tetano
Editor, Senior Moderator
Infect Disord Drug Targets
. 2022 Mar 25.
doi: 10.2174/1871526522666220325121403. Online ahead of print.
Knockdown of FLT4, Nup98, and Nup205 cellular genes effectively suppresses the reproduction of influenza virus strain A/WSN/1933 (H1N1) in vitro
Evgeny Pashkov[SUP] 1 [/SUP], Ekaterina Korchevaya[SUP] 2 [/SUP], Evgeny Faizuloev[SUP] 2 [/SUP], Artem Rtishchev[SUP] 3 [/SUP], Bogdan Cherepovich[SUP] 3 [/SUP], Elizaveta Bystritskaya[SUP] 4 [/SUP], Alexander Sidorov[SUP] 5 [/SUP], Alexander Poddubikov[SUP] 6 [/SUP], Anatoly Bykov[SUP] 7 [/SUP], Yuliya Dronina[SUP] 7 [/SUP], Oxana Svitich[SUP] 1 [/SUP], Vitaliy Zverev[SUP] 8 [/SUP]
Affiliations
Abstract
Background: Influenza is one of the most common infectious diseases, which affect the lower respiratory tract, and can lead to serious complications including death. It is known that currently available therapeutic agents and vaccines do not provide 100% protection against influenza viruses. The development of drugs based on the RNA interference mechanism in the context of this problem is a promising area. This paper aims to assess the effect of FLT4, Nup98, and Nup205 cellular gene knockdown on the reproduction of influenza A virus in human lung cell culture.
Materials and methods: Influenza virus strain A/WSN/1933 (St. Jude's Children's Research Hospital, USA) was used in this work as well as A549 cell culture (human lung adenocarcinoma, ATCC® CCL-185, USA) and MDCK cell culture (dog kidney cells, Institut Pasteur, France). Small interfering RNAs (siRNAs) (Syntol, Russia) were synthesized for targeting of the FLT4, Nup98, and Nup205 genes. Lipofectamin 2000 (Invitrogen, USA) was used for transfection. After 4 hours, the transfected cells were infected with the influenza virus at MOI = 0.1. Virus-containing fluid was collected within three days from the moment of transfection and the intensity of viral reproduction was assessed by CPE titration and hemagglutination reactions. Viral RNA concentration was determined by RT-PCR. Mann-Whitney U test was used for statistical analysis.
Results: In cells treated with siRNA for FLT4, Nup98, and Nup205 genes, there was a significant decrease in the expression of target genes and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) at MOI = 0.1, although the cell survival rate did not decrease significantly. On the first day, the viral titer in cells treated with declared siRNA was lower, on average, by 1 Lg, and on the second and third days, by 2.2-2.3 Lg, compared to cells treated with nonspecific siRNA. During RT-PCR, a significant decrease in the concentration of viral RNA with Nup98.1 and Nup205 siRNA was detected: up to 190 times and 30 times on the first day; 26 and 29 times on the second day; 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies has decreased 23, 18, and 16 times on the first, second, and third days. Similar results were obtained while determining the hemagglutinating activity of the virus. The hemagglutinating activity decreased mostly (by 16 times) in cells treated with Nup205 and FLT4.2 siRNAs on the third day. In cells treated with FLT4.1, Nup98.1, and Nup98.2 siRNAs, the hemagglutinating activity decreased by 8 times.
Conclusion: We identified a number of genes such as FLT4, Nup98, and Nup205, the decrease in the expression of which can effectively suppress viral reproduction. The original siRNA sequences were also obtained. These results are important for the creation of therapeutic and prophylactic agents, whose action is based on the RNA interference mechanism.
Keywords: Influenza A virus; RNA interference; genes; messenger RNA; small interfering RNAs; transfection.
. 2022 Mar 25.
doi: 10.2174/1871526522666220325121403. Online ahead of print.
Knockdown of FLT4, Nup98, and Nup205 cellular genes effectively suppresses the reproduction of influenza virus strain A/WSN/1933 (H1N1) in vitro
Evgeny Pashkov[SUP] 1 [/SUP], Ekaterina Korchevaya[SUP] 2 [/SUP], Evgeny Faizuloev[SUP] 2 [/SUP], Artem Rtishchev[SUP] 3 [/SUP], Bogdan Cherepovich[SUP] 3 [/SUP], Elizaveta Bystritskaya[SUP] 4 [/SUP], Alexander Sidorov[SUP] 5 [/SUP], Alexander Poddubikov[SUP] 6 [/SUP], Anatoly Bykov[SUP] 7 [/SUP], Yuliya Dronina[SUP] 7 [/SUP], Oxana Svitich[SUP] 1 [/SUP], Vitaliy Zverev[SUP] 8 [/SUP]
Affiliations
- PMID: 35339191
- DOI: 10.2174/1871526522666220325121403
Abstract
Background: Influenza is one of the most common infectious diseases, which affect the lower respiratory tract, and can lead to serious complications including death. It is known that currently available therapeutic agents and vaccines do not provide 100% protection against influenza viruses. The development of drugs based on the RNA interference mechanism in the context of this problem is a promising area. This paper aims to assess the effect of FLT4, Nup98, and Nup205 cellular gene knockdown on the reproduction of influenza A virus in human lung cell culture.
Materials and methods: Influenza virus strain A/WSN/1933 (St. Jude's Children's Research Hospital, USA) was used in this work as well as A549 cell culture (human lung adenocarcinoma, ATCC® CCL-185, USA) and MDCK cell culture (dog kidney cells, Institut Pasteur, France). Small interfering RNAs (siRNAs) (Syntol, Russia) were synthesized for targeting of the FLT4, Nup98, and Nup205 genes. Lipofectamin 2000 (Invitrogen, USA) was used for transfection. After 4 hours, the transfected cells were infected with the influenza virus at MOI = 0.1. Virus-containing fluid was collected within three days from the moment of transfection and the intensity of viral reproduction was assessed by CPE titration and hemagglutination reactions. Viral RNA concentration was determined by RT-PCR. Mann-Whitney U test was used for statistical analysis.
Results: In cells treated with siRNA for FLT4, Nup98, and Nup205 genes, there was a significant decrease in the expression of target genes and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) at MOI = 0.1, although the cell survival rate did not decrease significantly. On the first day, the viral titer in cells treated with declared siRNA was lower, on average, by 1 Lg, and on the second and third days, by 2.2-2.3 Lg, compared to cells treated with nonspecific siRNA. During RT-PCR, a significant decrease in the concentration of viral RNA with Nup98.1 and Nup205 siRNA was detected: up to 190 times and 30 times on the first day; 26 and 29 times on the second day; 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies has decreased 23, 18, and 16 times on the first, second, and third days. Similar results were obtained while determining the hemagglutinating activity of the virus. The hemagglutinating activity decreased mostly (by 16 times) in cells treated with Nup205 and FLT4.2 siRNAs on the third day. In cells treated with FLT4.1, Nup98.1, and Nup98.2 siRNAs, the hemagglutinating activity decreased by 8 times.
Conclusion: We identified a number of genes such as FLT4, Nup98, and Nup205, the decrease in the expression of which can effectively suppress viral reproduction. The original siRNA sequences were also obtained. These results are important for the creation of therapeutic and prophylactic agents, whose action is based on the RNA interference mechanism.
Keywords: Influenza A virus; RNA interference; genes; messenger RNA; small interfering RNAs; transfection.