Figure 2

Autophagy promotes TMUV replication. A HEK293T cells were treated with RAPA (200 nM), CQ (40 µM) or 3-MA (5 mM) for 6 h to induce or inhibit autophagy, cells treated with equal volume DMSO were used as the negative control and untreated cells were used as the mock control. Then, the cells were infected with TMUV for 12, 24, and 48 h. Cell lysates were harvested at the indicated times and analyzed via Western blotting with anti-LC3, anti-NS3 and anti-β-actin antibodies. B Normalized NS3/β-actin intensity band ratio from the data in A. C HEK293T cells were treated with RAPA (200 nM), CQ (40 µM) or 3-MA (5 mM) for 6 h alone or in combination, and then, the cells were infected with TMUV for 48 h. Cell lysates were harvested and analyzed via Western blotting with anti-LC3, anti-NS3 and anti-β-actin antibodies. D Normalized NS3/β-actin, LC3-II/β-actin, and LC3-II/LC3-I intensity band ratios from the data in C. E HEK293T cells were treated with RAPA (200 nM), CQ (40 µM) or 3-MA (5 mM) for 6 h to induce or inhibit autophagy, and then, the cells were infected with TMUV for 12, 24, and 48 h. Cell lysates were harvested at the indicated times for qPCR to test the relative expression of the E gene. The expression of the E gene in the mock group was set as 1. Student’s t test and one-way ANOVA were performed to determine statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).