Abstract
Influenza viruses replicate primarily in the lung tissue of different host species. For efficient replication the virus utilizes host factors that are expressed in target cells. Cell-penetrating peptide-conjugated Morpholino oligomers (PPMOs) designed to target viral proteins have shown promising results as potential antiviral drugs in tissue culture and animal models. However, since viruses tend to have high rates of mutations, targeting viral proteins may result in viral escape mutants. An alternative approach to inhibit virus replication with PPMOs is to target host factors that are required for virus replication. Delivery of PPMO through the intranasal route has been shown to be effective in knockdown of host factors or microbial genes leading to protection against respiratory pathogens and reduced microbial burden. In addition, protective host innate antiviral immune responses in the lung can be studied by knockdown of immune signaling factors using PPMOs. Here we describe a successful approach using PPMOs to knockdown either proviral or antiviral host factors leading to changes in influenza virus replication in the lungs of mice, providing a tool to investigate immune responses and host–virus interactions in vivo.
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References
Moulton HM (2013) In vivo delivery of morpholino oligos by cell-penetrating peptides. Curr Pharm Des 19(16):2963–2969
Yin H, Boisguerin P, Moulton HM, Betts C, Seow Y, Boutilier J, Wang Q, Walsh A, Lebleu B, Wood MJ (2013) Context dependent effects of chimeric peptide morpholino conjugates contribute to dystrophin exon-skipping efficiency. Mol Ther Nucleic Acids 2:e124. doi:10.1038/mtna.2013.51
Stein DA (2008) Inhibition of RNA virus infections with peptide-conjugated morpholino oligomers. Curr Pharm Des 14(25):2619–2634
Heald AE, Charleston JS, Iversen PL, Warren TK, Saoud JB, Al-Ibrahim M, Wells J, Warfield KL, Swenson DL, Welch LS, Sazani P, Wong M, Berry D, Kaye EM, Bavari S (2015) AVI-7288 for Marburg virus in nonhuman primates and humans. N Engl J Med 373(4):339–348. doi:10.1056/NEJMoa1410345
Rajsbaum R, Versteeg GA, Schmid S, Maestre AM, Belicha-Villanueva A, Martinez-Romero C, Patel JR, Morrison J, Pisanelli G, Miorin L, Laurent-Rolle M, Moulton HM, Stein DA, Fernandez-Sesma A, tenOever BR, Garcia-Sastre A (2014) Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKepsilon kinase-mediated antiviral response. Immunity 40(6):880–895
Tripathi S, Pohl MO, Zhou Y, Rodriguez-Frandsen A, Wang G, Stein DA, Moulton HM, DeJesus P, Che J, Mulder LC, Yanguez E, Andenmatten D, Pache L, Manicassamy B, Albrecht RA, Gonzalez MG, Nguyen Q, Brass A, Elledge S, White M, Shapira S, Hacohen N, Karlas A, Meyer TF, Shales M, Gatorano A, Johnson JR, Jang G, Johnson T, Verschueren E, Sanders D, Krogan N, Shaw M, Konig R, Stertz S, Garcia-Sastre A, Chanda SK (2015) Meta- and orthogonal integration of influenza “OMICs” data defines a role for UBR4 in virus budding. Cell Host Microbe 18(6):723–735
Geller BL, Marshall-Batty K, Schnell FJ, McKnight MM, Iversen PL, Greenberg DE (2013) Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo. J Infect Dis 208(10):1553–1560
Gabriel G, Nordmann A, Stein DA, Iversen PL, Klenk HD (2008) Morpholino oligomers targeting the PB1 and NP genes enhance the survival of mice infected with highly pathogenic influenza A H7N7 virus. J Gen Virol 89(Pt 4):939–948
Lupfer C, Stein DA, Mourich DV, Tepper SE, Iversen PL, Pastey M (2008) Inhibition of influenza A H3N8 virus infections in mice by morpholino oligomers. Arch Virol 153(5):929–937
Lai SH, Stein DA, Guerrero-Plata A, Liao SL, Ivanciuc T, Hong C, Iversen PL, Casola A, Garofalo RP (2008) Inhibition of respiratory syncytial virus infections with morpholino oligomers in cell cultures and in mice. Mol Ther 16(6):1120–1128
Paessler S, Rijnbrand R, Stein DA, Ni H, Yun NE, Dziuba N, Borisevich V, Seregin A, Ma Y, Blouch R, Iversen PL, Zacks MA (2008) Inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers. Virology 376(2):357–370
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25(4):402–408
Szretter KJ, Balish AL, Katz JM (2006) Influenza: propagation, quantification, and storage. Curr Protoc Microbiol. Chapter 15:Unit 15G 11
Acknowledgements
Work in the Rajsbaum lab (UTMB) is supported by the Institute for Human Infections and Immunity (IHII), and the Department of Microbiology and Immunology at the University of Texas Medical Branch (UTMB), Galveston. We also would like to thank Dr. Adolfo Garcia-Sastre (Icahn School of Medicine at Mount Sinai, NY) and his lab, where the original work was performed, as well as Dr. Hong Moulton (Oregon State University) for the design, synthesis, and general advice of the PPMOs.
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Rajsbaum, R. (2017). Intranasal Delivery of Peptide-Morpholinos to Knockdown Influenza Host Factors in Mice. In: Moulton, H., Moulton, J. (eds) Morpholino Oligomers. Methods in Molecular Biology, vol 1565. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6817-6_16
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DOI: https://doi.org/10.1007/978-1-4939-6817-6_16
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