A Preliminary Study On The Inhibition Of Host Innate Immunity By Protein DDX5

Host cell DEAD-box(DDX)family proteins are highly conserved and numerous RNA helicases,which are mainly involved in the transcriptional regulation of host cell genes.However,recent studies have shown that DDX family proteins are also involved in the innate immune response of the host and play an important role in the production of type I interferon(IFN-I)and inflammation.DDX5 protein is an important member of the DDX family,which not only plays a role in cell growth and development and tumorigenesis,but also affects virus replication in the process of virus infection.However,its mechanism is not clear.In our previous studies,we found that chicken Marek's virus can regulate the host DDX5 protein to inhibit the host innate immune response and promote the growth of the virus on chicken embryo fibroblasts,which indicates that DDX5 plays an important role in the process of avian innate immunity.However,it has not been reported whether DDX5 plays the same role in mammalian innate immunity.In order to further confirm that DDX5 protein regulates the innate immune response in mammals.In this study,vesicular stomatitis virus((VSV))was used to infect mouse fibroblasts with DDX5 gene knockout or overexpression,respectively.By measuring the transcription levels of IFN-?and IL-6 m RNA induced by virus,the contents of IFN-?and IL-6 in cell supernatant and their effects on virus replication,it was found that DDX5significantly inhibited the production of IFN-?and IL-6 after virus infection and enhanced virus replication.Studies have shown that DDX5 acts as a negative antiviral regulator in innate immunity to inhibit the production of IFN-?and IL-6 after virus infection and promote virus replication.DDX5 gene deletion mice were constructed by CRISPR/Cas9technique,and their offspring and wild type mice were challenged with VSV and Sendai virus(Se V).The secretion of IFN-?and IL-6 in macrophage culture supernatant and serum of two kinds of mice were measured by ELISA,and the replication titers of virus in internal organs(lung,liver and spleen)of mice were determined,and histopathological analysis was carried out at the same time.Compared with wild type offspring mice,the production of IFN-?and IL-6 in serum of DDX^5+/-mice increased significantly,and DDX5^+/-mice had less pulmonary interstitial pneumonia,mild liver injury and less spleen pathological changes.Studies have shown that DDX5 protein can promote virus replication and enhance virus-induced tissue damage through negative regulation of innate immunity in mice.In order to further explore the mechanism of action,the host protein target protein METTL3,interacting with DDX5 was screened and identified by Pull down and Co-IP experiments.By analyzing the domain of DDX5/METTL3,the functional domains of DDX5 truncated protein and METTL3 truncated protein interaction were constructed respectively.It was found that the molecular interaction between DDX5 and METTL3requires the N-terminal domain of METTL3 and the P68HR domain of DDX5.It has been confirmed that DDX5 interacts with the N-terminal of METTL3 through its P68HR region.In summary,we demonstrated for the first time that DDX5 protein negatively regulates the production of IFN-?and IL-6 in mammalian cells,which interacts with the N-terminal region of host cell METTL3 protein through the P68HR domain.The results not only provide a basis for the study of the mechanism of DDX5 in the innate immunity of viral infection,but also provide a potential target for the development of new antiviral drugs.