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. 2020 Jan 2;12(1):52. doi: 10.3390/v12010052

Figure 5.

Figure 5

Effect of PB2-R251K on polymerase activity, RNA synthesis, and protein expression. (A) Polymerase activity was analyzed in 293T cells co-transfected with pPolI-NP-luc, pPL-TK, and expression plasmids encoding the MS285 PB1, PA, and NP genes and a PB2 clones (WT or R251K) at 33 °C or 37 °C. Relative polymerase luciferase activity compared to r/MS285 polymerase complex. (B) A549 cells were infected at an MOI of 1 with r/MS285 and r/MS285-251K viruses and cultured at 37 °C. After 24 hpi, the relative levels of viral NP segment viral RNA (vRNA), complement RNA (cRNA), and messenger RNA (mRNA) were quantified by strand-specific real-time RT-qPCR and normalized against 18S rRNA levels. The vRNA, cRNA, and mRNA values were expressed relative to the results for r/MS285. (C) A549 cells were infected at an MOI of 1 with r/MS285 and r/MS285-251K viruses and cultured at 37 °C. Cell lysates were prepared at 3, 6, 9, and 12 hpi to evaluate the expression levels of NP and PB2 by Western blotting. GAPDH was used as a loading control. In (A,B), the results are expressed as the means ± SD (n = 3), and the statistical significance was calculated using one-way ANOVA. ** p < 0.01, *** p < 0.001.