Chemical intervention of influenza virus mRNA nuclear export
Fig 5
Compound 2 inhibits viral mRNA nuclear export.
(A) Structure of compound 2. (B) RNA-FISH and smRNA-FISH followed by fluorescence microscopy were performed in cells treated with 0.1% DMSO or 2.5 μM compound 2 to detect poly(A) RNA and GAPDH mRNA, respectively, in uninfected cells. (C-F) Total fluorescence intensity or nuclear to cytoplasmic fluorescence intensity (N/C ratio) were quantified for poly(A) RNA and GAPDH mRNA in the absence or presence of compound 2. For C (C, n = 174 cells; Compound 2, n = 181), D (C, n = 172 cells; Compound 2, n = 181 cells), E (C, n = 166 cells; Compound 2, n = 181 cells), and F (C, n = 151 cells; Compound 2, n = 160 cells). (G) Cells were treated as in B except that smRNA-FISH was performed with probes to detect M mRNA in cells infected with WSN at MOI 2 for 8 h. (H,I) Total fluorescence intensity or nuclear to cytoplasmic fluorescence intensity (N/C ratio) were quantified for M mRNA in the absence or presence of compound 2. For H (C, n = 91 cells; Compound 2, n = 104 cells) and I (C, n = 101 cells; Compound 2, n = 95 cells). (J) Cells were treated as in G except that smRNA-FISH was performed with probes to detect HA mRNA. (K,L) Total fluorescence intensity or nuclear to cytoplasmic fluorescence intensity (N/C ratio) were quantified for HA mRNA in the absence or presence of compound 2. For K (C, n = 104 cells; Compound 2, n = 137 cells) and L (C, n = 101 cells; Compound 2, n = 126 cells). (M) Cells were treated as in G except that smRNA-FISH was performed with probes to detect NS mRNA. (N,O) Total fluorescence intensity or nuclear to cytoplasmic fluorescence intensity (N/C ratio) were quantified for M mRNA in the absence or presence of compound 2. For N (C, n = 96 cells; Compound 2, n = 135 cells), and O (C, n = 106 cells; Compound 2, n = 113 cells). At least three independent experiments were performed for each imaging analysis. (P,Q) Relative mRNA ratios of M2 to M1 (P) and NS2 to NS1 (Q) were determined by qPCR from RNA obtained from cells infected as in G and treated with 0.1% DMSO, 1 μM, or 2.5 μM compound 2. The nuclear speckle assembly factor SON was knocked down with siRNAs as positive control for inhibition of M1 to M2 mRNA splicing. Three independent experiments were performed. C, control. (R) Cellular ATP levels were measured in cells treated with 0.1% DMSO or 2.5 μM of compound 2 at 24 h. Four independent experiments were performed and each contained 6 technical replicates. Graphs shows data points and mean +/- SD. *p<0.05; ***p<0.001, ****p<0.0001.