Molecular Mechanisms Underlying The Modulation Of Virus Assembly And IL-1β Production By The H240R Protein Of African Swine Fever Virus

African swine fever(ASF)is an acute,febrile,and fatal hemorrhagic disease caused by African swine fever virus(ASFV),which infects wild and domestic pigs with 100% morbidity and mortality.Currently,prevention and control of ASF are mainly dependent on diagnosing,stamping-out policy and strict biosecurity measures due to no commercial vaccines or effective drugs.ASFV encodes more than 160 proteins,which are mainly involved in viral genome replication,damage repair,transcription,virus assembly,immune responses and so on.ASFV encodes 68 structural proteins,which are components of the virion and play important roles in the life cycle,immune response and virulence of ASFV.p72,p54,p17 and p B438 L are involved in the process of assembly in the life cycle of ASFV to affect viral morphogenesis.Therefore,the functions of one-third of structural proteins remain unknown.Focusing on this scientific issue,we previously resolved the ASFV capsid structure using the cryogenic electron microscopy and revealed that the H240 protein(p H240R)might be localized on the capsid of ASFV based on bioinformatics analysis.To the best of our knowledge,there are no relevant studies on the biological function of p H240 R.This study aims to dissect the biological function of p H240 R in ASFV-infected target cells.To investigate the function of p H240 R in ASFV infection,the gene deletion or acquisition strategy combined with immunoelectron microscopy was used to analyze the role of p H240 R in the life cycle of ASFV.First,our study found that p H240 R is a relatively conserved protein with 85 to100% homology by comparing the amino acid sequences of p H240 R from different isolates.The m RNA transcription kinetics showed that H240 R was a late transcription gene.p H240 R was localized in the cytoplasm and colocalized with the major capsid protein p72 in the virus factory in ASFV-infected porcine alveolar macrophages(PAMs)by confocal microscopy.The result of immunoelectron microscopy showed that p H240 R was located on the capsid of ASFV virion,indicating that it is a capsid protein of ASFV.Using a recombinant ASFV,ASFV-ΔH240R,with the H240 R gene deleted from the wild-type ASFV(ASFV-WT)genome,the results indicated that titers of infectious progeny virus were reduced by approximately 2.0 logs compared with those of ASFV-WT.The genome copy numbers and titers of progeny virions of the ASFV-ΔH240R-or ASFV-WT-infected PAMs were measured at different time points.The results showed that there were no significant differences in the genome copy numbers.Intriguingly,infectious progeny virus production was significantly difference in cell-free particles released from the ASFV-ΔH240R-or ASFV-WT-infected PAMs,indicating that the PAMs infected with ASFV-ΔH240R produced larger amounts of noninfectious particles.The noninfectious particle-to-titter ratios(genome copy number/titer)for ASFV-ΔH240R were 50 times higher than those for ASFV-WT.The data showed that the defective phenotype of virions produced by ASFV-ΔH240R was not due to a defect in attachment,internalization or genome replication.However,the assembly sites contained precursor viral membranes and only a few mature icosahedral particles,while numerous tubular structures and bilobulate structures were found in the viral factories in the ASFV-ΔH240R-infected PAMs by immunoelectron microscopy.The outermost layer of the icosahedral capsids and the aberrant structures were recognized by anti-p72 antibodies conjugated with gold,indicating that these abnormal structures are ASFV.Moreover,these different abnormal structures were released from PAMs by budding through the plasma membrane,which occurred in a manner similar to that of ASFV-WT.These results indicate that the deletion of the H240 R gene affected the formation of icosahedral symmetry of ASFV and produced a large number of noninfectious progeny virions to reduce the replication efficiency of ASFV-ΔH240R in PAMs.To investigate the phenotypic changes of H240 R gene deletion on ASFV-infected target cell,RNA-sequence analysis was used to characterize differentially expressed genes in the ASFV-ΔH240R-or ASFV-WT-infected PAMs.The data showed that IL-1β,TNF-α,and IL-6 were highly transcribed in the ASFV-ΔH240R-infected PAMs compared with ASFV-WT,indicating that host immune response is activated.The production of IL-1β depends on nuclear factor-kappa B(NF-κB)and inflammasome signaling pathway,which play important roles in the process of antiviral infection.The m RNA transcription of pro-IL-1β,and phosphorylated p65 and IκBα were increased upon ASFV-ΔH240R infection compared with ASFV-WT,indicating that ASFV-ΔH240R infection induced activation of the NF-κB signaling pathway.Furthermore,p H240 R significantly inhibited the activation of NF-κB mediated by IκB kinases(IKK)by interacting with NEMO to inhibit its expression,thereby inhibiting pro-IL-1β transcription.Caspase 1 activity and IL-1βexpression were inhibited under the downregulation of NLRP3 by small interference RNA,which promoted the replication of ASFV-ΔH240R in PAMs,suggesting that the activation of inflammasome by ASFV-ΔH240R depends on NLRP3.Mechanistically,p H240 R significantly inhibited IL-1β production as demonstrated by GLuc reporter activity in the presence of NLRP3 inflammasome components.Moreover,p H240 R significantly inhibited the formation of ASC spots in the presence of nigericin and lipopolysaccharide treatment by confocal microscopy.Coimmunoprecipitation(Co-IP)and confocal microscopy assays showed that p H240 R interacted and colocalized with NLRP3 in the cytoplasm,but m RNA transcription level and protein expression of NLRP3 were not affected by p H240 R.To investigate the effect of p H240 R on NLRP3 oligomerization,four truncated NLRP3 mutants(NLRP3 D1,NLRP3 D2,NLRP3 D3,NLRP3 D4)were constructed and the colocalization was examined by confocal microscopy.The results showed NLRP3-D1 mutant containing PRR domain alone presented oligomer in HEK293 T cytoplasm.p H240 R significantly inhibited the oligomerization of NLRP3-D1 and colocalized with NLRP3-D1 in the cytoplasm,indicating that p H240 R inhibits the oligomerization of NLRP3 PRR domain,thereby inhibiting IL-1β maturation.In summary,our study showed for the first time that p H240 R is a capsid protein that interacts with the major capsid protein p72.p H240 R directs the morphogenesis of ASFV toward the icosahedral capsid in the process of virus assembly.In addition,pro-inflammatory cytokine IL-1βis produced in ASFV-ΔH240R-infected PAMs.Further studies showed that p H240 R interacts with NEMO to inhibit the activity of NF-κB signal pathway to inhibit pro-IL-1β transcription,and binds to NLRP3 to inhibit its oligomerization,further inhibiting the maturation of IL-1β.Our study elucidates the role of p H240 R in the replication cycle of ASFV and the molecular mechanism of inhibiting IL-1β production,which may provide a new target for the development of vaccines and drugs for the prevention and control of ASF.