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Knockdown of FLT4, Nup98, and Nup205 cellular genes as a suppressor for the viral activity of Influenza A/WSN/33 (H1N1) in A549 cell culture

https://doi.org/10.32362/2410-6593-2021-16-6-476-489

Abstract

Objectives. To evaluate the effect of cellular genes FLT4, Nup98, and Nup205 on the reproduction of the influenza A virus in A549 human lung cancer cell line.

Methods. The work was carried out using the equipment of the center for collective use of the I.I. Mechnikov Research Institute of Vaccines and Sera (Russia). The virus-containing fluid was collected within three days from the moment of transfection and infection and the intensity of viral reproduction was assessed by viral titration and hemagglutination reaction. The viral RNA concentration was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). To calculate statistically significant differences between groups, the nonparametric Mann–Whitney test was used.

Results. In cells treated with small interfering RNAs (siRNAs) targeted at FLT4, Nup98, and Nup205 genes, a significant decrease in their expression and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) was observed at a multiplicity of infection (MOI) = 0.1. Additionally, it was found that a decrease in the expression of target genes using siRNA does not lead to a significant decrease in cell survival. The viral titer in cells treated with siRNA FLT4.2, Nup98.1, and Nup205 on the first day was lower by an average of 1.0 lg, and on the second and third days, by 2.2–2.3 lg, compared to cells treated with nonspecific siRNA. During real-time RT-PCR, a significant decrease in the concentration of viral RNA was observed with siRNA Nup98.1 (up to 190 times) and Nup205 (up to 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 decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained when determining the hemagglutinating activity of the virus. The hemagglutinating activity on the third day most strongly decreased in cells treated with siRNA Nup205 and FLT4.2 (16 times). In cells treated with siRNA FLT4.1, Nup98.1, and Nup98.2, hemagglutinating activity decreased by 8 times.

Conclusions. In the present study, three cellular genes (FLT4, Nup98, and Nup205) were identified—the decrease in the expression of which effectively suppresses viral reproduction— and the original siRNA sequences were obtained. The results obtained are important for creating therapeutic and prophylactic medication, whose action is based on the RNA interference mechanism.

About the Authors

E. A. Pashkov
I.M. Sechenov First Moscow State Medical University (Sechenov University); I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Evgeny A. Pashkov - Postgraduate Student, Department of Microbiology, Virology and Immunology, I.M. Sechenov FMSMU (Sechenov University); Junior Researcher, Laboratory of Molecular Immunology, FSBSI “I. Mechnikov Research Institute of Vaccines and Sera”

8, Trubetskaya ul., Moscow, 119991; 5А, Malyi Kazennyi pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



E. B. Faizuloev
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Evgeny B. Faizuloev - Cand. Sci. (Biol.), Head of the Laboratory of Molecular Virology.

5А, Malyi Kazennyi pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



E. R. Korchevaya
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Ekaterina R. Korchevaya - Junior Researcher, Laboratory of Molecular Virology.

5А, Malyi Kazennyi pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



A. A. Rtishchev
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Artem A. Rtishchev - Junior Researcher, Laboratory of RNA viruses.

5А, Malyi Kazennyi pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



B. S. Cherepovich
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Bogdan S. Cherepovich - Junior Researcher, Laboratory of RNA viruses.

5А, Malyi Kazennyy pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



А. V. Sidorov
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Alexander V. Sidorov - Cand. Sci. (Biol.), Head of the Laboratory of DNA viruses.

5А, Malyi Kazennyi pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



A. V. Poddubikov
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Alexander A. Poddubikov - Cand. Sci. (Biol.), Head of the Laboratory of Microbiology of Opportunistic Pathogenic Bacteria.

5А, Malyi Kazennyi pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



Е. P. Bystritskaya
I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Elizaveta P. Bystritskaya - Junior Researcher, Laboratory of Molecular Immunology.

5А, Malyi Kazennyi pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



Yu. E. Dronina
I.M. Sechenov First Moscow State Medical University (Sechenov University); N.F. Gamaleya National Research Center for Epidemiology and Microbiology
Russian Federation

Yuliya E. Dronina - Cand. Sci. (Med.), Associate Professor, Department of Microbiology, Virology and Immunology, I.M. Sechenov FMSMU (Sechenov University); Senior Researcher, Laboratory of Legionellosis, N.F. Gamaleya NRC EM (The Gamaleya National Center).

8, Trubetskaya ul., Moscow, 119991; 18, Gamaleya ul., Moscow, 123098


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



A. S. Bykov
I.M. Sechenov First Moscow State Medical University (Sechenov University)
Russian Federation

Anatoly S. Bykov - Dr. Sci. (Med.), Professor, Department of Virology and Immunology.

8, Trubetskaya ul., Moscow, 119991


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



O. А. Svitich
I.M. Sechenov First Moscow State Medical University (Sechenov University); I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Oxana A. Svitich - Corresponding Member of the Russian Academy of Sciences, Dr. Sci. (Med.), Head FSBSI “I. Mechnikov Research Institute of Vaccines and Sera,” Head of the Laboratory of Molecular Immunology, FSBSI “I. Mechnikov Research Institute of Vaccines and Sera”; Professor, Department of Microbiolody, Virology and Immunology, I.M. Sechenov FMSMU (Sechenov University).

8, Trubetskaya ul., Moscow, 119991; 5А, Malyi Kazennyi pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



V. V. Zverev
I.M. Sechenov First Moscow State Medical University (Sechenov University); I. Mechnikov Research Institute of Vaccines and Sera
Russian Federation

Vitaliy V. Zverev - Full Member of the Russian Academy of Sciences, Dr. Sci. (Biol.), Scientific Director FSBSI “I. Mechnikov Research Institute of Vaccines and Sera”; Head of the Department of Microbiolody, Virology and Immunology, I.M. Sechenov FMSMU (Sechenov University).

8, Trubetskaya ul., Moscow, 119991; 5А, Malyi Kazennyi pereulok, Moscow, 105064


Competing Interests:

The authors declare no obvious and potential conflicts of interest related to the publication of this article



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Supplementary files

1. Influence of siRNA on the expression of genes FLT4, Nup98, and Nup205.
Subject
Type Исследовательские инструменты
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Indexing metadata ▾
  • Three cellular genes (FLT4, Nup98, and Nup205) have been identified, the decrease in the expression of which can suppress viral reproduction—a promising opportunity.
  • Original siRNA sequences for these genes were obtained.
  • Low cytotoxicity of siRNA was shown according to the results obtained from the MTT assay.
  • siRNAs that reduce the activity of the genes suppress viral reproduction in vitro assessed via viral titration, real-time RT-PCR, and HA.
  • Data were obtained on the correlation between decreased expression of cellular genes and decreased viral reproduction.

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For citations:


Pashkov E.A., Faizuloev E.B., Korchevaya E.R., Rtishchev A.A., Cherepovich B.S., Sidorov А.V., Poddubikov A.V., Bystritskaya Е.P., Dronina Yu.E., Bykov A.S., Svitich O.А., Zverev V.V. Knockdown of FLT4, Nup98, and Nup205 cellular genes as a suppressor for the viral activity of Influenza A/WSN/33 (H1N1) in A549 cell culture. Fine Chemical Technologies. 2021;16(6):476-489. https://doi.org/10.32362/2410-6593-2021-16-6-476-489

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