 176–188.

    Article  PubMed  CAS  Google Scholar 

  2. Attoui H, Fang Q, Jaafar F M, et al. 2002. Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae). J Gen Virol, 83: 1941–1951.

    PubMed  CAS  Google Scholar 

  3. Bagasra O, Prilliman K R. 2004. RNA interference: the molecular immune system. J Mol Histol, 35: 545–553.

    Article  PubMed  CAS  Google Scholar 

  4. Begemann G. 2008. MicroRNAs and RNA interference in zebrafish development. Zebrafish, 5(2): 111–119.

    Article  PubMed  CAS  Google Scholar 

  5. Blevins T, Rajeswaran R, Shivaprasad PV, et al. 2006. Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res, 34: 6233–6246.

    Article  PubMed  CAS  Google Scholar 

  6. Cao M, Ren H, Pan X, et al. 2004. Inhibition of EGFP expression by siRNA in EGFP-stably expressing Huh-7 cells. J Virol Methods, 119: 189–194.

    Article  PubMed  CAS  Google Scholar 

  7. Chang J, Provost P, Taylor J M. 2003. Resistance of human hepatitis delta virus RNAs to dicer activity. J Virol, 77(22): 11910–11917.

    Article  PubMed  CAS  Google Scholar 

  8. Csorba T, Burgyán J. 2011. Gel mobility shift assays for RNA binding viral RNAi suppressors. Methods Mol Biol, 721:245–252.

    Article  PubMed  CAS  Google Scholar 

  9. Dlakic M. 2006. DUF283 domain of Dicer proteins has a double-stranded RNA-binding fold. Bioinformatics, 22: 2711–2714.

    Article  PubMed  CAS  Google Scholar 

  10. Francki R I B, Fauquet C M, Knudson D L, et al. 1991. Classification and Nomenclature of Viruses: Fifth Report of the International Committee on Taxonomy of Viruses. New York: Springer-Verlag, 2.

    Google Scholar 

  11. Galiana-Arnoux D, Dostert C, Schneemann A, et al. 2006. Essential function in vivo for Dicer-2 in host defense against RNA viruses in dro-sophila. Nat Immunol, 7: 590–597.

    Article  PubMed  CAS  Google Scholar 

  12. Giraldez A J, Giraldez R M, Cinalli M E, et al. 2005. MicroRNAs regulate brain morphogenesis in zebrafish. Science, 308: 833–838.

    Article  PubMed  CAS  Google Scholar 

  13. Hammond S M. 2005. Dicing and slicing: the core machinery of the RNA interference pathway. FEBS Lett, 579: 5822–5829.

    Article  PubMed  CAS  Google Scholar 

  14. He Y, Xu H, Yang Q, et al. 2011. The use of an in vitro microneutralization assay to evaluate the potential of recombinant VP5 protein as an antigen for vaccinating against Grass carp reovirus. Virol J, 8: 132.

    Article  PubMed  CAS  Google Scholar 

  15. Ibrahim F, Rohr J, Jeong W, et al. 2006. Untemplated oligoadenylation promotes degradation of RISC-cleaved transcripts. Science, 314: 1893.

    Article  PubMed  CAS  Google Scholar 

  16. Jacobs B L, Langland J O. 1996. When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. Virology, 219(2): 339–349.

    Article  PubMed  CAS  Google Scholar 

  17. Ji W T, Chulu J L, Lin F L, et al. 2008. Suppression of protein expression of three avian reovirus S-class genome segments by RNA interference. Vet Microbiol, 129(3–4): 252–261.

    Article  PubMed  CAS  Google Scholar 

  18. Kobayashi T, Chappell J D, Danthi P, et al. 2006. Gene-specific inhibition of reovirus replication by RNA interference. J Virol, 80(18): 9053–9063.

    Article  PubMed  CAS  Google Scholar 

  19. Kobayashi T, Ooms L S, Chappell J D, et al. 2009. Identification of functional domains in reovirus replication proteins muNS and mu2. J Virol, 83(7): 2892–906.

    Article  PubMed  CAS  Google Scholar 

  20. Li B, Fan Y D, Li Y Q, et al. 2009. Highly efficient inhibition on replication of grass carp reovirus mediated by chemically synthesized small interfering RNAs. Chin J Virol, 25(5): 388–394. (in Chinese)

    CAS  Google Scholar 

  21. Liao S, Chen Y, Du F, et al. 2010. Resistance of siGCRV transgenic rare minnow (Gobiocypris Rarus) to grass carp reovirus. Acta Hydrobiol Sin, 34(4): 837–842. (in Chinese)

    Article  CAS  Google Scholar 

  22. Lu L, Ho Y, Kwang J. 2006. Suppression of porcine arterivirus replication by baculovirus-delivered shRNA targeting nucleoprotein. Biochem Biophys Res Commun, 340(4): 1178–1183.

    Article  PubMed  CAS  Google Scholar 

  23. Ma J, Wang W, Zeng L, et al. 2011. Inhibition of the replication of grass carp reovirus in CIK cells with plasmid-transcribed shRNAs. J Virol Methods, 175(2): 182–187.

    Article  PubMed  CAS  Google Scholar 

  24. Matsumoto G, Kushibiki T, Kinoshita Y, et al. 2006. Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma. Cancer Sci, 97: 313–321.

    Article  PubMed  CAS  Google Scholar 

  25. Moissiard G, Voinnet O. 2004. Viral suppression of RNA silencing in plants. Mol Plant Pathol, 5(1): 71–82.

    Article  PubMed  CAS  Google Scholar 

  26. Moissiard G, Voinnet O. 2006. RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc Natl Acad Sci USA, 103: 19593–19598.

    Article  PubMed  CAS  Google Scholar 

  27. Parker G S, Eckert D M, Bass B L. 2006. RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. RNA, 12: 807–818.

    Article  PubMed  CAS  Google Scholar 

  28. Patrick J, Paddison P J, Caudy A A, et al. 2002. Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A, 99(3): 1443–1448.

    Article  Google Scholar 

  29. Qiu T, Lu R, Zhang J, et al. 2001. Complete nucleotide sequence of the S10 genome segment of grass carp reovirus (GCRV). Dis Aquat Org, 44: 69–74.

    Article  PubMed  CAS  Google Scholar 

  30. Qu S, Zhang X, Zheng C, et al. 2000. Apoptosis of the cell line from grass carp (CIK) induced by fish reovirus. Acta Hydrobiol Sin, 24(6): 616–620. (in Chinese)

    Google Scholar 

  31. Su J, Zhu Z, Wang Y. 2008. Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus. Fish Shellfish Immunol, 25: 106–113.

    Article  PubMed  CAS  Google Scholar 

  32. Takeda A, Tsukuda M, Mizumoto H, et al. 2005. A plant RNA virus suppresses RNA silencing through viral RNA replication. EMBO J, 24(17): 3147–3157.

    Article  PubMed  CAS  Google Scholar 

  33. Tuschl T, Zamore P D, Lehmann R, et al. 1999. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev, 13: 3191–3197.

    Article  PubMed  CAS  Google Scholar 

  34. Wang L, Zhou J Y, Yao J H, et al. 2010. U6 promoter-driven siRNA injection has nonspecific effects in zebrafish. Biochem. Biophys._Res Commun, 391(3): 1363–1368.

    Article  PubMed  CAS  Google Scholar 

  35. Wang T, Liu P, Chen H. 1994. Preliminary study on the susceptibility of Gobiocypris rarus to hemorrhagic virus of grass carp (GCHV). Acta Hydrobiol Sin, 18: 144–149. (in chinese)

    Google Scholar 

  36. Wang X H, Aliyari R, Li W X, et al. 2006. RNA interference directs innate immunity against viruses in adult Drosophila. Science, 312: 452–454.

    Article  PubMed  CAS  Google Scholar 

  37. Weber F, Wagner V, Rasmussen S B, et al. 2006. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol, 80 (10): 5059–5064.

    Article  Google Scholar 

  38. Zhang C, Wang Q, Shi C, et al. 2010. Molecular analysis of grass carp reovirus HZ08 genome segments 1–3 and 5–6. Virus Genes, 41(1): 102–104.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Key Laboratory of Aquatic Genetic Resources and Utilization /Ministry of Agriculture, Shanghai Ocean University, Shanghai, 201306, China

    Shuai Guo, Dan Xu, Tu Wang, Jia-le Li & Li-qun Lu

  2. Department of Laboratory Medicine, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080, China

    Hong-xu Xu

Authors
  1. Shuai Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hong-xu Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jia-le Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Li-qun Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Li-qun Lu.

Additional information

Foundation items: The Shanghai committee of Science and Technology (Grant No.10PJ1404800) and the National Natural Science Foundation of China (Grant No. 31072244).

These authors contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Guo, S., Xu, D., Xu, Hx. et al. Suppression of RNA interference pathway in vitro by Grass carp reovirus. Virol. Sin. 27, 109–119 (2012). https://doi.org/10.1007/s12250-012-3230-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12250-012-3230-4

Key words

  • Grass carp reovirus (GCRV)
  • RNA interference (RNAi)
  • Dicer
  • Northern blot