Figure 4. Dapivirine inhibited the nuclear entry of vRNP complex at the early stage of viral replication.

(A) Retention of influenza vRNPs in cytoplasm by dapivirine. A549 cells infected with the A/WSN/33 (H1N1) virus (MOI = 30) were treated with DMSO or 30 μM of dapivirine. After fixation at indicated times (2, 4, and 6 h p.i.), cells were stained with mouse anti-influenza A NP antibody, rabbit anti-M1, and DAPI to determine the viral NP, M1 protein, and nucleus, respectively. (B) CC₅₀ of dapivirine against A549 cell line with a 6 h incubation time. The concentration at 30 μM was indicated with an arrow. The CC₅₀ value is the mean of two independent experiments ± standard deviation. (C–F) Dapivirine reduced the levels of viral RNAs and proteins. A549 cells infected with the A/WSN/33 (H1N1) virus (MOI = 1) were treated with DMSO and 10 or 30 μM of dapivirine. At 6 h p.i., cells were harvested and total viral RNA was extracted and quantified by RT-qPCR (C and D). Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of RNA level is the mean of two independent experiments ± standard deviation. Viral protein levels were quantified by western blot (E) and immunofluorescence (F). (G–I) Mini-genome assay. A549 cells were transfected with a combination of plasmids for minigenome assay (see in Materials and methods). Dapivirine was added to the culture medium at 2 h post transfection. After 22 h incubation, cells were harvest and the activities of Firefly (G) and Renilla (H) luciferases were measured using the dual luciferease kit from Promega. Renilla was served as an internal control to normalize the transfection efficiency (I). Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of luciferase signal is the mean of two independent experiments ± standard deviation.