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Investigation of the anti-influenza activity of siRNA complexes against the cellular genes FLT4, Nup98, and Nup205 in vitro

https://doi.org/10.32362/2410-6593-2022-17-2-140-151

Abstract

Objectives. Evaluation of changes in the viral activity of influenza A/WSN/33 after complex knockdown of combinations of cellular genes FLT4, Nup98 and Nup205 in human lung cell culture A549.

Methods. The work was carried out using the equipment of the Center for Collective Use of the I. Mechnikov Research Institute of Vaccines and Sera, Russia. The authors performed transfection of combinations of small interfering ribonucleic acid (siRNA) complexes that cause simultaneous disruption of the expression of cellular genes FLT4, Nup98, and Nup205. Within three days from the moment of transfection and infection, the supernatant fluid and cell lysate were taken for subsequent viral reproduction intensity determination using the titration method for cytopathic action. The dynamics of changes in the concentration of viral ribonucleic acid (vRNA) was determined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). The nonparametric Mann–Whitney test was used to calculate statistically significant differences between groups.

Results. Using all of the combinations of siRNA complexes, cell viability did not decrease below the threshold level of 70%. In cells treated with complex FLT4.2 + Nup98.1 + Nup205 at the multiplicity of infection (MOI) equal to 0.1, a significant decrease in viral reproduction by 1.5 lg was noted on the first day in relation to nonspecific and viral controls. The use of siRNA complexes at MOI 0.01 resulted in a more pronounced antiviral effect. The viral titer in cells treated with siRNA complexes FLT4.2 + Nup98.1 and Nup98.1 + Nup205 decreased by 1.5 lg on the first day. In cells treated with complexes FLT4.2 + Nup205 and FLT4.2 + Nup98.1 + Nup205, it decreased by 1.8 and 2.0 lg on the first day and by 1.8 and 2.5 lg on the second day, respectively, in relation to nonspecific and viral controls. When conducting real-time RT-PCR, a significant decrease in the concentration of vRNA was noted. At MOI 0.1, a 295, 55, and 63-fold decrease in the viral load was observed with the use of siRNA complexes FLT4.2 + Nup98.1, Nup98.1 + Nup205, and FLT4.2 + Nup98.1 + Nup205, respectively. On the second day, a decrease in vRNA was also observed in cells treated with complex A. A 415-fold decrease in vRNA on the third day was noted in cells treated with complex FLT4.2 + Nup205. At MOI 0.01, the concentration of vRNA decreased 9.5 times when using complex B relative to nonspecific and viral control.

Conclusions. The study showed a pronounced antiviral effect of siRNA combinations while simultaneously suppressing the activity of cellular genes (FLT4, Nup98, and Nup205), whose expression products are playing important role in the viral reproduction process, and obtained original designs of siRNA complexes. The results obtained are of great importance for the creation of emergence prophylactic and therapeutic drugs, whose action is based on the mechanism of RNA interference.

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; Junior Researcher, Laboratory of Molecular Immunology

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


Competing Interests:

The authors declare no conflicts of interest



M. O. Korotysheva
I.M. Sechenov First Moscow State Medical University (Sechenov University)
Russian Federation

Maria O. Korotysheva, Student, International School «Medicine of the Future»

8, Trubetskaya ul., Moscow


Competing Interests:

The authors declare no conflicts of interest



A. V. Pak
I.M. Sechenov First Moscow State Medical University (Sechenov University)
Russian Federation

Anastasia V. Pak, Student, Institute of Clinical Medicine

8, Trubetskaya ul., Moscow


Competing Interests:

The authors declare no conflicts of interest



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 conflicts of interest



A. 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 conflicts of interest



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 conflicts of interest



E. 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 conflicts of interest



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; Senior Researcher, Laboratory of Legionellosis

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


Competing Interests:

The authors declare no conflicts of interest



V. K. Solntseva
I.M. Sechenov First Moscow State Medical University (Sechenov University)
Russian Federation

Viktoriia K. Solntseva, Cand. Sci. (Med.), Senior Lecturer, A.A. Vorobiev Department of Microbiology, Virology and Immunology

8, Trubetskaya ul., Moscow, 119991


Competing Interests:

The authors declare no conflicts of interest



T. A. Zaiceva
I.M. Sechenov First Moscow State Medical University (Sechenov University)
Russian Federation

Tatyana A. Zaiceva, Cand. Sci. (Med.), Senior Lecturer, A.A. Vorobiev Department of Microbiology, Virology and Immunology

8, Trubetskaya ul., Moscow, 119991


Competing Interests:

The authors declare no conflicts of interest



E. P. Pashkov
I.M. Sechenov First Moscow State Medical University (Sechenov University)
Russian Federation

Evgeny P. Pashkov, Dr. Sci. (Med.), Professor, A.A. Vorobiev Department of Microbiology, Virology and Immunology

8, Trubetskaya ul., Moscow, 119991


Competing Interests:

The authors declare no conflicts of interest



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 conflicts of interest



O. A. 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 of the Federal State Budgetary Scientific Institution «I. Mechnikov Research Institute of Vaccines and Sera», Head of the Laboratory of Molecular Immunology; Professor, Department of Microbiolody, Virology and Immunology

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


Competing Interests:

The authors declare no conflicts of interest



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; Head of the Department of Microbiolody, Virology and Immunology

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


Competing Interests:

The authors declare no conflicts of interest



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Supplementary files

1. Influence of siRNAs complexes (А – FLT4.2 + Nup98.1; B – Nup98.1 + Nup205; C – FLT4.2 + Nup205; D – FLT4.1 + Nup98.1 + Nup205) directed to the FLT4, Nup98, and Nup205 genes on the reproduction of the influenza virus
Subject
Type Исследовательские инструменты
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Indexing metadata ▾
  • The low cytotoxicity of siRNA complexes for cells was shown according to the MTT test results when the expression of several genes was simultaneously suppressed.
  • siRNA complexes that simultaneously reduce the activity of two or more of these genes suppress viral reproduction in vitro, assessed using viral titration by the cytopathic effect and real time RT-PCR.
  • Data were obtained on the correlation between a decrease in the expression of several cellular genes simultaneously and a decrease in viral reproduction.

Review

For citations:


Pashkov E.A., Korotysheva M.O., Pak A.V., Faizuloev E.B., Sidorov A.V., Poddubikov A.V., Bystritskaya E.P., Dronina Yu.E., Solntseva V.K., Zaiceva T.A., Pashkov E.P., Bykov A.S., Svitich O.A., Zverev V.V. Investigation of the anti-influenza activity of siRNA complexes against the cellular genes FLT4, Nup98, and Nup205 in vitro. Fine Chemical Technologies. 2022;17(2):140-151. https://doi.org/10.32362/2410-6593-2022-17-2-140-151

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