Figure 5. Cellular antiviral mechanism of R151785.

(A) R151785 blocks intracellular PA-PB1 interaction. HEK 293T cells were transfected with the PA-GFP-expressing plasmid alone or along with PB1-expressing plasmid. DMSO or R151785 (30 μM) was added into the cell culture at 6 h post-transfection. At 30 h post-transfection, cells were fixed and stained with DAPI for imaging. Bar = 10 μm. (B-E) Time-of-addition experiment. MDCK cells were infected with A/California/ 07/2009 (H1N1) (MOI = 0.01) at −2 h. After 1-hour incubation at 4 °C for attachment and another 1 h at 37 °C for viral entry, cells were washed with PBS buffer and incubated with DMEM at 37 °C for 12 h, then viruses in culture supernatant were harvested for titration by plaque assay. Oseltamivir carboxylate (1 μM) (C), Baloxavir (30 nM) (D), or R151785 (15 μM) (E) was present during the time as illustrated in (B). ND means No Drug. Data represents mean ± SE from three independent experiments. Asterisks indicate statistically significant difference in comparison with ND (**, p < 0.01, ***, p < 0.001; student’s t-test). (F and G) R151785 reduces viral RNA (F) and protein (G) levels. MDCK cells were infected with A/WSN/33 (H1N1) virus (MOI = 1) and followed by treatment with DMSO, 3, 10 or 30 μM of R151785 for 6 h. Total RNA or protein was extracted for detection by RT-qPCR or western blot. Nucleozin (10 μM) was included as a control. Data represents mean ± SE from three independent experiments. Asterisks indicate statistically significant difference in comparison with DMSO control. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (student’s t-test). (H) R151785 inhibits influenza replication. MDCK cells were infected with A/WSN/33 (H1N1) virus at MOI of 1. Cells were fixed at 6 h p. i. and stained with mouse anti-influenza A NP antibody, rabbit anti-M1 and DAPI to determine the NP, M1, and nucleus, respectively. Bar = 25 μm.