Fig 1.
ER stress are activated in DEF cells upon DEV infection.
(A)The ER ultrastructure in DEV-infected DEF cells under electron microscopy.Untreated cells were used as negative controls. Black arrows indicate the ER. Dilated ERs exist in DEV-treated cells. Scale bars: 1 μm. (B) DEF cells were infected with DEV at an MOI of 1 for 36h. Autolysosomes labeled with p62 antibodies were observed by immunoelectron microscopy. The arrow indicates colloidal gold dots representing p62. Scale bar: 500 nm. Untreated cells were used as negative controls. (C)DEF cells were infected with DEV at an MOI of 1 for 36 h. Endoplasmic reticulums labeled with GRP78 antibodies were observed by immunoelectron microscopy. The arrow indicates colloidal gold dots representing GRP78. Scale bar: 500 nm. Untreated cells were used as negative controls. (D) DEF cells were mock-treated or infected with DEV at a MOI of 1 and collected at 36, 48 and 60 hours post-infection and subjected to a western blot with antibodies against GRP78, LC3, β-actin (loading control), or DEV gB protein, as indicated. Thapsigargin (Tg) as a ER inducer. (E) Intensity band ratio of LC3-II to LC3-I and GRP78 to β-actin.
Fig 2.
PERK and IRE1 pathways was activated under ER stress and was involved in DEV-induced autophagy.
(A) DEF cells were infected with DEV at a MOI of 1 or treated with 300 nM Tg for 24 hours, and whole-cell protein extracts were collected at 36, 48, and 60 hours post-infection and then subjected to immunoblotting analysis of GRP78, p-PERK, p-eIF2α, LC3, ATF4, p-IRE1 and β-actin using the indicated antibodies. Tg is an inducer of ER stress. (B) Ratios of GRP78 to β-actin, p-PERK to PERK, p-eIF2α to eIF2α, ATF4 to actin, and p-IRE1 to IRE1, and LC3-II to LC3-I. (C) Activation of the IRE1-XBP1 signaling pathway. Total RNA was isolated from DEF cells, and RT-PCR was performed using XBP1-specific primers. PCR products were digested with PstI. Spliced XBP1 (s) products, 549 bp; Unspliced XBP1 (u) products, 276 bp; GAPDH was used as the loading control. (D) Inactivation of the ATF6 signaling pathway. DEF cells were infected with DEV at a MOI of 1 or treated with 300 nM Tg for 24 hours, and whole-cell protein extracts were collected at 36, 48, and 60 hours post-infection and then subjected to immunoblotting analysis of ATF6 and β-actin using the indicated antibodies.
Fig 3.
Effects of PERK-eIF2α and IRE1-XBP1 pathways on DEV replication.
(A) DEF cells were transfected with a Negative Control siRNA (siNC) or siRNAs directed against PERK or IRE1 (100 pmol/ml), followed by DEV infection. Whole-cell protein extracts were collected at 48 hours post-infection and then subjected to immunoblotting analysis of PREK, p-PERK, eIF2α p-eIF2α, IRE1, p-IRE1, LC3, and β-actin using the indicated antibodies. (B) Ratio data of LC3-II to LC3-I in treated DEF cells. (C) CQ was added to each sample in presented treated DEF cells.Whole-cell protein extracts were collected at 48 hours post-infection and then subjected to immunoblotting analysis of PREK, p-PERK, eIF2α p-eIF2α, IRE1, p-IRE1, LC3, and β-actin using the indicated antibodies. (D) Ratio data of LC3-II to LC3-I in treated DEF cells. (E) The virus yields were determined at 48 hours post-infection and expressed as TCID50 per 0.1 ml.
Fig 4.
siRNAs or drugs had no effect on cell viability.
siRNA and NC-siRNA were transfected or drugs treated for 48 hours, cell viability was tested using a WST-1 Cell Proliferation and Cytotoxicity kit, and absorbent density at 450 nm was expressed as relative cell viability.