Fig. 2

USUV infection and USUV NS4A overexpression induce the formation of autophagosomes. A Huh7 cells were infected with USUV (MOI = 1) and fixed at 24 hpi. Cells were stained for the orthoflavivirus envelope protein (green) and LC3 (red). Nuclei were visualized by Hoechst 33,258. Scale bars (white) in the bottom right corner represent 10 μm. Total number of LC3-puncta were determined in three independent images of the mock-infected and USUV-infected cells. Representative images are shown. B Huh7 cells were infected with USUV (MOI = 1) and protein lysates were collected at the indicated times. LC3, p62, and GAPDH levels were then analyzed by western blot. Numbers between the panels represent the band intensity ratio of LC3-II or p62 to GAPDH and are normalized to mock-infected cells at 8 hpi. C-D Huh7 cells (C) or A549 cells (D) were infected with USUV (MOI = 1) for 48 h. 6 h before harvest the cells were treated with 100 nM bafilomycin A1 (BafA1) or left untreated. Samples were immunoblotted for LC3 and GAPDH. Numbers between the panels represent the band intensity ratio of LC3-II to GAPDH and are normalized to the untreated mock-infected cells. E H1299 cells were co-transfected with LC3-GFP (green) and the plasmid encoding HA-tagged USUV NS4A or the empty vector control (EV). Cells were fixed at 24 hpt and stained for the HA tag (red). Scale bars (white) in the bottom right corner represent 10 μm. F 293T cells were transfected with different USUV protein expression plasmids, and treated with DMSO or BafA1 (100 nM) for 6 h before harvesting. At 24 hpt lysates were collected and immunoblotted for LC3, V5 (NS1, NS2A, NS2B3, NS3, NS4B, and NS5), HA (NS2B, and NS4A), and GAPDH. Numbers between the panels represent the band intensity ratio of LC3-II to GAPDH and are normalized to the empty vector (EV) transfected cells