Amphotericin b effect on the sensitivity to influenza infection of WI-38 VA-13 cells with IFITM3 gene knockout

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Abstract

BACKGROUND: The application of CRISPR/Cas9 is one of the most rapidly developing areas in biotechnology. This method was used to obtain clones of а human origin cell line with knockout of one or more genes of the IFITM family, representing host restriction factors for influenza infection. Amphotericin B has previously been shown to promote influenza infection by blocking IFITM3 function.

AIM: The aim of this study was to evaluate the effect of amphotericin B on the sensitivity of IFITM knockout cells to influenza A virus infection.

MATERIALS AND METHODS: WI-38 VA-13 cells and mutant clones with IFITM3 knockout (F3 clone) or IFITM1, IFITM3 knockout (clone E12) were infected with influenza virus A/PR/8/34 (H1N1) in the presence or absence of amphotericin B. Forty-four hours after infection, the culture medium was taken to determine the infectious activity of the virus by titration in the MDCK cell culture, as well as the hemagglutinating activity of the virus. The infected cells were stained with fluorescently labeled antibodies against the viral NP protein, and the number of NP-positive cells was determined by flow cytometry.

RESULTS: The addition of amphotericin B increased the hemagglutinating and infectious activity of the virus in WI-38 VA-13cells, while the difference was insignificant for clones with IFITM gene knockout. A similar dependency was obtained for the percent of infected cells.

CONCLUSIONS: Mutant cells with a knockout of one or several genes of the IFITM family were equally susceptible to influenza infection regardless of the addition of amphotericin B, which confirms the crucial importance of a defect in the IFITM3 protein in increasing the permissiveness of cells to influenza A virus.

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About the authors

Kira S. Koryabina

Smorodintsev Research Institute of Influenza; St. Petersburg State Institute of Technology

Author for correspondence.
Email: kira336@yandex.ru

student

Russian Federation, Saint Petersburg

Mariya V. Sergeeva

Smorodintsev Research Institute of Influenza; Institute of Chemical Biology and Fundamental Medicine of SB RAS

Email: mari.v.sergeeva@gmail.com
ORCID iD: 0000-0003-0411-9896
SPIN-code: 9720-5812

PhD (Biol.), Leading Researcher at the Laboratory of Vector Vaccines

Russian Federation, Saint Petersburg; Novosibirsk

Andrey B. Komissarov

Smorodintsev Research Institute of Influenza; Institute of Chemical Biology and Fundamental Medicine of SB RAS

Email: a.b.komissarov@gmail.com
ORCID iD: 0000-0003-1733-1255
SPIN-code: 3792-8290

PhD (Biol.), Head of the Laboratory of Molecular Virology

Russian Federation, Saint Petersburg; Novosibirsk

Nataliya V. Eshchenko

Institute of Chemical Biology and Fundamental Medicine of SB RAS

Email: eschenko96@gmail.com

Researcher at the Laboratory of Genomic Editing

Russian Federation, Novosibirsk

Grigoriy A. Stepanov

Institute of Chemical Biology and Fundamental Medicine of SB RAS

Email: stepanovga@niboch.nsc.ru
ORCID iD: 0000-0003-3393-3192
SPIN-code: 8587-2044

PhD (Chem.), Senior Researcher, Head of the Laboratory of Genomic Editing

Russian Federation, Novosibirsk

References

  1. Feeley EM, Sims JS, John SP, et al. IFITM3 inhibits influenza A virus infection by preventing cytosolic entry. PLoS Pathog. 2011;7(10):e1002337. doi: 10.1371/journal.ppat.1002337
  2. Lin TYu, Chin CR, Everitt AR, et al. Amphotericin B increases influenza A virus infection by preventing IFITM3-mediated restriction. Cell Rep. 2013;5(4):895–908. doi: 10.1016/j.celrep.2013.10.033
  3. Stepanov GA, Sergeeva MV, Mozhaeva AE, et al. Sozdanie i izuchenie kletochnykh linii s CRISPR/Cas9-napravlennym nokautom individual’nykh genov dlya povyshennoi produktsii virusa grippa. Proceedings of The Vserossiiskaya mul’tikonferentsiya s mezhdunarodnym uchastiem “Biotekhnologiya – meditsine budushchego”; 2019 2 June – 2 July; Novosibirsk. Novosibirsk; 2019. P. 215. (In Russ.)

Supplementary files

Supplementary Files
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1. Fig. 1. The yield of the A/PR8 virus in the infected original WI-38 VA-13 cells and mutant clones E12 and F3: а — viral infectious activity; b — viral hemagglutinating activity. AmpB — amphotericin B

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2. Fig. 2. The number of infected mutant and original cells 48 hours after infection with A/PR8 virus as determined by staining for viral nucleoprotein. AmpB — amphotericin B

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Copyright (c) 2021 Koryabina K.S., Sergeeva M.V., Komissarov A.B., Eshchenko N.V., Stepanov G.A.

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