| How host cells defend against viral infection is a key question in biology.Innate immunity is the first barrier against viral infection,which is initiated by pattern recognition receptors（PRRs）-mediated recognition of pathogen-associated molecular patterns（PAMPs）,including viral nucleic acids.Upon viral infection,viral nucleic acids activate a series of signal cascades to induce expression of interferons（IFNs）,chemokines and inflammatory cytokines,which eliminat viral invasion by inhibiting virus replication and inducing apoptosis of infected cells.Pattern recognition receptors that recognize cytoplasmic nucleic acids mainly include toll-like receptor 3/7/8/9（TLR3/7/8/9）,retinoic acid-inducible gene I（RIG-I）-like receptors and DNA sensor cyclic GMP-AMP synthase（cGAS）.Among them,cGAS-mediated innate immune response plays a crucial role in host defense against DNA viruses.cGAS recognizes both viral DNA and the mislocalized self-DNA in a sequence-independent manner,and then catalyzes synthesis of cyclic GMP-AMP（cGAMP）from ATP and GTP.The cGAMP further binds to and activates ER-associated membrane protein STING/MITA to trigger antiviral innate immune response.In past years,the mechanisms of cGAS activation have been gradually revealed.However,how cGAS activity is regulated has not been fully understood.How viruses invade host cells has been well elucidated in past decades.Except for some enveloped viruses that are released into the cytoplasm by direct fusion of their own envelopes with the plasma membranes,non-enveloped viruses and most enveloped viruses invade host cells through endocytosis and then are transported to endosomes via vesicular trafficking.The invaded viruses achieve endosomal escape for successful viral replication,which depends on the acidic environment of endosomes,or otherwise are digested in the lysosomes.These processes are extremely similar to that of transfection of nuclear acids by the commonly used agents lipofectamine 2000（LF2000）and polyethylenimine（PEI）.In recent years,some studies have reported that invasion of viruses or foreign substances could be sensed by the immune surveillance machinery of host cells.However,the underlying mechanisms are still poorly understood.It has been well established that tyrosine kinases play critical roles in mediating downstream signaling cascades upon ligand-receptor interaction on the cell surface.To explore the potential plasma membrane-associated molecular events that regulate cGAS-STING/MITA-mediated innate immune responses during viral endocytosis,we screened a group of tyrosine kinase inhibitors to examine their effects on expression of Ifnb1 gene induced by several different DNA stimuli.The inhibitors of spleen tyrosine kinase（SYK）specifically inhibited the transcription of antiviral genes induced by HSV-1 and HT-DNA that are endocytosed into cytoplasm,but not by endogenous mitochondrial DNA（mt DNA）.Consistently,SYK-deficiency significantly inhibited endocytosed foreign DNA-triggered transcription of antiviral genes and the phosphorylation of key molecules in the cGAS-STING/MITA-mediated innate immune signaling.Furthermore,SYK also played an important role in the innate immune response against DNA viruses in vivo.Syk inhibitor treatment significantly inhibited HSV-1-induced secretion of antiviral cytokines in the serum of mice,as well as caused higher viral titers in the mouse brains and higher mortality of mice.Further studies showed that SYK interacted with cGAS and phosphorylated human cGAS Y214/215（corresponding to mouse c Gas Y200/201）.Mutation of Y214/215 to F or SYK inhibition suppressed the activity of cGAS by dampening the formation of the liquid-liquid separation structure of“cGAS-DNA”.Importantly,we further identified the vacuolar ATPase（V-ATPase）which mediated the activation of SYK during endocytosis and acted as a scaffold protein to promote the interaction between SYK and cGAS.V-ATPase inhibitor treatment inhibited endocytosed foreign DNA-triggered transcription of antiviral genes and phosphorylation of SykY519/520,c GasY201 and Irf3S388.Finally,we showed that the V-ATPase-SYK axis also played a key role in the LF2000-induced innate immune response.Collectively,we demonstrated that the V-ATPase-SYK axis-mediated cGAS priming and DNA ligand binding are two necessary but independent signals,and the combination of these two signals induces an efficient and potent innate immune response.In summary,we propose a“two-signal”model for effective cGAS activation,including V-ATPase-SYK-mediated priming signal and nucleic acid DNA stimulation signal during endocytosis of virus or foreign DNA.Mechanically,viral infection or exogenous DNA transfection triggers recruitment of tyrosine kinases SYK and cGAS to endosomal V-ATPase.V-ATPase mediates SYK activation and acts as a scaffold protein to promote interaction between SYK and cGAS,which facilitates phosphorylation of human cGAS Y214/215（corresponding to mouse c Gas Y200/201）by SYK and primes cGAS activity.When bound to DNA,the primed cGAS promotes robust cGAMP production and STING/MITA-mediated innate immune response.This study renews our understanding of intracellular nucleic acid sensor-mediated innate immunity and provides a direct link between viral endocytosis and innate immune surveillance machinery,which may provide new insights for drug design of infectious and autoimmune diseases. |
