Determination of cold-adapted influenza virus (Orthomyxoviridae: Alphainfluenzavirus) polymerase activity by the minigenome method with a fluorescent protein

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Abstract

Introduction. Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier.

Objective. The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method.

Materials and methods. Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct.

Results. Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase.

Conclusion. Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.

About the authors

Pavel A. Ivanov

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, the Russian Ministry of Health

Email: ivanovpa@mail.ru
ORCID iD: 0000-0002-7105-7579

PhD, senior researcher, Laboratory of Virus Physiology, the Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russia

Russian Federation, 123098, Moscow

Aleksandr V. Lyashko

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, the Russian Ministry of Health

Email: lyaalex@bk.ru
ORCID iD: 0000-0001-5714-9461

junior researcher, Laboratory of Virus Physiology, the Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russia

Russian Federation, 123098, Moscow

Vladimir Y. Kost

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: goron.dekar@gmail.com
ORCID iD: 0000-0003-1703-2685

researcher, Laboratory of Molecular Toxinology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia

Russian Federation, 117997, Moscow

Natalia F. Lomakina

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, the Russian Ministry of Health

Author for correspondence.
Email: nflomakina@yandex.ru
ORCID iD: 0000-0003-2638-4244

PhD, senior researcher, the N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, the Russian Ministry of Health, Moscow, Russia

Russian Federation, 123098, Moscow

Artyom A. Rtishchev

Mechnikov Research Institute for Vaccines and Sera

Email: rtishchevartyom@gmail.com
ORCID iD: 0000-0002-4212-5093

researcher, Laboratory of genetics of RNA viruses, Mechnikov Research Institute for Vaccines and Sera, Moscow, Russia

Russian Federation, 105064, Moscow

Nataliya I. Bunkova

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, the Russian Ministry of Health

Email: nbounkova@mail.ru
ORCID iD: 0009-0007-8846-4633

PhD, senior researcher, Laboratory of Immunobiotechnology, the Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russia

Russian Federation, 123098, Moscow

Tatiana A. Timofeeva

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, the Russian Ministry of Health

Email: timofeeva.tatyana@inbox.ru
ORCID iD: 0000-0002-8991-8525

PhD, leading researcher, head of Laboratory of Virus Physiology, the Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russia

Russian Federation, 123098, Moscow

Marina A. Balanova

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, the Russian Ministry of Health

Email: mbalanova@yandex.ru
ORCID iD: 0000-0003-2727-7221

researcher, Laboratory of Virus Physiology, the Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russia

Russian Federation, 123098, Moscow

Stepan A. Ionov

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, the Russian Ministry of Health; Mendeleev University of Chemical Technology

Email: stephan.ionov@yandex.ru
ORCID iD: 0009-0005-3393-0399

laboratory technican, Laboratory of Virus Physiology, the Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russia; student, FSBEI HE Mendeleev University of Chemical Technology, Moscow, Russia

Russian Federation, 123098, Moscow; 125047, Moscow

Dmitry V. Gorikov

N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, the Russian Ministry of Health; Mendeleev University of Chemical Technology

Email: gorikov.dmitry@mail.ru
ORCID iD: 0009-0002-5159-8738

laboratory technican, Laboratory of Virus Physiology, the Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Moscow, Russia; student, FSBEI HE Mendeleev University of Chemical Technology, Moscow, Russia

Russian Federation, 123098, Moscow; 125047, Moscow

Stanislav G. Markushin

Mechnikov Research Institute for Vaccines and Sera

Email: s.g.markushin@rambler.ru
ORCID iD: 0000-0003-0994-5337

DSc, head of Laboratory of genetics of RNA viruses, Mechnikov Research Institute for Vaccines and Sera, Moscow, Russia

Russian Federation, 105064, Moscow

References

  1. Krammer F., Smith G.J.D., Fouchier R.A.M., Peiris M., Kedzierska K., Doherty P.C., et al. Influenza. Nat. Rev. Dis. Primers. 2018; 4(1): 3. https://doi.org/10.1038/s41572-018-0002-y
  2. Alexandrova G.I., Smorodintsev A.A. Obtaining of an additionally attenuated vaccinating cryophilic influenza strain. Rev. Roum. Inframicrobiol. 1965; 2(3): 179–86.
  3. Maassab H.F. Adaptation and growth characteristics of influenza virus at 25 degrees C. Nature. 1967; 213(76): 612–4. https://doi.org/10.1038/213612a0
  4. Maassab H.F., Bryant M.L. The development of live attenuated cold-adapted influenza virus vaccine for humans. Rev. Med. Virol. 1999; 9(4): 237–44. https://doi.org/10.1002/(sici)1099-1654(199910/12)9:4%3C237::aid-rmv252%3E3.0.co;2-g
  5. Ambrose C.S., Luke C., Coelingh K. Current status of live attenuated influenza vaccine in the United States for seasonal and pandemic influenza. Influenza Other Respir. Viruses. 2008; 2(6): 193–202. https://doi.org/10.1111/j.1750-2659.2008.00056.x
  6. Rudenko L., Yeolekar L., Kiseleva I., Isakova-Sivak I. Development and approval of live attenuated influenza vaccines based on Russian master donor viruses: Process challenges and success stories. Vaccine. 2016; 34(45): 5436–41. https://doi.org/10.1016/j.vaccine.2016.08.018
  7. Caspard H., Mallory R.M., Yu J., Ambrose C.S. Live-attenuated influenza vaccine effectiveness in children from 2009 to 2015-2016: a systematic review and meta-analysis. Open Forum Infect. Dis. 2017; 4(3): ofx111. https://doi.org/10.1093/ofid/ofx111
  8. Hoffmann E., Neumann G., Kawaoka Y., Hobom G., Webster R.G. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl Acad. Sci. USA. 2000; 97(11): 6108–13. https://doi.org/10.1073/pnas.100133697
  9. Hoffmann E., Krauss S., Perez D., Webby R., Webster R.G. Eight-plasmid system for rapid generation of influenza virus vaccines. Vaccine. 2002; 20(25-26): 3165–70. https://doi.org/10.1016/s0264-410x(02)00268-2
  10. Neumann G., Horimoto T., Kawaoka Y. Reverse genetics of influenza viruses – applications in research and vaccine design. In: Klenk H.D., Matrosovich M.N., Stech J., eds. Avian Influenza. Volume 27. Basel: Karger; 2008: 118–33.
  11. Horimoto T., Takada A., Fujii K., Goto H., Hatta M., Watanabe S., et al. The development and characterization of H5 influenza virus vaccines derived from a 2003 human isolate. Vaccine. 2006; 24(17): 3669–76. https://doi.org/10.1016/j.vaccine.2005.07.005
  12. Kittel C., Sereinig S., Ferko B., Stasakova J., Romanova J., Wolkerstorfer A., et al. Rescue of influenza virus expressing GFP from the NS1 reading frame. Virology. 2004; 324(1): 67–73. https://doi.org/10.1016/j.virol.2004.03.035
  13. Te Velthuis AJW, Long JS, Barclay WS. Assays to measure the activity of influenza virus polymerase. In: Yamauchi Y., eds. Influenza Virus. Methods in Molecular Biology, Volume 1836. New York: Humana Press; 2018. https://doi.org/10.1007/978-1-4939-8678-1_17
  14. Cox N.J., Kitame F., Kendal A.P., Maassab H.F., Naeve C. Identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, A/Ann Arbor/6/60 (H2N2). Virology. 1988; 167(2): 554–67.
  15. Rodriguez L., Blanco-Lobo P., Reilly E.C., Maehigashi T., Nogales A., Smith A., et al. Comparative study of the temperature sensitive, cold adapted and attenuated mutations present in the master donor viruses of the two commercial human live attenuated influenza vaccines. Viruses. 2019; 11(10): 928. https://doi.org/10.3390/v11100928
  16. Isakova-Sivak I., Chen L.M., Matsuoka Y., Voeten J.T., Kiseleva I., Heldens J.G., et al. Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2). Virology. 2011; 412(2): 297–305. https://doi.org/10.1016/j.virol.2011.01.004
  17. Ghendon Y.Z., Markushin S.G., Tsfasman T.M., Akopova I.I., Ahmatova N.K., Koptiaeva I.B. New cold-adapted donor strains for live influenza vaccine. Voprosy virusologii. 2013; 58(1): 11–7. https://elibrary.ru/pwjuoj (in Russian)
  18. Markushin S.G., Tsfasman T.M., Terekhov A.V., Lisovskaya K.V., Akopova I.I. Cold-adapted A/Krasnodar/101/35/59 (H2N2) strain-a promising strain-donor of attenuation for procuration of live influenza vaccines. Zhurnal mikrobiologii, epidemiologii i immunobiologii. 2015; (5): 27–32. https://elibrary.ru/zqjycx (in Russian)
  19. Markushin S.G., Kost V.Yu., Akopova I.I., Koptiaeva I.B., Lisovskaya K.V., Pereversev A.D., et al. The investigation of the possibility of site-specific mutagenesis using in construction of live influenza vaccines. Epidemiologiya i vaktsinoprofilaktika. 2014; (6): 100–3. https://elibrary.ru/tenlph (in Russian)
  20. Kost V., Tsfasman T., Terekhov A., Koptiaeva I., Lisovskaja K., Markushin S. Attenuation of the cold-adapted (ca) A/Krasnodar/101/35/1959 (H2N2) influenza strain: Role of the Ile147Thr mutation in the PB1 gene. IJSRM.Human. 2017; 6(2): 96–114.
  21. Kost V.Yu., Rtischev A.A., Mintaev R.R., Akopova I.I., Lisovskaya K.V., Markushin S.G. Study of the biological properties of attenuated variants of the virulent A/WSN/33 strain of influenza virus, obtained by the site-specific mutagenesis of PB2-gene. Zhurnal mikrobiologii, epidemiologii i immunobiologii. 2019; (2): 68–76. https://doi.org/10.36233/0372-9311-2019-2-68-76 https://elibrary.ru/wrchzu (in Russian)
  22. Stech J., Stech O., Herwig A., Altmeppen H., Hundt J., Gohrbandt S., et al. Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification. Nucleic Acids Res. 2008; 36(21): e139. https://doi.org/10.1093/nar/gkn646
  23. Jeong J.Y., Yim H.S., Ryu J.Y., Lee H.S., Lee J.H., Seen D.S., et al. One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl. Environ. Microbiol. 2012; 78(15): 5440–3. https://doi.org/10.1128/aem.00844-12
  24. Terekhov A.V., Tsfasman T.M., Markushin S.G., Kopt’ayeva I.B., Lisovskaya K.V., Kost V.Yu. Study att-reassortant phenotype between virulent strain of influenza A(H1N1)/WSN/33 and cold-adapted vaccine strain of influenza A(H2N2)/Krasnodar/101/35/59. Epidemiologiya i vaktsinoprofilaktika. 2013; (5): 41–7. https://elibrary.ru/refcoh (in Russian)
  25. Perez J.T., García-Sastre A., Manicassamy B. Insertion of a GFP reporter gene in influenza virus. Curr. Protoc. Microbiol. 2013; Chapter 15: 15G4. https://doi.org/10.1002/9780471729259.mc15g04s29

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2. Fig. 1. NS1GFPNS2 reporter construct (control). a – modified segment 8 of A/Krasnodar/101/35/59 (H2N2) influenza virus is inserted in the plasmid which includes the NS1 gene (with a removed splicing site for NS2), the green fluorescent protein gene (GFP), and spliced NS2 gene. Cellular RNA polymerase II uses the CMV promoter of the NS1GFPNS2 reporter to synthesize mRNA (mRNA) followed by translation of NS2 and fused NS1-GFP proteins; b – expression of the fused NS1-GFP protein in transfected Cos-1 cells cultured at 33 °C (left) and 37 °C (right). The scale ruler is 10 microns.

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3. Fig. 2. The dCMV reporter construct for evaluating the transcriptional activity of viral RNA-dependent RNA polymerase (RdRp). The CMV promoter has been removed from a plasmid carrying inserted NS1GFPNS2 sequence. On one chain of the inserted double-stranded DNA fragment, cellular RNA polymerase I (PolI) synthesizes virionic RNA (vRNA) from the PolI promoter. Then, the viral RNA polymerase RdRp, expressed by the minigenome, uses this vRNA for synthesis of mRNA that translates NS1-GFP and NS2 proteins.

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4. Fig. 3. The dCMVrev reporter construct for evaluating the replicative and transcriptional activity of viral RNA-dependent RNA polymerase (RdRp). An inverted double-stranded DNA fragment NS1GFPNS2 is inserted in the plasmid in which the CMV promoter is deleted. Cellular RNA polymerase I (PolI) synthesizes a complementary NS1GFPNS2 chain (cRNA) of an inserted construct. Viral RNA-dependent RNA polymerase (RdRp) replicates viral RNA (vRNA) from cRNA. Then RdRp uses vRNA as a template for synthesis of mRNA for following translation of NS1-GFP and NS2 proteins.

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5. Fig. 4. Expression of NS1-GFP in Cos-1 cells after transfection with a reporter construct without a CMV promoter (dCMV) together with the minigenome of A/Krasnodar/101/35/59 (H2N2) virus (bottom row), or A/Puerto Rico/8/34 (H1N1) (top row) at 33 °C (left column) and 37 °C (right column). The scale ruler is 10 microns.

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6. Fig. 5. Expression of NS1-GFP in Cos-1 cells after cotransfection with a reporter construct carrying an inverted NS1GFPNS2 sequence and deleted a CMV promoter (dCMVrev) together with the minigenome of A/Krasnodar/101/35/59 (H2N2) (bottom row) or A/Puerto Rico/8/34 (H1N1) (top row) viruses at 33 °C (left column) and 37 °C (right column). The scale ruler is 10 microns.

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7. Fig. 6. NS1-GFP fluorescence in conventional units (Arbitrary Units – A.U., Y-axis) at a temperature of 33 °C (black column) and 37 °C (gray column) in Cos-1 cells transfected by reporter constructs together with the virus minigenome of A/Puerto Rico/8/34 (PR8) or A/Krasnodar/101/35/59 (Krasnodar). In each column, the arithmetic mean is given according to the results of three independent experiments with a standard deviation. The dCMV construct characterizes polymerase activity mainly during transcription, and dCMVrev characterizes polymerase activity during replication and transcription. * – significant difference at p < 0.008. Designations: CMV/NS1GFPNS2 – NS1GFPNS2 reporter construct with CMV promoter (control); dCMV is a reporter construct of NS1GFPNS2 without a CMV promoter; dCMVrev is a reporter construct without a CMV promoter with an inverted NS1GFPNS2 sequence.

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Copyright (c) 2023 Ivanov P.A., Lyashko A.V., Kost V.Y., Lomakina N.F., Rtishchev A.A., Bunkova N.I., Timofeeva T.A., Balanova M.A., Ionov S.A., Gorikov D.V., Markushin S.G.

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