The Molecular Mechanism Of African Swine Fever Virus I10L Protein In Inhibition Of Autophagy To Promote Viral Replication

African swine fever(ASF)is a highly infectious swine disease caused by African swine fever virus(ASFV)infection,leading to significant losses in swine industry.Currently,there is no safe and effective commercial vaccine available for ASF.With a large genome,ASFV encodes over 160 proteins and the functions of half of these proteins remain unknown.To develop ASF vaccines,it is critical to completely understand the functions of ASFV-encoded proteins.Previous reports have shown that ASFV may inhibit the host antiviral immune response for immune evasion by various strategies.However,it is not fully understood whether other proteins encoded by ASFV are involved in regulating the host antiviral immune response.To identify ASFV proteins that regulate host innate immune,we constructed 46 expression vectors of ASFV proteins and verified expression in HEK293 T cells.Among them,11 ASFV-encoded proteins are able to inhibit IFN expression induced by c GAS-STING signaling.Specifically,the suppressive roles of I215 L and I226 R are the most striking ones,suggesting that both ASFV I215 L and I226 R function as negative regulators of host antiviral viral immune responses.Mechanistically,I215 L inhibits the phosphorylation of TBK1 and IRF3 and the activation of IFNβ promoter mediated by expression of c GAS-STING,STING or TBK1.These results suggest that I215 L may target TBK1 to regulate host antiviral immune responses.In contrast,while I226 R inhibits c GAS-induced activation of IFNβ promoter,it did not affect STING-,TBK1-,and IRF3-5D-induced activation of IFNβ promoter activation.Consistently,I226 R also does not impair c GAMP-induced IFN expression.This data suggests that I226 R may target c GAS to regulate the host antiviral immune response.Considering that autophagy is crucial to eliminate pathogens from infected cells,we also investigate the role of ASFV-encoded proteins on autophagy.We examined the effects of 20 nonstructural proteins of ASFV on autophagy.The results show that ASFV-encoded protein I10 L expression caused the accumulation of autophagy-related proteins LC3 and p62 in a timeand dose-dependent manner,indicating that I10 L is an important autophagy regulating protein.In addition,treatment with both the autophagy inhibitor CQ or Baf-A1 and the autophagy inducer INK128 or EBSS treatments does not enhance the accumulation of LC3 and p62 caused by I10 L,suggesting that I10 L inhibits autophagy.Furthermore,immunofluorescence and transmission electron microscopy also reveal that I10 L caused the accumulation of autophagosomes.To investigate whether I10 L inhibits autophagy by blocking autophagosomelysosome fusion,we used m RFP-GFP-LC3 assays to measure autophagic maturation.Our findings indicate that I10 L inhibits the fusion of autophagosomes and lysosomes.Immunofluorescence and immunoprecipitation assays demonstrate that the transmembrane domain(TM domain)of I10 L was the primary functional domain responsible for its inhibitory effect on autophagy.In addition,the results show that the interaction between I10 L and RAB7 protein occurs in the TM domain of I10 L.To further demonstrate the mechanism of I10 L,we test the formation of RAB7-HOPS and SNAREs complex.The results show that I10 L effectively decreases the interaction between RAB7 and the HOPS subunit VPS39,thus preventing HOPS target the lysosome and forming a functional complex with RAB7.After the RAB7-HOPS tethering is accomplished,the assembly of the SNARE complex is the final step in autophagosome and lysosome membrane fusion.Immunoprecipitation and immunofluorescence analyses show that I10 L disrupting the interaction with SNAP29 and STX17,suggesting I10 L hinders the assembly of the STX17-SNAP29-VAMP8.Additionally,I10 L significantly reduces the interaction with SNAP29 and STX6,leading to the failure of STX6-SNAP29 complex formation.Furthermore,the results indicate that ASFV-ΔI10L replication was significantly reduced,compared to ASFV-WT.Furthermore,ASFV-ΔI10L was found to significantly weaken the inhibitory effect of autophagy.Collectively,these results suggests that I10 L promotes ASFV replication by inhibiting autophagy.In summary,11 ASFV proteins was identified to negatively regulate c GAS-STINGmediated host antiviral immune responses with I215 L and I226 R are two representative proteins.More improtatly,ASFV-encoded I10 L blocks autophagy process and impedes the fusion of autophagosomes and lysosomes by interacting with RAB7.The above findings aid in comprehending the immune escape mechanism of ASFV and offer new insights into the pathogenicity of ASFV and the development of ASF vaccines.