Study On The Role Of STING In Autophagy Induced By Foot-and-Mouth Disease Virus Infection And Its Molecular Mechanism

Stimulator of interferon genes(STING)is an important innate immunity-related adaptor protein that located in the endoplasmic reticulum of mammalian cells.The canonical role of STING is to restrict DNA virus infection via activating the interferon signaling pathway;however,recent studies have found that STING is also involved in limiting RNA virus infection,and its mechanism beyond this remains unclear.Foot-and-mouth disease virus(FMDV)is a positive,single-stranded RNA virus that is the causative agent of the acute,hot,and highly contagious disease of foot-and-mouth disease(FMD).In this study FMDV was used as the model virus to explore the role and molecular mechanism of STING during FMDV infection.It was found that FMDV infection caused the degradation of STING protein in host PK-15 cells.Inhibition of autophagy with CQ and Baf-A1 can inhibit FMDV-mediated degradation of STING.Knockdown of the key autophagy-related gene 5(ATG5)or ATG7 inhibits STING degradation and viral replication;knockdown of the reticulophagy receptor FAM134 B inhibits STING degradation and viral replication,indicating that FMDV induces the selective reticulophagy to degrade STING and promote viral replication.Previous studies have found that FMDV activates autophagy through the protein kinase R(PKR)-like endoplasmic reticulum kinase(PERK)-eukaryotic initiation factor 2 alpha(e IF2?)pathway of the endoplasmic reticulum stress unfolded protein response(UPR).To further explore the molecular mechanism of how FMDV induced the reticulophagy,PERK was silenced or knockouted using either RNAi or CRISPR-Cas9 technology.It was found that the FMDV-infected PERK-deficient cells could not activate reticulophagy,STING does not go through degradation,and viral replication is inhibited;Knockdown of the UPR sensor protein activating transcription factor 6(ATF6)or using the specific inhibitor 4?8c to inhibit the activation of the sensor protein inositol requiring enzyme 1(IRE1)does not affect the reticulophagy activation,STING degradation and viral replication,indicating that FMDV infection specifically activates the PERK-mediated integrated stress response(ISR)to induce reticulophagy.Moreover,using RNAi and CRISPR-Cas9 technologies to silence or knockout the gene of STING,it was found that the absence of STING inhibits viral replication,PERK activation,reticulophagy induction and STING degradation,which indicates that STING is the necessary upstream molecule to activate PERK to induce reticulophagy.Moreover,immunoprecipitation results showed that STING interacted with PERK.In order to investigate the upstream signaling events of STING activation,RNAi technology was used to inhibit the expression of cellular pattern recognition receptors cyclic GMP-AMP synthase(c GAS),retinoic acid inducible gene-I(RIG-I)and melanoma differentiation associated gene 5(MDA5),respectively.The results showed that FMDV infection could not activate the reticulophagy to degrade STING in RIG-I deficient cells,indicating that RIG-I is the upstream signaling molecule for STING activation.To explore the mechanism by which STING activates reticulophagy,truncated STING 1-340,which can not induce interferon production,was reinstituted into STING knockout cells.FMDV infection can still activate reticulophagy to degrade STING,indicating that the autophagy induction function of STING is independent of its interferon-inducing function.Using H-151 specifically inhibits the activation of human STING that has been reinstituted in STING knockout cells and FMDV infection can still activate reticulophagy to degrade STING and viral replication is not affected,suggesting that the trafficing and activation of STING is not required for reticulophagy induction.The mutants STING E69 A and STING C148 A were reinstituted in STING knockout cells respectively.And it was turned out that FMDV can induce reticulophagy and STING degradation in STING E69 A reinstituted cells,while the activation of reticulophagy and STING degradation was blocked in STING C148 A reinstituted cells,implying that the activation of reticulophagy depends on the polymerization of STING but independent of its phosphorylation.This study clarified the molecular mechanisms of STING-mediated autophagy in regulating FMDV replication,enriched the regulatory mechanisms of STING in modulating RNA virus infection,and uncovered the important role of STING in linking natural immune responses and cellular adaptive stress responses.