Figure 7: Pharmaceutical drugs that inhibit cytoskeletal dynamics attenuate the number of TNTs and cell-to-cell spread of the viral genome.

(A) A549 cells were incubated for 6 h with or without cytochalasin D and nocodozole, and stained for actin (Red) and Tubulin (Green) filaments before imaging and recording in 3D by confocal microscopy. Shown are the maximum intensity projections of the focal planes of a representative image stack from control and drug-treated cells. Arrows indicate TNTs observed in control (DMSO-treated) cells. TNTs were not observed in cells treated with either drug compared to the untreated-control. For each time point > 1000 cells were analyzed, and the experiment was repeated three times. Scale bar: 10 μm. (B) NS1-GFP infected cells were incubated with uninfected RFP-A549 cells, in the presence of neutralizing antibodies (2000 HI units), Oseltamivir (100 μM) and Cytochalasin D (20 μM). After 8 h of incubation, the cells in the R-and R/G-quadrants and G-quadrant were sorted. The sorted cells were cultured in the presence of Cytochalasin D (20 μM) for an additional 6 h, after which RNA was extracted and the expression of vRNA for NP and NS1 were analyzed using RT-PCR. The graph shows the expression of vRNA genes, NP and NS1 and data are shown as mean ± SD. β-Actin was used as a loading control. ANOVA was performed to compare the expression in cells in the G-quadrant versus cells in the R- and R/G-quadrants, and p values < 0.05 are indicated with an asterisk.