Caspase-Dependent DDX21 Cleavage Regulates Antiviral Innate Immunity

The DEAD/H-box family RNA helicase is an important class of helicases.DDX21 is a member of the DEAD box family.It is mainly located in the nucleus and plays an important role in cellular RNA processing.Recently it has been shown that DDX21 can regulate the antiviral innate immunity.DDX21can regulate innate immunity by the formation of DDX1-DDX21-DHX36-TRIF complex in bone marrow-derived myeloid dendritic cells（mDCs）.After dengue virus infection,DDX21 migrates from the nucleus into the cytoplasm and regulates the immune response.However,it is unknown whether DDX21 has other innate immune regulatory mechanisms.To explore the new mechanism which DDX21 regulate the natural immune regulation,we conducted the following research:1.DDX21 promotes virus infection by regulating IFN pathway.To observe the role of DDX21 in antiviral immune response,DDX21 gene was silenced followed by virus infection.Western blot,TCID50,ELISA and RT-PCR were used to identify the effects of DDX21 knockdown on viral replication and interferon production.The results indicate that DDX21 knockdown promotes the production of interferon and inhibits viral replication.However,the overexpression of DDX21 had no effect on interferon production and viral replication.2.The cleavage of DDX21 by virus infection and treatment with RNA/DNA ligands.In order to study the modification of DDX21 after viral infection,different types of cell were infected by different viruses or nucleic acid mimics.Western blot analysis showed that both endogenous and exogenous DDX21 were cleaved after virus infection and treatments of DNA/RNA ligands.3.DDX21 was cleaved in a Caspase-3/6-dependent manner.To explore the cleavage mechanism of DDX21,we first used inhibitors to treat virus-infected cells.Results showed that caspase inhibitor z-VAD-FMK significantly impaired the cleavage of DDX21.Then we constructed a series of DDX21deletion and mutant plasmids,and proved that DDX21 was cleaved at D¹²⁶ in response to virus infection.By analyzing and experimentally verifying the sequence of the sites,we proved that the cleavage of DDX21 was mediated by Caspase-3/6.4.DDX21 was cleaved and translocated from nucleus to cytoplasm in response to virus infection.In order to observe the translocation of DDX21 after cleavage,we detected the localization of endogenous and exogenous DDX21 by laser confocal method and found that DDX21 migrated to the cytoplasm after virus infection which was mediated by its cleavage.5.DDX21 cleavage lead to the inhibition of IFN-βsignaling pathway.In order to investigate the effect of DDX21 cleavage on interferon production,we compared the effects of DDX21 cleavage and non-cleavage on interferon production.The results showed that DDX21 cleavage inhibited the production of interferon.6.DDX21 is capable of binding to RNA via its C-terminus.To further investigate how the cleavage of DDX21 regulates the production of interferon,we used CO-IP and RNA pull down methods to detect the interaction of DDX21 with itself and RNA.The results showed that DDX21 can interact with itself and RNA via its C terminus.In this paper,we have demonstrated that DDX21 plays an important role in antiviral innate immunity.More importantly,we first confirm the cleavage of DDX21 upon virus infection and the DDX21 cleavage was involved in its translocation and binding to dsRNA.This provides a theoretical basis for further research on the regulation mechanism of DDX21 on antiviral innate immunity.