Study On The Evasion And Mechanism Of ASFV Protein A528R In Porcine Innate Immune TLR8 Signaling Pathway

African swine fever(ASF)is a highly contagious and fatal infectious disease caused by African swine fever virus(ASFV),and its natural hosts are domestic pigs and wild boars.ASFV has a fast epidemic rate and can cause persistent infection in the host without showing obvious disease symptoms,making it difficult to prevent and control.The ASFV genome is about 170-193kb,including 150-167 open reading frames(ORFs),encoding nearly 200 proteins.The virus has a large genome and a complex immune evasion mechanism,and there is no effective vaccine so far.Most of the encoded proteins of ASFV have unknown functions,and their study can further to deepen our understanding of ASFV immune evasion and pathogenic mechanisms,and has potential significance for the development of new vaccines.Innate immune Toll-like receptor 8(TLR8)is a member of the Toll-like receptor family.After being activated by the corresponding pathogen-associated molecular patterns(PAMP),it releases a variety of inflammatory cytokines including interleukins,mediates antibacterial and antiviral responses.Given the complexity of the structure of ASFV,multiple innate immune recognition receptors,including TLR8,may be involved in the viral infection recognition and immunity.This paper mainly focuses on the immunosuppression and mechanism of ASFV protein on the TLR8 signaling pathway.In this study,the regulatory role of ASFV 137 ORF in porcine TLR8 signal pathway was detected by TLR8-NF-κB stability reporter cell line,and the A528R protein which had the most significant inhibitory effect on TLR8-NF-κB signal was screened.The target of A528R in TLR8-NF-κB signal pathway was detected by double luciferase reporting assay.The results showed that A528R could inhibit the downstream NF-κB signal caused by the expression of main signal proteins(TLR8,MyD88,IKK-β and NF-κB p65)in TLR8-NF-κB signal pathway,although it does not rule out the possibility that A528R has multiple action targets in TLR8NF-κB signal pathway.However,it is speculated that p65 is the key target for A528R to play an inhibitory role in TLR8-NF-κB signal pathway.Indeed,A528R can inhibit p65 phosphorylation,nuclear translocation,nuclear translocation and cell signaling activity.The cellular localization of A528R and p65 in porcine alveolar macrophages(PAM)was analyzed by indirect immunofluorescence,and it was found that there was obvious co-localization between the two.Co-immunoprecipitation(Co-IP)results showed that A528R interacted with p65 protein.Further,by constructing mutants of A528R and p65,the interaction site between A528R and p65 was explored.The results showed that the Rel Homology Domain(RHD)of p65 mediates its interaction with A528R;5 of A528R Both ankyrin repeats(ANKs)affect their interaction with p65.The TLR8 signaling pathway has significant antibacterial and antiviral effects upon activation.A528R interfered with the anti-bacterial and antiviral effects of R848/TLR8 signal,but the inhibitory effect of A528R on the anti-infective effect of the TLR8 pathway decreased after its ANK site mutation.To sum up,this study proved that ASFV A528R protein interacts with p65 protein in TLR8 signal pathway through its ANK site,inhibits the TLR8 signal pathway and its mediated antiviral and anti-bacterial effects,and plays an important role in ASFV immune escape,which will provide theoretical clues for the prevention and treatment of ASFV and vaccine development.