The effect of the PKR inhibitor, 2-aminopurine, on the replication of influenza A virus, and segment 8 mRNA splicing

Figures

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  Figure 1. (a) Schematic presentation of NS1 and NS2 mRNA. A dot indicates a 5′ capp, the small blocks show the untranslated regions, and bigger boxes the coding region for each protein, line – intron, and A(n) – poly-A tail. (b) Structure of the NS1 mRNA fragment, which can fold into a pseudoknot. (b) is done based on the study of Jiang et al. [14].

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  Figure 2. IAV replication in cells treated with 2-AP. HEK-293T (left panel), MDCK 2 (middle panel), and A549 (right panel) were infected with influenza virus A/PuertoRico/8/34 at an MOI 0.01 and treated with different 2-AP amounts, i.e., 5, 2.5, and 0.5 mM. The copy number of viral RNA in supernatant was determined by RT qPCR at 24 h post-treatment. The results of representative experiments performed with triplicate samples are shown. Error bars indicate standard deviations (SD). Similar results were obtained in three independent experiments. A, B – values with different superscript letters differ p < 0.05.

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  Figure 3. NS1 and NS2 expression (left panel), as well as NS2/NS1 ratio (right panel), in cells treated with 2-AP. HEK-293T, MDCK 2, and A549 were infected with influenza virus A/PuertoRico/8/34 at an MOI 0.01 and treated with different 2-AP amounts, i.e., 5, 2.5, and 0.5 mM. Concentration of NS1 and NS2 in cell lysates was determined by RT qPCR (left panel). The NS2/NS1 ratio was calculated based on the mean SQ. The results of representative experiments performed with triplicate samples are shown. Error bars indicate SD. Similar results were obtained in three independent experiments. A, B – values with different superscript letters differ p < 0.05.

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  Figure 4. Inhibition of PKR activity and its effect on expression of IAV NS1 and NS2 in cells treated with 2-AP. HEK-293T (right panel), MDCK 2 (middle panel), and A549 (right panel) were infected with influenza virus A/PuertoRico/8/34 at an MOI 0.01 and treated with different 2-AP amounts, i.e., 5, 2.5, and 0.5 mM. The inhibition of PKR was demonstrated by the expression of p-eIF2α, a factor which was shown to be phosphorylated by PKR. The expression of NS1 and NS2 in the cell lysates was visualized with specific antibodies. The results of representative experiments are shown. Similar results were obtained in two independent experiments.