Role Of Dead-box RNA Helicase DDX25 And DDX3X During Dengue Virus Infection

Dengue virus(DENV), transmitted by Aedes aegypti and Aedes albopictus, is the most widely distributed insect-borne virus in the world. It is a single strand RNA virus. Human beings, primate animals and mosquitos are the natural host of DENV. The severity of diseases can range from asymptomatic to life-threatening dengue hemorrhagic fevers(DHF) or dengue shock syndromes(DSS). Millions of DENV infections occur each year, but the mechanism of DENV infection is still not clear. DENV has 4 different serotypes. Infection with one type usually gives lifelong immunity to that type, but only short-term immunity to the others. Subsequent infection with a different type increases the risk of severe complications. For that reason, DENV vaccine development is challenging.Dead-box RNA Helicases belong to the large SF2 helicase superfamily. They participate in various processes of RNA metabolism. A number of DEAD-box helicases are involved in anti-viral immunity. In this project, an RNAi screening of 40 human DDX genes on DENV infected HEK293 T cells were performed to investigate which DDXs are involved in DENV replication. DDX3 X and DDX25 were selected as our research objects. The result showed that DENV replication was significantly upregulated in DDX3 X si RNA-treated cells, but downregulated in DDX25 si RNA-treated cells.In order to further study the antiviral effect of DDX3 X, we overexpressed DDX3 X in HEK293 T cells, then infected the cells with DENV. The result showed that the replication of DENV was signaficantly reduced in DDX3 X overeexpressed cells. Further study showed that, during the early stage of DENV infection, the expression of IFN- β was reduced in DDX3 X silenced cells. While when DDX3 X was overexpressed, the production of IFN-βwas increased. DDX3 X contributed to the transcription of IFN- β in DENV infected 293 T cells. DDX3 X promotes the dimerization and phosphorylation of IRF3 and the transcription activity of NFκB induced by RIG-I and DENV. Our results suggested that DDX3 X may contribute to early IFN-β production through activating both IRF3 and NFκB branches, thereby inhibits DENV replication.Different from the previous reports which showed that DDX25 is a tissue specific RNA helicase, we found that DDX25 is expressed in various human tissues and cell lines. What’s more, DDX25 was distributed in cytoplasm and nucleus. The expression of DDX25 was significantly increased after DENV infection in epithelial cells. Silencing DDX25 could inhibit the replication of DENV, while overexpressing DDX25 helped the viral replication. Further study showed that interfering with DDX25 in 293 T cells, the transcription of IFN-β was highly increased. While, overexpressing of DDX25 may cause a decrease of IFN-β transcription. In addition, DDX25 could inhibit the transcription of IFN-β induced by Se V, VSV and poly I:C. Further study suggested that DDX25 inhibited the transcription of IFN-β induced by overexpression of RIG-I, MDA5, MAVS, TBK1 and IRF3. At the same time, DDX25 inhibited the transcription activity of NFκB induced by overexpression of RIG-I, IKKα and P65 subunit. Therefore, we proposed that DDX25 could inhibit both IRF3 and NFκB activity in IFN-β signaling pathway, thereby, enhance DENV infection.In conclusion, we have shown the anti DENV activity of DDX3 X, and for the first time, found that DDX25 could inhibit IFN-β signaling pathway. These study further enriched the knowledge on the roles of Dead-box RNA helicases in the anti-viral innate immunity, and could also provide new targets for antiviral drugs.