Effect of antiviral siRNAs on the production of cytokines in vitro
https://doi.org/10.32362/2410-6593-2022-17-5-384-393
Abstract
Objectives. To evaluate the dynamics of the expression level of IL-1β and IL-28β (IFN-λ3) genes as a result of complex knockdown of some cellular genes, whose expression products play an important role in the reproduction of the influenza virus.
Methods. Following the collection of virus-containing liquid and cell lysate within three days from the moment of transfection and infection, the intensity of viral reproduction was assessed using the cytopathic effect titration method. The concentration of viral ribonucleic acid (vRNA) and change in the expression of IL-1β and IL-28β (IFN-λ3) were determined by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). The nonparametric Mann–Whitney test was used to statistically calculate significant differences between groups.
Results. The use of each small interfering ribonucleic acid (siRNA) complex led to a decrease in viral reproduction on the first day at the multiplicity of infection (MOI) of 0.001. The use of complex A (FLT4.2 + Nup98.1) and D (FLT4.2 + Nup98.1 + Nup205) led to a decrease in viral titer by 2.8 lgTCID50/mL and by 2.1 lgTCID50/mL relative to the use of nonspecific L2 siRNA and viral control (p ≤ 0.05). Transfection of complexes B (Nup98.1 + Nup205) and C (FLT4.2 + Nup205) also reduced the viral titer by 1.5 lgTCID50/mL and 1.8 lgTCID50/mL relative to nonspecific L2 siRNA and viral control (p ≤ 0.05). When conducting real-time RT-qPCR, a significant decrease in the concentration of viral RNA was also noted. When using complexes B, C, and D, the concentration of vRNA decreased on the first day by 14.5, 4.1, and 15 times, respectively. On the second day, a decrease in vRNA was observed in cells with B and D complexes by 17.1 and 18.3 times (p ≤ 0.05). Along with a decrease in the viral titer and vRNA, an increase in the expression of the IL-1β and IL-28β genes was observed on the first day when using all siRNA complexes relative to nonspecific and viral controls (p ≤ 0.05). On the second day, an increase was also observed in cells with A and D complexes, while on the third day, there was an increase in the expression of these genes in cells with complex D (p ≤ 0.05).
Conclusions. The use of siRNA complexes is shown to have a pronounced antiviral effect while simultaneously suppressing the activity of cellular genes (FLT4, Nup98 and Nup205). In parallel, the transfection of complexes that block the formation of expression products necessary for viral reproduction is demonstrated to lead to an increase in the level of expression of the IL-1β and IL-28β genes. These results indicate not only that the use of siRNA has antiviral activity, but also immunomodulatory activity, which can contribute to a more effective immune response of the body.
Keywords
About the Authors
A. V. PakRussian Federation
Anastasia V. Pak, Student, Institute of Clinical Medicine
8, Trubetskaya ul., Moscow, 119991
E. A. Pashkov
Russian Federation
Evgeny A. Pashkov, Postgraduate Student, A.A. Vorobiev Department of Microbiology, Virology and Immunology; Junior Researcher, Laboratory of Molecular Immunology
8, Trubetskaya ul., Moscow, 119991
5А, Malyi Kazennyi pereulok, Moscow, 105064
RSCI SPIN-code 4933-1128
N. D. Abramova
Russian Federation
Natalia D. Abramova, Junior Researcher, Laboratory of Molecular Immunology
5А, Malyi Kazennyi pereulok, Moscow, 105064
RSCI SPIN-code 1763-8942
A. V. Poddubikov
Russian Federation
Alexander A. Poddubikov, Cand. Sci. (Biol.), Head of the Laboratory of Microbiology of Opportunistic Pathogenic Bacteria
5А, Malyi Kazennyi pereulok, Moscow, 105064
RSCI SPIN-code 9658-1
F. G. Nagieva
Russian Federation
Firaya G. Nagieva, Dr. Sci. (Med.), Associate Professor, Head of the Laboratory of Hybrid Cell Cultures
5А, Malyi Kazennyi pereulok, Moscow, 105064
RSCI SPIN-code 5897-3591
E. A. Bogdanova
Russian Federation
Ekaterina A. Bogdanova, Cand. Sci. (Med.), Associate Professor, A.A. Vorobiev Department of Microbiology, Virology and Immunology
8, Trubetskaya ul., Moscow, 119991
RSCI SPIN-code 7250-5808
E. P. Pashkov
Russian Federation
Evgeny P. Pashkov, Dr. Sci. (Med.), Professor, A.A. Vorobiev Department of Microbiology, Virology and Immunology
8, Trubetskaya ul., Moscow, 119991
O. A. Svitich
Russian Federation
Oxana A. Svitich, Corresponding Member of the Russian Academy of Sciences, Dr. Sci. (Med.), Head of the Federal State Budgetary Scientific Institution, Head of the Laboratory of Molecular Immunology, Federal State Budgetary Scientific Institution; Professor, A.A. Vorobiev Department of Microbiology, Virology and Immunology
5А, Malyi Kazennyi pereulok, Moscow, 105064
8, Trubetskaya ul., Moscow, 119991
RSCI SPIN-code 8802-5569
V. V. Zverev
Russian Federation
Vitaliy V. Zverev, Full Member of the Russian Academy of Sciences, Dr. Sci. (Biol.), Scientific Director of the Federal State Budgetary Scientific Institution; Head of the A.A. Vorobiev Department of Microbiology, Virology and Immunology
5А, Malyi Kazennyi pereulok, Moscow, 105064
8, Trubetskaya ul., Moscow, 119991
RSCI SPIN-code 2122-1808
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Supplementary files
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1. Influence of siRNAs complexes (А: FLT4 + Nup98; Β: Nup98 + Nup205; C: FLT4 + Nup205; D: FLT4 + Nup98 + Nup205) directed to the FLT4, Nup98, and Nup205 genes on the reproduction of the influenza virus. | |
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- The use of siRNA complexes leads to a decrease in viral reproduction with a multiplicity of infection of 0.001 according to real-time reverse transcription quantitative polymerase chain reaction and titration by cytopathic effect.
- When genes whose expression products play a role in viral reproduction are blocked, the level of pro-inflammatory cytokines increases.
- The data obtained indicate that the use of siRNA causes not only an antiviral effect, but also an immunomodulatory effect.
- The correlation between reduced expression of cellular genes, reduced viral reproduction, and an increase in the level of pro-inflammatory cytokines was revealed.
Review
For citations:
Pak A.V., Pashkov E.A., Abramova N.D., Poddubikov A.V., Nagieva F.G., Bogdanova E.A., Pashkov E.P., Svitich O.A., Zverev V.V. Effect of antiviral siRNAs on the production of cytokines in vitro. Fine Chemical Technologies. 2022;17(5):384-393. https://doi.org/10.32362/2410-6593-2022-17-5-384-393