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Infectious Disorders - Drug Targets

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ISSN (Print): 1871-5265
ISSN (Online): 2212-3989

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Research Article

Knockdown of FLT4, Nup98, and Nup205 Cellular Genes Effectively Suppresses the Reproduction of Influenza Virus Strain A/WSN/1933 (H1N1) In vitro

Author(s): Evgeny Pashkov*, Ekaterina Korchevaya, Evgeny Faizuloev, Artem Rtishchev, Bogdan Cherepovich, Elizaveta Bystritskaya, Alexander Sidorov, Alexander Poddubikov, Anatoly Bykov, Yuliya Dronina, Oxana Svitich and Vitaliy Zverev

Volume 22, Issue 5, 2022

Published on: 19 April, 2022

Article ID: e250322202629 Pages: 9

DOI: 10.2174/1871526522666220325121403

Price: $65

Abstract

Background: Influenza is one of the most common infectious diseases, which affects the lower respiratory tract, and can lead to serious complications, including death. It is known that currently available therapeutic agents and vaccines do not provide 100% protection against influenza viruses. The development of drugs based on the RNA interference mechanism in the context of this problem is a promising area. This paper aims to assess the effect of FLT4, Nup98, and Nup205 cellular gene knockdown on the reproduction of the influenza A virus in human lung cell culture.

Materials and Methods: Influenza virus strain A/WSN/1933 (St. Jude's Children's Research Hospital, USA) was used in this work as well as A549 cell culture (human lung adenocarcinoma, ATCC® CCL- 185, USA) and MDCK cell culture (dog kidney cells, Institut Pasteur, France). Small interfering RNAs (siRNAs) (Syntol, Russia) were synthesized for targeting the FLT4, Nup98, and Nup205 genes. Lipofectamin 2000 (Invitrogen, USA) was used for transfection. After 4 hours, the transfected cells were infected with the influenza virus at MOI = 0.1. Virus-containing fluid was collected within three days from the moment of transfection, and the intensity of viral reproduction was assessed by CPE titration and hemagglutination reactions. Viral RNA concentration was determined by RT-PCR. Mann- Whitney U test was used for statistical analysis.

Results: In cells treated with siRNA for FLT4, Nup98, and Nup205 genes, there was a significant decrease in the expression of target genes and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) at MOI = 0.1, although the cell survival rate did not decrease significantly. On the first day, the viral titer in cells treated with declared siRNA was lower, on average, by 1 Lg, and on the second and third days, by 2.2-2.3 Lg, compared to cells treated with nonspecific siRNA. During RT-PCR, a significant decrease in the concentration of viral RNA with Nup98.1 and Nup205 siRNA was detected: up to 190 times and 30 times on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies has been decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained while determining the hemagglutinating activity of the virus. The hemagglutinating activity decreased mostly (by 16 times) in cells treated with Nup205 and FLT4.2 siRNAs on the third day. In cells treated with FLT4.1, Nup98.1, and Nup98.2 siRNAs, the hemagglutinating activity decreased by 8 times.

Conclusion: We identified a number of genes, such as FLT4, Nup98, and Nup205, whose expression can efficiently suppress viral reproduction when their expression is decreased. The original siRNA sequences were also obtained. These results are important for the creation of therapeutic and prophylactic agents, whose action is based on the RNA interference mechanism.

Keywords: Influenza A virus, RNA interference, genes, messenger RNA, small interfering RNAs, transfection.

Graphical Abstract

[1]
Hussain M, Galvin HD, Haw TY, Nutsford AN, Husain M. Drug resistance in influenza A virus: the epidemiology and management. Infect Drug Resist 2017; 10: 121-34.
[http://dx.doi.org/10.2147/IDR.S105473] [PMID: 28458567]
[2]
Peasah SK, Azziz-Baumgartner E, Breese J, Meltzer MI, Widdowson M-A. Influenza cost and cost-effectiveness studies globally-a review. Vaccine 2013; 31(46): 5339-48.
[http://dx.doi.org/10.1016/j.vaccine.2013.09.013] [PMID: 24055351]
[3]
Rezkalla SH, Kloner RA. Influenza-related viral myocarditis. WMJ 2010; 109(4): 209-13.
[PMID: 20945722]
[4]
Nguyen JL, Yang W, Ito K, Matte TD, Shaman J, Kinney PL. Seasonal influenza infections and cardiovascular disease mortality. JAMA Cardiol 2016; 1(3): 274-81.
[http://dx.doi.org/10.1001/jamacardio.2016.0433] [PMID: 27438105]
[5]
Ekstrand JJ. Neurologic complications of influenza. Semin Pediatr Neurol 2012; 19(3): 96-100.
[http://dx.doi.org/10.1016/j.spen.2012.02.004] [PMID: 22889537]
[6]
Edet A, Ku K, Guzman I, Dargham HA. Acute influenza encephalitis/encephalopathy associated with influenza A in an incompetent adult. Case Rep Crit Care 2020; 2020: 6616805.
[http://dx.doi.org/10.1155/2020/6616805] [PMID: 33425396]
[7]
Err H, Wiwanitkit V. Emerging H6N1 influenza infection: renal problem to be studied. Ren Fail 2014; 36(4): 662.
[http://dx.doi.org/10.3109/0886022X.2014.883934] [PMID: 24502265]
[8]
Morens DM, Taubenberger JK, Fauci AS. Predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness. J Infect Dis 2008; 198(7): 962-70.
[http://dx.doi.org/10.1086/591708] [PMID: 18710327]
[9]
Metersky ML, Masterton RG, Lode H, File TM Jr, Babinchak T. Epidemiology, microbiology, and treatment considerations for bacterial pneumonia complicating influenza. Int J Infect Dis 2012; 16(5): e321-31.
[http://dx.doi.org/10.1016/j.ijid.2012.01.003] [PMID: 22387143]
[10]
Vanderbeke L, Spriet I, Breynaert C, Rijnders BJA, Verweij PE, Wauters J. Invasive pulmonary aspergillosis complicating severe influenza: epidemiology, diagnosis and treatment. Curr Opin Infect Dis 2018; 31(6): 471-80.
[http://dx.doi.org/10.1097/QCO.0000000000000504] [PMID: 30299367]
[11]
van der Vries E, Schutten M, Fraaij P, Boucher C, Osterhaus A. Influenza virus resistance to antiviral therapy. Adv Pharmacol 2013; 67: 217-46.
[http://dx.doi.org/10.1016/B978-0-12-405880-4.00006-8] [PMID: 23886002]
[12]
Han J, Perez J, Schafer A, et al. Influenza virus: Small molecule therapeutics and mechanisms of antiviral resistance. Curr Med Chem 2018; 25(38): 5115-27.
[http://dx.doi.org/10.2174/0929867324666170920165926] [PMID: 28933281]
[13]
Looi QH, Foo JB, Lim MT, Le CF, Show PL. How far have we reached in development of effective influenza vaccine? Int Rev Immunol 2018; 37(5): 266-76.
[http://dx.doi.org/10.1080/08830185.2018.1500570] [PMID: 30252547]
[14]
Pleguezuelos O, James E, Fernandez A, et al. Efficacy of FLU-v, a broad-spectrum influenza vaccine, in a randomized phase IIb human influenza challenge study. NPJ Vaccines 2020; 5(1): 22.
[http://dx.doi.org/10.1038/s41541-020-0174-9] [PMID: 32194999]
[15]
Wang F, Chen G, Zhao Y. Biomimetic nanoparticles as universal influenza vaccine. Smart Mater Med 2020; 1: 21-3.
[http://dx.doi.org/10.1016/j.smaim.2020.03.001] [PMID: 33349811]
[16]
Smith M. Vaccine safety: medical contraindications, myths, and risk communication. Pediatr Rev 2015; 36(6): 227-38.
[http://dx.doi.org/10.1542/pir.36.6.227] [PMID: 26034253]
[17]
Wang J, Wu Y, Ma C, et al. Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus. Proc Natl Acad Sci USA 2013; 110(4): 1315-20.
[http://dx.doi.org/10.1073/pnas.1216526110] [PMID: 23302696]
[18]
Leneva IA, Russell RJ, Boriskin YS, Hay AJ. Characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol. Antiviral Res 2009; 81(2): 132-40.
[http://dx.doi.org/10.1016/j.antiviral.2008.10.009] [PMID: 19028526]
[19]
Hurt AC, Ernest J, Deng Y-M, et al. Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa. Antiviral Res 2009; 83(1): 90-3.
[http://dx.doi.org/10.1016/j.antiviral.2009.03.003] [PMID: 19501261]
[20]
Hurt AC. The epidemiology and spread of drug resistant human influenza viruses. Curr Opin Virol 2014; 8: 22-9.
[http://dx.doi.org/10.1016/j.coviro.2014.04.009] [PMID: 24866471]
[21]
Lampejo T. Influenza and antiviral resistance: an overview. Eur J Clin Microbiol Infect Dis 2020; 39(7): 1201-8.
[http://dx.doi.org/10.1007/s10096-020-03840-9] [PMID: 32056049]
[22]
Fire AZ. Gene silencing by double-stranded RNA (Nobel Lecture). Angew Chem Int Ed 2007; 46(37): 6966-84.
[http://dx.doi.org/10.1002/anie.200701979] [PMID: 17722137]
[23]
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391(6669): 806-11.
[http://dx.doi.org/10.1038/35888] [PMID: 9486653]
[24]
Wang Q-C, Nie Q-H, Feng Z-H. RNA interference: antiviral weapon and beyond. World J Gastroenterol 2003; 9(8): 1657-61.
[http://dx.doi.org/10.3748/wjg.v9.i8.1657] [PMID: 12918096]
[25]
McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002; 3(10): 737-47.
[http://dx.doi.org/10.1038/nrg908] [PMID: 12360232]
[26]
Estrin MA, Hussein ITM, Puryear WB, Kuan AC, Artim SC, Runstadler JA. Host-directed combinatorial RNAi improves inhibition of diverse strains of influenza A virus in human respiratory epithelial cells. PLoS One 2018; 13(5): e0197246.
[http://dx.doi.org/10.1371/journal.pone.0197246] [PMID: 29775471]
[27]
Janssen HLA, Reesink HW, Lawitz EJ, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med 2013; 368(18): 1685-94.
[http://dx.doi.org/10.1056/NEJMoa1209026] [PMID: 23534542]
[28]
Qureshi A, Tantray VG, Kirmani AR, Ahangar AG. A review on current status of antiviral siRNA. Rev Med Virol 2018; 28(4): e1976.
[http://dx.doi.org/10.1002/rmv.1976] [PMID: 29656441]
[29]
Hoy SM. Patisiran: First global approval. Drugs 2018; 78(15): 1625-31.
[http://dx.doi.org/10.1007/s40265-018-0983-6] [PMID: 30251172]
[30]
Givosiran. In: LiverTox: Clinical and Research Information on Drug- Induced Liver Injury. Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases 2012. Available from: http://www.ncbi.nlm.nih.gov/books/NBK555105/
[31]
Lesch M, Luckner M, Meyer M, et al. RNAi-based small molecule repositioning reveals clinically approved urea-based kinase inhibitors as broadly active antivirals. PLoS Pathog 2019; 15(3): e1007601.
[http://dx.doi.org/10.1371/journal.ppat.1007601] [PMID: 30883607]
[32]
Karlas A, Machuy N, Shin Y, et al. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 2010; 463(7282): 818-22.
[http://dx.doi.org/10.1038/nature08760] [PMID: 20081832]
[33]
Arenas-Hernandez M, Vega-Sanchez R. Housekeeping gene expression stability in reproductive tissues after mitogen stimulation. BMC Res Notes 2013; 6(1): 285.
[http://dx.doi.org/10.1186/1756-0500-6-285] [PMID: 23875991]
[34]
Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2^(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath 2013; 3(3): 71-85.
[PMID: 25558171]
[35]
Lee HK, Loh TP, Lee CK, Tang JW-T, Chiu L, Koay ES-C. A universal influenza A and B duplex real-time RT-PCR assay. J Med Virol 2012; 84(10): 1646-51.
[http://dx.doi.org/10.1002/jmv.23375] [PMID: 22930514]
[36]
Ge Q, McManus MT, Nguyen T, et al. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci USA 2003; 100(5): 2718-23.
[http://dx.doi.org/10.1073/pnas.0437841100] [PMID: 12594334]
[37]
Ramakrishnan MA. Determination of 50% endpoint titer using a simple formula. World J Virol 2016; 5(2): 85-6.
[http://dx.doi.org/10.5501/wjv.v5.i2.85] [PMID: 27175354]
[38]
Eierhoff T, Hrincius ER, Rescher U, Ludwig S, Ehrhardt C. The epidermal growth factor receptor (EGFR) promotes uptake of influenza A viruses (IAV) into host cells. PLoS Pathog 2010; 6(9): e1001099.
[http://dx.doi.org/10.1371/journal.ppat.1001099] [PMID: 20844577]
[39]
Shaw ML, Stertz S. Role of host genes in influenza virus replication. Curr Top Microbiol Immunol 2018; 419: 151-89.
[http://dx.doi.org/10.1007/82_2017_30] [PMID: 28643205]
[40]
Watanabe T, Watanabe S, Kawaoka Y. Cellular networks involved in the influenza virus life cycle. Cell Host Microbe 2010; 7(6): 427-39.
[http://dx.doi.org/10.1016/j.chom.2010.05.008] [PMID: 20542247]
[41]
Sanjuán R, Domingo-Calap P. Mechanisms of viral mutation. Cell Mol Life Sci 2016; 73(23): 4433-48.
[http://dx.doi.org/10.1007/s00018-016-2299-6] [PMID: 27392606]
[42]
Presloid JB, Novella IS. RNA Viruses and RNAi: Quasispecies implications for viral escape. Viruses 2015; 7(6): 3226-40.
[http://dx.doi.org/10.3390/v7062768] [PMID: 26102581]
[43]
Das AT, Brummelkamp TR, Westerhout EM, et al. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol 2004; 78(5): 2601-5.
[http://dx.doi.org/10.1128/JVI.78.5.2601-2605.2004] [PMID: 14963165]
[44]
Rupp JC, Locatelli M, Grieser A, et al. Host cell copper transporters CTR1 and ATP7A are important for Influenza A virus replication. Virol J 2017; 14(1): 11.
[http://dx.doi.org/10.1186/s12985-016-0671-7] [PMID: 28115001]
[45]
Wang R, Zhu Y, Zhao J, et al. Autophagy promotes replication of influenza A virus in vitro. J Virol 2019; 93(4): e01984-18.
[http://dx.doi.org/10.1128/JVI.01984-18] [PMID: 30541828]

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