Fig. 3

The pro-viral effect of ADAR1p110 on EV-D68 is related to its deaminase domain. (A) Schematic structural illustration of ADAR1p110 and its three key amino acid sites for the enzyme activity in the deaminase domain. (B, C) 293 T cells were transfected with plasmids M07, M07-HA-ADAR1p110, M07-HA-ADAR1p110-H910A, p110-C996A and p110-C1036A, separately. 48 h later, cells were infected with EV-D68 at MOI of 0.2. After 24 h infection, the expression levels of HA-ADAR1, ADAR1 mutants and VP1 in cell lysates were detected by western blotting and the relative molecular mass was marked besides each band. VP1/beta-actin of each group were quantified as described above. (D) The original sequence of viral 5’-UTR was presented in the box. (E) 293 T cells were transfected with either plasmid expressing HA-ADAR1p110 or the control plasmid M07. 48 h later, the cells were infected with EV-D68 at MOI of 0.2. The cells were collected at 24 h post-infection, and total cellular RNA was extracted for RT-qPCR. Mutations in viral 5’-UTR were determined by Sanger sequencing. DNA sequencing chromatograms of PCR products are shown. (F) After 15 generations of persistent infection in ADAR1 knockdown (ADAR1 KD) 293 T or normal 293 T cells, the 5’-UTR mutation of EV-D68 was determined as described above. DNA sequencing chromatograms of RT-qPCR products are shown. All data above are shown as mean ± SEM of n = 3 replicates, student’s t-test: **p < 0.01;***p < 0.001; NS, not significant