Skip to main content

Advertisement

SpringerLink
Log in

Inhibition of influenza A virus replication by RNA interference targeted against the PB1 subunit of the RNA polymerase gene

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

Influenza (flu) pandemics have posed a great threat to human health in the last century. However, current vaccination strategies and antiviral drugs provide limited protection. RNA interference (RNAi) is an effective means of suppressing influenza virus replication. PB1 is the critical protein subunit of the influenza virus RNA polymerase. The gene encoding this protein, PB1, is highly conserved among different subtypes of IAV and was therefore chosen as the target in this study. The oligonucleotide, PB1-shRNA, contains a 21-bp siRNA corresponding to nucleotides 1,632 to 1,652 of PB1 linear vRNA with BamHI or EcoRI restriction enzyme sites incorporated at the ends. The PB1-shRNA oligonucleotide was directionally cloned into the RNAi-ready pSIREN-shuttle vector. The correct structure of the resulting pSIREN/PB1 plasmid was confirmed by restriction endonuclease digestion. Madin-Darby canine kidney (MDCK) cells were transfected with pSIREN/PB1 and subsequently infected with IAV at an MOI of 0.1 (A/PR/8/34, H1N1). The virus titer in cell culture supernatants was determined 48 hours later, and it was found that virus growth was inhibited by more than 50-fold relative to controls. Furthermore, embryonated eggs and mice were inoculated with liposome-encapsulated pSIREN/PB1 and then challenged with the A/PR/8/34 virus. The results showed at least a 100-fold inhibition in virus replication in egg allantoic fluid and a survival rate of between 50% and 100% in experimental mice. This study demonstrates that PB1-shRNA expressed by the recombinant plasmid pSIREN/PB1 inhibits influenza A virus replication both in vitro and in vivo. These observations provide a foundation for the development of a new and efficient treatment of influenza infections.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Tumpey TM, Basler CF, Aguilar PV et al (2005) Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 310(5745):77–80

    Article  PubMed  CAS  Google Scholar 

  2. Fouchier R, Kuiken T, Rimmelzwaan G et al (2005) Global task force for influenza. Nature 435:419–420

    Article  PubMed  CAS  Google Scholar 

  3. Lewis DB (2006) Avian flu to human influenza. Annu Rev Med 57:139–154

    Article  PubMed  CAS  Google Scholar 

  4. Gambotto A, Barratt-Boyes SM, de Jong MD et al (2008) Human infection with highly pathogenic H5N1 influenza virus. Lancet 371(9622):1464–1475

    Article  PubMed  CAS  Google Scholar 

  5. Neumann G, Noda T, Kawaoka Y (2009) Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459:931–939

    Article  PubMed  CAS  Google Scholar 

  6. Smith GJ, Vijaykrishna D, Bahl J et al (2009) Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459:1122–1125

    Article  PubMed  CAS  Google Scholar 

  7. Naffakh N, Werf SV (2009) An outbreak of Swine-origin influenza A (H1N1) virus with evidence for human to human transmission. Microbes Infect 11:725–728

    Article  PubMed  Google Scholar 

  8. Wright PF, Neumann G, Kawaoka Y (2007) Orthomyxoviruses. In: Knipe DM, Howley P, Griffin DE, Lamb RA, Martin MA (eds) Fields virology, 5th edn. Lippincott Williams & Wilkins, New York, pp 1692–1740

    Google Scholar 

  9. Gu J, Xie Z, Gao Z et al (2007) H5N1 infection of the respiratory tract and beyond: a molecular pathology study. Lancet 370(9593):1137–1145

    Google Scholar 

  10. Lowen AC, Mubareka S, Tumpey TM et al (2006) The guinea pig as a transmission model for human influenza viruses. Proc Natl Acad Sci USA 103:9988–9992

    Article  PubMed  CAS  Google Scholar 

  11. Ottolini MG, Blanco JCG, Eichelberger MC et al (2005) The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis. J Gen Virol 86:2823–2830

    Article  PubMed  CAS  Google Scholar 

  12. Grimm D, Kay MA (2007) Combinatorial RNAi: a winning strategy for the race against evolving targets. Am Soc Gene Therapy 15(5):878–888

    CAS  Google Scholar 

  13. Kim D, Rossi J (2008) RNAi Mechanisms and applications. Biotechniques 44(5):613–616

    Article  PubMed  CAS  Google Scholar 

  14. Zhang P, Wang J, Wan JY et al (2010) Screening efficient siRNA in vitro as the candidate genes for chicken anti-avian influenza virus H5N1 breeding. Mol Biol 44(1):37–44

    Article  CAS  Google Scholar 

  15. Hao L, Sakurai A, Watanabe T et al (2008) Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature 454(7206):890–893

    Article  PubMed  CAS  Google Scholar 

  16. Takahashi T, Murakami K, Nagakura M et al (2008) Sulfatide is required for efficient replication of influenza A virus. J Virol 82(12):5940–5950

    Article  PubMed  CAS  Google Scholar 

  17. Kai Z, Hongxuan H, Mingxing D (2008) RNA interference of avian influenza virus H5N1 by inhibiting viral mRNA with siRNA expression plasmids. J Biotechnol 135:140–144

    Article  Google Scholar 

  18. Hongbo Z, Meilin J, Zhengjun Y (2007) Effective small interfering RNAs targeting matrix and nucleocapsid protein gene inhibit influenza A virus replication in cells and mice. Antiviral Res 76:186–193

    Article  Google Scholar 

  19. Sui HY, Zhao GY, Huang JD (2009) Small interfering RNA targeting m2 gene induces effective and long term inhibition of influenza A virus replication. PLoS ONE 4(5):e5671. doi:10.1371

    Article  PubMed  Google Scholar 

  20. Reynolds A, Leake D, Boese Q et al (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330

    Article  PubMed  CAS  Google Scholar 

  21. Liu F, Song YK, Liu D (1999) Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Therapy 6:1258–1266

    Article  PubMed  CAS  Google Scholar 

  22. Tamura S, Ito Y, Asanuma H et al (1992) Cross-protection against influenza virus infection afforded by trivalent inactivated vaccines inoculated intranasally with cholera toxin B subunit. J Immunol 149:981–988

    PubMed  CAS  Google Scholar 

  23. WHO. Influenza Fact Sheet No. 211. http://www.who.int/mediacentre/factsheets/fs211/en/

  24. Smith NM, Bresee JS, Shay DK et al (2006) Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). Advisory Committee on Immunization Practices. MMWR Recomm Rep 55:1–42

    PubMed  Google Scholar 

  25. Leroux-Roels I, Borkowski A, Vanwolleghem T et al (2007) Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised controlled trial. Lancet 370:580–589

    Article  PubMed  CAS  Google Scholar 

  26. Tompkins SM, Lo CY, Tumpey TM et al (2004) Protection against lethal influenza virus challenge by RNA interference in vivo. PNAS 101:8682–8686

    Article  PubMed  CAS  Google Scholar 

  27. Lee NS, Dohjima T, Bauer G et al (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechol 19:500–505

    Google Scholar 

  28. He G, Zhang S, Wang C et al (2005) Study on time and dose of RNA interference induced by short hairpin loop RNA. Adv Biochem Biophys 32:258–266

    CAS  Google Scholar 

  29. Zhang G, Gao X, Song YK et al (2004) Hydroporation as the mechanism of hydrodynamic delivery. Gene Therapy 11:675–682

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported financially by the Ministry of Education Foundation of China and the National Natural Science Foundation of China (No. 30973235) and the China Medical Board (CMB) Foundation from Sichuan University.

Author information

Authors and Affiliations

  1. Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, 610041, Sichuan, China

    Wanyi Li, Xiaofan Yang, Baoning Wang, Yuan Yang, Zhonghua Jiang & Mingyuan Li

  2. Department of Microbiology, Guiyang Medical College, Guiyang, 550004, China

    Yan Jiang

  3. State Key Laboratory of Oral Diseases (Sichuan University), Chengdu, 610041, Sichuan, China

    Mingyuan Li

Authors
  1. Wanyi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaofan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yan Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Baoning Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhonghua Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mingyuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mingyuan Li.

Additional information

W. Li and X. Yang contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, W., Yang, X., Jiang, Y. et al. Inhibition of influenza A virus replication by RNA interference targeted against the PB1 subunit of the RNA polymerase gene. Arch Virol 156, 1979–1987 (2011). https://doi.org/10.1007/s00705-011-1087-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-011-1087-8

Keywords

Search

Navigation