Fig. 3

USUV replication is not influenced by the canonical autophagy pathway. A-B A549, Huh7, and MEF cells were infected with USUV (MOI = 1). After the 1-hour virus adsorption period, cells were treated with DMSO (negative control), rapamycin (1 μm), wortmannin (1 μm), or bafilomycin A1 (100nM). At 24 hpi, the supernatants were collected to determine virus titers by plaque assay (A) and the cell viability was measured by MTS (B). Means ± standard deviation of 2–3 independent experiments are shown and statistical significance relative to DMSO-treated cells is indicated (* p < 0.05, ** p < 0.01, **** p < 0.0001). C-H wildtype (WT) MEFs, MEFs with knockouts (KO) for the autophagy related genes ATG3 (C-D), ATG5 (E-F), and ATG13 (G-H), and the corresponding cells with reintroduced genes (RSC) were infected with USUV (MOI = 3). At 24 and 48 hpi, the supernatants were collected to determine virus titers by plaque assay. Means ± standard deviation of 2–4 independent experiments are shown and statistical significance relative to wildtype cells is indicated (* p < 0.05). D, F, H Knockout of the autophagy related genes and the reintroduction of the genes were validated by RT-qPCR (D) or western blot analysis (F, H)