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Inhibition of Respiratory Syncytial Virus Replication by Simultaneous Targeting of mRNA and Genomic RNA Using Dual-Targeting siRNAs

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Abstract

We attempted to generate siRNAs with two active strands, which can simultaneously knock down the expression of mRNA and viral genomic RNA. In this study, short hairpin RNAs (shRNAs) against N and F genes were used. Expression of F and N mRNA transcripts as well as genomic RNA was determined with relative real-time RT-PCR. The RSV load in infected cell culture supernatant was determined by absolute quantitative real-time PCR. We found that (i) in the presence of shRNA-N, a greater reduction in viral genomic RNA was found; (ii) the level of expression at MOI 0.01 was reduced more than MOI 0.1; (iii) reduction in N transcript was greater than F; and (iv) finally, in combination pre-treatment with two shRNAs, the reduction was not significant as compared to single shRNA transfection. shRNAs also inhibited the production of RSV progeny as shown by viral load in infected HEp-2 cells. (i) Virus load reduction was greater at MOI 0.01 than 0.1 and (ii) significant load reduction was not seen with combination shRNA pre-treatment. The antiviral potency was also confirmed by plaque assay and western blot analysis. Our results provided further evidence that RNAi could be a powerful treatment option against respiratory viruses.

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References

  1. Tapia, L. I., Shaw, C. A., Aideyan, L. O., Jewell, A. M., Dawson, B. C., Haq, T. R., et al. (2014). Gene sequence variability of the three surface proteins of human respiratory Syncytial virus (HRSV) in texas. PLoS ONE, 9, e90786.

    Article  Google Scholar 

  2. Malekshahi, S. S., Azad, T. M., Yavarian, J., Shahmahmoodi, S., Naseri, M., & Rezaei, F. (2010). Molecular detection of respiratory viruses in clinical specimens from children with acute respiratory disease in Iran. Pediatric Infectious Disease Journal, 29, 931–933.

    Article  Google Scholar 

  3. Murray, J., Saxena, S., & Sharland, M. (2014). Preventing severe respiratory syncytial virus disease: Passive, active immunisation and new antivirals. Archives of Disease in Childhood, 99, 469–473.

    Article  Google Scholar 

  4. Peter, L., & Collins, R. A. K. (2013). Fields Virology, chapter 38. Respiratory syncytial virus and metapneumovirus (6th ed.). Philidelphia: Lippincott Williams & Wilkins.

    Google Scholar 

  5. Aamir, U. B., Alam, M. M., Sadia, H., Zaidi, S. S., & Kazi, B. M. (2013). Molecular characterization of circulating respiratory syncytial virus (RSV) genotypes in Gilgit-Baltistan province of Pakistan during 2011 to 2012 Winter Season. PLoS One, 8, e74018.

    Article  CAS  Google Scholar 

  6. Boivin, G., Caouette, G., Frenette, L., Carbonneau, J., Ouakki, M., & De Serres, G. (2008). Human respiratory syncytial virus and other viral infections in infants receiving palivizumab. Journal of Clinical Virology, 42, 52–57.

    Article  CAS  Google Scholar 

  7. Zhu, Q., Patel, N. K., McAuliffe, J. M., Zhu, W., Wachter, L., McCarthy, M. P., et al. (2012). Natural polymorphisms and resistance-associated mutations in the fusion protein of respiratory syncytial virus (RSV): Effects on RSV susceptibility to palivizumab. Journal of Infectious Disease, 205, 635–638.

    Article  CAS  Google Scholar 

  8. Vig, K., Lewis, N., Moore, E. G., Pillai, S., Dennis, V. A., & Singh, S. R. (2009). Secondary RNA structure and its role in RNA interference to silence the respiratory syncytial virus fusion protein gene. Molecular Biotechnology, 43, 200–211.

    Article  CAS  Google Scholar 

  9. Tian, J., Huang, K., Krishnan, S., Svabek, C., Rowe, D. C., Brewah, Y., et al. (2013). RAGE inhibits human respiratory syncytial virus syncytium formation by interfering with F-protein function. Journal of General Virology, 94, 1691–1700.

    Article  CAS  Google Scholar 

  10. Singleton, R., Etchart, N., Hou, S., & Hyland, L. (2003). Inability to evoke a long-lasting protective immune response to respiratory syncytial virus infection in mice correlates with ineffective nasal antibody responses. Journal of Virology, 77, 1131–11303.

    Article  Google Scholar 

  11. Eckardt-Michel, J., Lorek, M., Baxmann, D., Grunwald, T., Keil, G. M., & Zimmer, G. (2008). The fusion protein of respiratory syncytial virus triggers p53-dependent apoptosis. Journal of Virology, 82, 3236–3249.

    Article  CAS  Google Scholar 

  12. Galloux, M., Tarus, B., Blazevic, I., Fix, J., Duquerroy, S., & Eléouët, J. F. (2012). Characterization of a viral phosphoprotein binding site on the surface of the respiratory syncytial nucleoprotein. Journal of Virology, 86, 8375–8387.

    Article  CAS  Google Scholar 

  13. Cowton, V. M., McGivern, D. R., & Fearns, R. (2006). Unravelling the complexities of respiratory syncytial virus RNA synthesis. Journal of General Virology, 87, 1805–1821.

    Article  CAS  Google Scholar 

  14. Ramilo, O. (2009). Evolution of prophylaxis: MoAb, siRNA, vaccine, and small molecules. Paediatrics Respiratory Review, 10, 23–25.

    Article  Google Scholar 

  15. Empey, K. M., Peebles, R. S., Jr., & Kolls, J. K. (2010). Pharmacologic advances in the treatment and prevention of respiratory syncytial virus. Clinical infectious disseas, 50, 1258–1267.

    Article  CAS  Google Scholar 

  16. Whittaker, R. K., & Whittaker, P. A. (2005). RNA interference: From gene silencing to gene-specific therapeutics. Pharmacology & Therapeutics, 107, 222–239.

    Article  Google Scholar 

  17. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., & Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498.

    Article  CAS  Google Scholar 

  18. Barik, S. (2004). Control of nonsegmented negative-strand RNA virus replication by siRNA. Virus Research, 102, 27–35.

    Article  CAS  Google Scholar 

  19. Wei, J. X., Yang, J., Sun, J. F., Jia, L. T., Zhang, Y., Zhang, H. Z., et al. (2009). Both strands of siRNA have potential to guide posttranscriptional gene silencing in mammalian cells. PLoS ONE, 4, e5382.

    Article  Google Scholar 

  20. Hutvagner, G. (2005). Small RNA asymmetry in RNAi: Function in RISC assembly and gene regulation. FEBS letter, 579, 5850–5857.

    Article  CAS  Google Scholar 

  21. Chang, C. I., Kang, H. S., Ban, C., Kim, S., & Lee, D. K. (2009). Dual-target gene silencing by using long, synthetic siRNA duplexes without triggering antiviral responses. Molecular Cell, 27, 689–695.

    Article  CAS  Google Scholar 

  22. Tiemann, K., Höhn, B., Ehsani, A., Forman, S. J., Rossi, J. J., & Saetrom, P. (2010). Dual-targeting siRNAs. RNA, 16, 1275–1284.

    Article  CAS  Google Scholar 

  23. McIntyre, G. J. A. N. D., & Fanning, G. C. (2006). Design and cloning strategies for constructing shRNA expression vectors. BMC Biotechnology, 6, 1.

    Article  Google Scholar 

  24. Hajeri, P. B. A. N. D., & Singh, S. K. (2009). siRNAs: Their potential as therapeutic agents–Part I. Designing of siRNAs. Drug discovery today, 14, 851–858.

    Article  CAS  Google Scholar 

  25. Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki-Hamazaki, H., Juni, A., et al. (2004). Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Research, 32, 936–948.

    Article  CAS  Google Scholar 

  26. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., & Khvorova, A. (2004). Rational siRNA design for RNA interference. Nature Biotechnology, 22, 326–330.

    Article  CAS  Google Scholar 

  27. Jagla, B., Aulner, N., Kelly, P. D., Song, D., Volchuk, A., Zatorski, A., et al. (2005). Sequence characteristics of functional siRNAs. RNA, 11, 864–872.

    Article  CAS  Google Scholar 

  28. Shatizadeh Malekshahi, S., Arefian, E., Salimi, V., Mokhtari Azad, T., & Yavarian, J. (2016). Potential siRNA molecules for nucleoprotein and M2/L overlapping region of respiratory syncytial virus: In silico design. Jundishapur Journal Microbiology, 9, e34304.

    Google Scholar 

  29. 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 Microbiology, 1, 34.

    Article  CAS  Google Scholar 

  30. Bitko, V., Musiyenko, A., Shulyayeva, O., & Barik, S. (2004). Inhibition of respiratory viruses by nasally administered siRNA. Natute medicine, 11, 50–55.

    Article  Google Scholar 

  31. Kong, X., Zhang, W., Lockey, R. F., Auais, A., Piedimonte, G., & Mohapatra, S. S. (2007). Respiratory syncytial virus infection in Fischer 344 rats is attenuated by short interfering RNA against the RSV-NS1 gene. Genetics Vaccines & Therapy, 5, 593–598.

    Google Scholar 

  32. Zhang, W., Yang, H., Kong, X., Mohapatra, S., San Juan-Vergara, H., Hellermann, G., et al. (2005). Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nature Medicine, 11, 56–62.

    Article  CAS  Google Scholar 

  33. Khaitov, M. R., Shilovskiy, I. P., Nikonova, A. A., Shershakova, N. N., Kamyshnikov, O. Y., & Babakhin, A. A. (2014). Mall interfering RNAs targeted to interleukin-4 and respiratory syncytial virus reduce airway inflammation in a mouse model of virus-induced asthma exacerbation. Human Gene Therapy, 25, 642–650.

    Article  CAS  Google Scholar 

  34. Loinger, A., Shemla, Y., Simon, I., Margalit, H., & Biham, O. (2010). Competition between small RNAs: A quantitative view. Biophysical Journal, 102, 1712–1721.

    Article  Google Scholar 

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Acknowledgments

We express our thanks to our colleagues in Virology Department, School of Public Health, Tehran University of Medical Sciences. This study was part of a PhD thesis supported by Tehran University of Medical Sciences (Grant No. 92-2-27-22689). We also thank Dr. Ebrahim Faghihloo (Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran) and Dr. Mohammad Shayestehpour (Virology Department, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran) for assisting with western blot experiments.

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Authors and Affiliations

  1. Virology Department, School of Public Health, Tehran University of Medical Science, Porsina Ave, Tehran, Iran

    Somayeh Shatizadeh Malekshahi, Vahid Salimi, Ghazal Fatemi-nasab, Sarvin Adjaminejad-Fard, Jila Yavarian & Talat Mokhtari-Azad

  2. Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran

    Ehsan Arefian

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  1. Somayeh Shatizadeh Malekshahi
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  2. Vahid Salimi
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  4. Ghazal Fatemi-nasab
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Correspondence to Talat Mokhtari-Azad.

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Malekshahi, S.S., Salimi, V., Arefian, E. et al. Inhibition of Respiratory Syncytial Virus Replication by Simultaneous Targeting of mRNA and Genomic RNA Using Dual-Targeting siRNAs. Mol Biotechnol 58, 767–775 (2016). https://doi.org/10.1007/s12033-016-9976-4

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