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Fig. 2 | Virology Journal

Fig. 2

From: ADAR1p110 promotes Enterovirus D68 replication through its deaminase domain and inhibition of PKR pathway

Fig. 2

ADAR1p110 but not ADAR1p150 promotes EV-D68 replication. (A) 293 T cells were transfected with either siADAR1 or siNC, respectively. 48 h later, the transfected cells were infected with EV-D68 at MOI of 0.2. At 24 h after infection, cells were lysed and the expression levels of ADAR1 and viral protein VP1 were determined by western blotting, the supernatant was collected for viral titer assay. (B) The amount of VP1/beta-actin in (A) were quantified as described above. (C) Viral titers of the cell supernatant from (A) were determined by TCID50. (D) 293 T cells were transfected with plasmids expressing DDK-ADAR1p150 (4ug) or vector pCAGGS, 24 h later, the transfected cells were infected with EV-D68 at MOI of 0.2. The cells were collected and the expression of VP1, DDK-ADAR1p150 and beta-actin were determined by western blotting after 24 h infection. (E) VP1/beta-actin of each group were quantified as described above. (F) 293 T cells were transfected with 2 or 4 μg of plasmids expressing HA-ADAR1p110 or empty vector M07, then the cells were infected with EV-D68 as above. HA-ADAR1p110 and VP1 were determined by western blotting. (G)VP1/beta-actin for groups in (F) were quantified as above. (H) The cell supernatant from (F) was collected and viral titers were determined by TCID50. (I) Either HA-ADAR1p110 or DDK-ADAR1p150 expression plasmids were transfected into Hela cells, separately. After 24 h post transfection, cells were infected with EV-D68 (MOI 1.0). At 24 h after infection, cells were fixed and HA-ADAR1p110 was stained with rabbit HA primary antibody and goat anti-rabbit AF488, DDK-ADAR1p150 was stained with rabbit DDK primary antibody and goat anti-rabbit AF488, viral RNAs were stained with mouse J2 antibody and goat anti-mouse AF568. Nuclei were counterstained with DAPI. Scale bar is 10 μm. (J, K) Hela cells were transfected with plasmids expressing HA-ADAR1p110. 48 h later, cells were infected with HPIV3 at MOI of 0.1 for 24 h. The HA-ADAR1 and viral protein HN levels were determined by western blotting and HN/beta-actin were quantified as above. (L, M) Hela cells were transfected with plasmids expressing DDK-ADAR1p150 and infected with HPIV3 as above, DDK-ADAR1p150 and HN in cell lysates were detected and HN/beta-actin were quantified as above. In all the panels for western blotting, the relative molecular mass was marked besides each band. All data above were shown as mean ± SEM of n = 3 replicates, student’s t-test: **p < 0.01; ***p < 0.001; NS, not significant

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