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

Background: Severe respiratory and neurological diseases caused byEV-D68 pose a serious threat to public health, but there are currently no effective drugs and vaccines. ADAR1 has been reported to have diverse biological functions in various viral infections, but its role in EV-D68 infection remains undetermined. Methods RD cells, 293T cells and Hela cells were used to evaluate the expression level of ADAR1 upon EV-D68 (Ferm strain) and HPIV3 (NIH47885) infection, respectively. The knockdown through siRNA and overexpression of either ADAR1p110 or ADAR1p150 in cell lines above were utilized to determine the role of the two proteins after viral infection. ADAR1p110 dsRBDs deleted mutation was generated by seamless clone kit. The expression patterns of ADAR1, EV-D68 VP1 protein and HPIV3 HN protein were identified by western blotting. TCID 50 was applied to detect viral titers. The transcription level of EV-D68 mRNA was analyzed by RT-qPCR and viral 5’UTR-mediated translation was analyzed by dual luciferase reporter gene. Conclusion Here, we found that the transcription and expression of ADAR1 was inhibited upon EV-D68 infection. RNA interference of endogenous ADAR1 decreased VP1 protein expression and viral titers, while overexpression of ADAR1p110 but not ADAR1p150 facilitated virus replication. Immunofluorescence assays showed that ADAR1p110 migrated from the nucleus to the cytoplasm after EV-D68 infection. Further, ADAR1p110 lost the pro-viral ability after mutating the key sites in the deaminase domain, and 5’-UTR sequencing of the viral genome revealed that ADAR1p110 probably plays a role in EV-D68 RNA editing. In addition, after knockdown of ADAR1, both p-PKR and p-eIF2α increased. Attenuated translation activity of viral genome 5’-UTR was also shown in dual-luciferase reporter gene assay. At last, deletion of ADAR1p110 dsRBDs increased PKR phosphorylation, which incurs a lower level of VP1 expression, indicating that the promotion of EV-D68 replication by ADAR1p110 is also related to the inhibition of PKR activation by its double stranded RNA binding domains (dsRBDs). Together, our work illustrates ADAR1p110 as a novel pro-viral factor of EV-D68 replication and provides a theoretical basis for EV-D68 antiviral research.