BACKGROUND: Influenza viruses are dangerous pathogens. Seventy-Seven genomes of recently emerged genotype 4 reassortant Eurasian avian-like H1N1 virus (G4-EA-H1N1) are currently available. We investigated the presence and variation of potential G-quadruplex forming sequences (PQS), which can serve as targets for antiviral treatment. RESULTS: PQS were identified in all 77 genomes. The total number of PQS in G4-EA-H1N1 genomes was 571. Interestingly, the number of PQS per genome in individual close relative viruses varied from 4 to 12. PQS were not randomly distributed in the 8 segments of the G4-EA-H1N1 genome, the highest frequency of PQS being found in the NP segment (1.39 per 1000 nt), which is considered a potential target for antiviral therapy. In contrast, no PQS was found in the NS segment. Analyses of variability pointed the importance of some PQS; even if genome variation of influenza virus is extreme, the PQS with the highest G4Hunter score is the most conserved in all tested genomes. G-quadruplex formation in vitro was experimentally confirmed using spectroscopic methods. CONCLUSIONS: The results presented here hint several G-quadruplex-forming sequences in G4-EA-H1N1 genomes, that could provide good therapeutic targets.

Department of Biology and Ecology Institute of Environmental Technologies Faculty of Science University of Ostrava 710 00 Ostrava Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 5 62500 Brno Czech Republic

Faculty of Chemistry Brno University of Technology Purkyňova 118 612 00 Brno Czech Republic

Institute of Biophysics of the Czech Academy of Sciences Královopolská 135 612 65 Brno Czech Republic

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Smith GJD, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG, et al. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza a epidemic. Nature. 2009;459(7250):1122–1125. doi: 10.1038/nature08182. PubMed DOI

Hoffmann M, Pöhlmann S. Cell entry of influenza a viruses: sweet talk between HA and CaV1.2. Cell Host Microbe. 2018;23(6):697–699. doi: 10.1016/j.chom.2018.05.019. PubMed DOI

Neumann G, Noda T, Kawaoka Y. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature. 2009;459(7249):931–939. doi: 10.1038/nature08157. PubMed DOI PMC

Zhang R, Xu C, Duan Z. Novel antigenic shift in HA sequences of H1N1 viruses detected by big data analysis. Infect Genet Evol. 2017;51:138–142. doi: 10.1016/j.meegid.2017.03.028. PubMed DOI

Mostafa A, Kanrai P, Ziebuhr J, Pleschka S. The PB1 segment of an influenza a virus H1N1 2009pdm isolate enhances the replication efficiency of specific influenza vaccine strains in cell culture and embryonated eggs. J Gen Virol. 2016;97(3):620–631. doi: 10.1099/jgv.0.000390. PubMed DOI

Lee J-Y, Ouh I-O, Cho S-D, Cho I-S, Park CK, Song J-Y. Complete genome sequence of H1N1 swine influenza virus from pigs in the Republic of Korea in 2016. Baltrus DA, editor. Microbiol Resour Announc 2018;7(23):e01229–e01218, e01229-18. PubMed PMC

Sullivan SJ, Jacobson RM, Dowdle WR, Poland GA. 2009 H1N1 Influenza. Mayo Clin Proc. 2010;85(1):64–76. doi: 10.4065/mcp.2009.0588. PubMed DOI PMC

Zimmer SM, Burke DS. Historical perspective — emergence of influenza a (H1N1) viruses. N Engl J Med. 2009;361(3):279–285. doi: 10.1056/NEJMra0904322. PubMed DOI

Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63(5):669–684. doi: 10.1084/jem.63.5.669. PubMed DOI PMC

Sun H, Xiao Y, Liu J, Wang D, Li F, Wang C, et al. Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection. Proc Natl Acad Sci U S A. 2020;29:201921186. PubMed PMC

Burge S, Parkinson GN, Hazel P, Todd AK, Neidle S. Quadruplex DNA: sequence, topology and structure. Nucleic Acids Res. 2006;34(19):5402–5415. doi: 10.1093/nar/gkl655. PubMed DOI PMC

Malgowska M, Czajczynska K, Gudanis D, Tworak A, Gdaniec Z. Overview of the RNA G-quadruplex structures. Acta Biochim Pol. 2016;63(4):609–21. PubMed

Varshney D, Spiegel J, Zyner K, Tannahill D, Balasubramanian S. The regulation and functions of DNA and RNA G-quadruplexes. Nat Rev Mol Cell Biol. 2020;21(8):459–474. doi: 10.1038/s41580-020-0236-x. PubMed DOI PMC

Ruggiero E, Richter SN. Viral G-quadruplexes: New frontiers in virus pathogenesis and antiviral therapy. In: Annual Reports in Medicinal Chemistry. Elsevier; 2020 [cited 2020 Jul 29]. p. S0065774320300142. PubMed PMC

Lavezzo E, Berselli M, Frasson I, Perrone R, Palù G, Brazzale AR, et al. G-quadruplex forming sequences in the genome of all known human viruses: A comprehensive guide. PLoS Comput Biol. 2018 13; 14(12). PubMed PMC

Lombardi EP, Londoño-Vallejo A. A guide to computational methods for G-quadruplex prediction. Nucleic Acids Res. 2020;48(3):1603. doi: 10.1093/nar/gkaa033. PubMed DOI PMC

Bedrat A, Lacroix L, Mergny J-L. Re-evaluation of G-quadruplex propensity with G4Hunter. Nucleic Acids Res. 2016;44(4):1746–1759. doi: 10.1093/nar/gkw006. PubMed DOI PMC

Gazanion E, Lacroix L, Alberti P, Gurung P, Wein S, Cheng M, et al. Genome wide distribution of G-quadruplexes and their impact on gene expression in malaria parasites. Di Antonio M, editor. PLoS Genet. 2020;16(7):e1008917. doi: 10.1371/journal.pgen.1008917. PubMed DOI PMC

Bartas M, Čutová M, Brázda V, Kaura P, Šťastný J, Kolomazník J, et al. The presence and localization of G-Quadruplex forming sequences in the domain of Bacteria. Molecules. 2019;24(9):1711. doi: 10.3390/molecules24091711. PubMed DOI PMC

Čutová M, Manta J, Porubiaková O, Kaura P, Šťastný J, Jagelská EB, et al. Divergent distributions of inverted repeats and G-quadruplex forming sequences in Saccharomyces cerevisiae. Genomics. 2019:S0888754319305269. PubMed

Brázda V, Kolomazník J, Lýsek J, Bartas M, Fojta M, Šťastný J, et al. G4Hunter web application: a web server for G-quadruplex prediction. Hancock J, editor. Bioinformatics. 2019;35(18):3493–3495. doi: 10.1093/bioinformatics/btz087. PubMed DOI PMC

Bartas M, Brázda V, Bohálová N, Cantara A, Volná A, Stachurová T, et al. In-depth Bioinformatic analyses of Nidovirales including human SARS-CoV-2, SARS-CoV, MERS-CoV Viruses Suggest Important Roles of Non-canonical Nucleic Acid Structures in Their Lifecycles. Front Microbiol. 2020;11:1583. doi: 10.3389/fmicb.2020.01583. PubMed DOI PMC

Di Antonio M, Ponjavic A, Radzevičius A, Ranasinghe RT, Catalano M, Zhang X, et al. Single-molecule visualization of DNA G-quadruplex formation in live cells. Nat Chem [Internet]. 2020 20. PubMed PMC

Prorok P, Artufel M, Aze A, Coulombe P, Peiffer I, Lacroix L, et al. Involvement of G-quadruplex regions in mammalian replication origin activity. Nat Commun. 2019;10(1):3274. doi: 10.1038/s41467-019-11104-0. PubMed DOI PMC

Artusi S, Perrone R, Lago S, Raffa P, Di Iorio E, Palù G, et al. Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells. Nucleic Acids Res. 2016;44(21):10343–10353. PubMed PMC

Callegaro S, Perrone R, Scalabrin M, Doria F, Palù G, Richter SN. A core extended naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication. Sci Rep. 2017;7(1):2341. doi: 10.1038/s41598-017-02667-3. PubMed DOI PMC

Métifiot M, Amrane S, Litvak S, Andreola M-L. G-quadruplexes in viruses: function and potential therapeutic applications. Nucleic Acids Res. 2014;42(20):12352–12366. doi: 10.1093/nar/gku999. PubMed DOI PMC

González VM, Martín ME, Fernández G, García-Sacristán A. Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses. Pharmaceuticals. 2016;9(4):78. PubMed PMC

Majerciak V, Zheng Z-M. Detection of Viral RNA Splicing in Diagnostic Virology. In: Tang Y-W, Stratton CW, editors. Advanced Techniques in Diagnostic Microbiology. Cham: Springer International Publishing; 2018. p. 345–402.

Renaud de la Faverie A, Guédin A, Bedrat A, Yatsunyk LA, Mergny J-L. Thioflavin T as a fluorescence light-up probe for G4 formation. Nucleic Acids Res. 2014;42(8):e65. doi: 10.1093/nar/gku111. PubMed DOI PMC

Vondrušková J, Kypr J, Kejnovská I, Fialová M, Vorlíčková M. Guanine quadruplex formation by RNA/DNA hybrid analogs of Oxytricha telomere G4T4G4 fragment. Biopolymers. 2008;89(10):797–806. doi: 10.1002/bip.21015. PubMed DOI

Brázda V, Laister RC, Jagelská EB, Arrowsmith C. Cruciform structures are a common DNA feature important for regulating biological processes. BMC Mol Biol. 2011;12(1):33. doi: 10.1186/1471-2199-12-33. PubMed DOI PMC

Ruggiero E, Richter SN. G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy. Nucleic Acids Res. 2018;46(7):3270–3283. doi: 10.1093/nar/gky187. PubMed DOI PMC

Shen L-W, Qian M-Q, Yu K, Narva S, Yu F, Wu Y-L, et al. Inhibition of influenza a virus propagation by benzoselenoxanthenes stabilizing TMPRSS2 gene G-quadruplex and hence down-regulating TMPRSS2 expression. Sci Rep. 2020;10(1):7635. doi: 10.1038/s41598-020-64368-8. PubMed DOI PMC

Eisfeld AJ, Neumann G, Kawaoka Y. At the Centre: influenza a virus ribonucleoproteins. Nat Rev Microbiol. 2015;13(1):28–41. doi: 10.1038/nrmicro3367. PubMed DOI PMC

Compans RW, Content J, Duesberg PH. Structure of the ribonucleoprotein of influenza virus. J Virol. 1972;10(4):795–800. doi: 10.1128/JVI.10.4.795-800.1972. PubMed DOI PMC

Hu Y, Sneyd H, Dekant R, Wang J. Influenza A Virus Nucleoprotein: A Highly Conserved Multi-Functional Viral Protein as a Hot Antiviral Drug Target. Curr Top Med Chem. 2017;17(20):2271–85. PubMed PMC

Kikin O, D’Antonio L, Bagga PS. QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences. Nucleic Acids Res. 2006; 34(Web Server):W676–W682. PubMed PMC

Dolinnaya NG, Ogloblina AM, Yakubovskaya MG. Structure, properties, and biological relevance of the DNA and RNA G-quadruplexes: overview 50 years after their discovery. Biochem Moscow. 2016;81(13):1602–1649. doi: 10.1134/S0006297916130034. PubMed DOI PMC

Hognon C, Miclot T, García-Iriepa C, Francés-Monerris A, Grandemange S, Terenzi A, et al. Role of RNA guanine Quadruplexes in Favoring the dimerization of SARS unique domain in coronaviruses. J Phys Chem Lett 2020;11(14):5661–5667. PubMed

Speranskaia AS, Mel’nikova NV, Belenkin MS, Dmitriev AA, Oparina NI, Kudriavtseva AV. Genetic diversity and evolution of the influenza C virus. Genetika. 2012;48(7):797–805. PubMed

Xu R, Ekiert DC, Krause JC, Hai R, Crowe JE, Wilson IA. Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science. 2010;328(5976):357–360. doi: 10.1126/science.1186430. PubMed DOI PMC

Kuenstling TE, Sambol AR, Hinrichs SH, Larson MA. Oligomerization of bacterially expressed H1N1 recombinant hemagglutinin contributes to protection against viral challenge. Sci Rep. 2018;8(1):11856. doi: 10.1038/s41598-018-30079-4. PubMed DOI PMC

Gómez-Puertas P, Albo C, Pérez-Pastrana E, Vivo A, Portela A. Influenza virus matrix protein is the major driving force in virus budding. J Virol. 2000;74(24):11538–11547. doi: 10.1128/JVI.74.24.11538-11547.2000. PubMed DOI PMC

Campbell PJ, Danzy S, Kyriakis CS, Deymier MJ, Lowen AC, Steel J. The M segment of the 2009 pandemic influenza virus confers increased neuraminidase activity, filamentous morphology, and efficient contact transmissibility to a/Puerto Rico/8/1934-based Reassortant viruses. J Virol. 2014;88(7):3802–3814. doi: 10.1128/JVI.03607-13. PubMed DOI PMC

Ji D, Juhas M, Tsang CM, Kwok CK, Li Y, Zhang Y. Discovery of G-quadruplex-forming sequences in SARS-CoV-2. Brief Bioinform. 2020:bbaa114. PubMed PMC

Sayers EW, Agarwala R, Bolton EE, Brister JR, Canese K, Clark K, et al. Database resources of the National Center for biotechnology information. Nucleic Acids Res. 2019;47(D1):D23–D28. doi: 10.1093/nar/gky1069. PubMed DOI PMC

Stothard P. The sequence manipulation suite: JavaScript programs for Analyzing and formatting protein and DNA sequences. BioTechniques. 2000;28(6):1102–1104. doi: 10.2144/00286ir01. PubMed DOI

Sievers F, Higgins DG. Clustal omega. Curr Protoc Bioinformatics. 2014;48:3.13.1–16. PubMed

Crooks GE. WebLogo: A Sequence Logo Generator. Genome Res. 2004;14(6):1188–1190. doi: 10.1101/gr.849004. PubMed DOI PMC

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