Molecular Mechanism Of Inhibiting CGAS-STING Pathway By African Swine Fever Virus E248R Protein

African swine fever(ASF)is a hemorrhagic and devastating disease of pigs caused by African swine fever virus(ASFV).It is a legally reported animal disease of the World Organization for Animal Health(OIE)and it is also a type of animal disease stipulated by the laws of China.ASFV is a nucleocytoplasmic large DNA virus(NCLDV)with complex immune evasion mechanisms,and no effective vaccine is available against the disease at present.the innate immune response is the first line of defense in mammals to defense against pathogenic microorganisms.Pathogen-associated molecular patterns(PAMPs)are molecules that can be recognized by natural immune cells.Pattern recognition receptors(PRRs)are the molecules to recognize pathogens by host cells.PRRs mainly include TLR,NLRs,RLRs,CLRs,and cGAS,especially the cGAS-STING signaling pathway is a classic DNA recognition pathway,which plays an important role in the natural immunity induced by DNA viruses.Previous studies showed that virulent strain Armenia/07 blocked the cGAS-STING pathway in the early stage of infection.Here,the strain Amenia/07 and the epidemic strain in China are both belong to genotype ?.In addition,previous research showed that infectivity of the E248R-deficient ASFV was reduced at least 100-fold.However,it was not clear whether the ASFV E248 R protein was involved in the regulation of the cGAS-STING pathway.We used dosed-dependent dual-Luciferase reporter assay experiments and found that E248 R negatively regulated cGAS-STING and HT-DNA mediated IFN-? production.Then we used RT-PCR technology to verify that E248 R inhibited the IFNB1 gene transcription.Besides,we found that E248 R interacted with STING by co-immunoprecipitation and confocal experiments.Overexpression E248 R and STING in HEK293 cells or E248 R in Hela cells,both exogenous and endogenous STING were degraded.Through drug treatment experiments,we found 3-MA treatment could inhibit the degradation of STING by E248 R,suggesting that STING was degraded through the autophagy pathway.Then,we screened out the ATG101,an autophagy-related protein,which could interact with E248 R via yeast two-hybrid experiment,and the interaction was also confirmed by co-immunoprecipitation.Besides,exogenous experiments confirmed that ATG101 degraded STING,as well as E248 R,which promoted the expression of ATG101 were in a dose-dependent manner.The co-immunoprecipitation experiment showed that ATG101,STING,and E248 R could interact with each other.In summary,we demonstrate here that E248 R negatively regulates the cGAS-STING pathway,and confirm that E248 R recruits ATG101 to degrade STING and negatively regulate type I IFN production.We first identify the function of E248 R in innate immunity,and the finding expands the understanding of the immune evasion mechanisms of ASFV.