Regulatory Mechanism Of CGAS And DDX3 On Innate Immunity Against Newcastle Disease Virus

Newcastle disease virus(NDV)can induce Newcastle disease(ND),which is highly contagious infectious,and has a serious impact on the development of poultry industry.Newcastle disease virus is not only used to target for the treatment of tumors,but also an effective vaccine carrier due to its replication efficiency in different cells.The replication efficiency of NDV in cells can be described from two aspects:1)Inhibit the synthesis of host protein and promoting the synthesis of virus m RNA and protein by utilizing host synthesis system;2)Escape from host antiviral immunity by some proteases encoded by itself or the host.In order to explore the mechanism of efficient replication of NDV,two important innate immunity molecular:cGAS(responsible for DNA sensing)and DDX3(involved in RNA sensing)interacting with proteins encoded by NDV were screened out by mass spectrometry and immunoprecipitation.cGAS is a key intracellular receptor responsible for sensing cytosolic DNA,which is mainly located in the cytoplasm.It can recognize exogenous DNA and self DNA released into the cytoplasm,such as mt DNA and nuclear DNA.Research in our laboratory had shown that mitochondria were significantly damaged in cells infected with NDV Herts/33.Through indirect immunofluorescence assay and LC-MS assay,we found that NDV Herts/33 infection could induce mt DNA released into the cytoplasm,the engagement of cGAS by mt DNA induced the formation of liquidlike droplets in which cGAS was activated and induces the synthesis of the second messenger cGAMP.Phase transition induced the formation of stress granules,and NDV infection induced the formation of viral SGs.G3BP1 is the marker protein of SGs.By constructing He La cell lines stably expressing G3BP1-GFP and H1299 cell lines knocking down G3BP1 gene,it was further found that G3BP1 enhanced the binding of cGAS with DNA,promoted the activation of cGAS and the syhthesis of cGAMP,which activate host antiviral innate immunity.And it was found that cGAS deficiency promote the replication of NDV and increase the virus titer.In addition,the study found that NDV Herts/33 infection could induce the second band of cGAS protein,but finally it was found that the second band could be the cGAS isoform2 through different assays.DDX3 is important for RNA recognition receptors,which assists RNA recognition receptors to recognize foreign RNA efficiently,activate the interferon pathway quickly and efficiently,and is crucial for the host to play an antiviral role.We detected viral proteins and interferon pathway related proteins during NDV infection after silencing DDX3 and expressing exogenous DDX3.We found that silencing DDX3 inhibits viral replication and promotes the expression of IFN-?and IFIT-1,and exogenous DDX3promotes viral replication,which indicate a pro-viral role of DDX3.Further study showed that virus infection induces DDX3 degradation and proteasome inhibitor MG132 could significantly inhibit DDX3degradation induced by virus infection.By constructing DDX3 deletion plasmids and lysine to arginine mutated plasmids,DDX3 K^138/162 was confirmed as the site of degradation.After infection,DDX3 was modified by ubiquitination and thus was degraded through proteasome.We confirmed that the cGAS-STING pathway is activated after infection and play an role in antiviral innate immunity.More importantly,we found that NDV infection might induces the expression of cGAS isoform2 which needs to be confirmed.DDX3 is degraded by proteasome through ubiquitination at K^138/162after infection,which attenuates antiviral natural immunity and enhances viral replication.This study provides theoretical basis for further research on the mechanism of host protein regulating and virus escaping antiviral innate immunity.