FIG 3.

Hepatitis delta virus replication induces autophagosome accumulation in Huh7 cells. (A) Huh7 cells were transfected as indicated or treated with rapamycin (200 nM) for 24 h. At 72 h posttransfection, cells were lysed, and the levels of LC3, HDAg, and β-actin were evaluated by Western blotting. (B) Huh7 cells were cotransfected with pCIneo or pSVLD3 and peGFP-LC3. As a positive control for autophagy induction, Huh7 cells cotransfected with pCIneo and peGFP-LC3 were treated with rapamycin (100 μM) for 3 h. After 72 h, cells were fixed and then stained with anti-HDAg antibody from HDV⁺ human serum (red). The nucleus was stained with DAPI (blue). (C) The number of GFP-LC3 puncta per cell was calculated from confocal images (n = 100). (D) Huh7 cells were transfected with pCIneo or pSVLD3 or treated with rapamycin (200 nM) for 24 h. At 3 and 9 days posttransfection, cells were lysed, and the levels of LC3, HDAg, and β-actin were evaluated by Western blotting. (E) Ratios of LC3II/β-actin.