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

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.

Keywords

• influenza a virus
• rna interference
• gene
• messenger rna
• small interfering rnas