The H240R Protein of African Swine Fever Virus Inhibits Interleukin 1β Production by Inhibiting NEMO Expression and NLRP3 Oligomerization

The H240R protein (pH240R), encoded by the H240R gene of African swine fever virus (ASFV), is a 241-amino-acid capsid protein. We previously showed that the deletion of H240R from the ASFV genome, creating ASFV-ΔH240R, resulted in an approximately 2-log decrease in infectious virus production compared with the wild-type ASFV strain (ASFV-WT), and ASFV-ΔH240R induced higher interleukin 1β (IL-1β) production in porcine alveolar macrophages (PAMs) than did ASFV-WT, but the underlying mechanism remains to be elucidated. Here, we demonstrate that the activation of the NF-κB signaling and NLRP3 inflammasome was markedly induced in PAMs upon ASFV-ΔH240R Infection compared with ASFV-WT. Moreover, pH240R inhibited NF-κB activation by interacting with NEMO and promoting the autophagy-mediated lysosomal degradation of NEMO, resulting in reduced pro-IL-1β transcription. Strikingly, NLRP3 deficiency in PAMs inhibited the ASFV-ΔH240R-induced IL-1β secretion and Caspase 1 activation, indicating an essential role of NLRP3 inflammasome activation during ASFV-ΔH240R replication. Mechanistically, pH240R interacted with NLRP3 to inhibit its oligomerization, leading to decreased IL-1β production. Furthermore, the inhibition of the NF-κB signaling and NLRP3 inflammasome activation promoted ASFV-ΔH240R replication in PAMs. Taken together, the results of this study reveal an antagonistic mechanism by which pH240R suppresses the host immune response by manipulating activation of the NF-κB signaling and NLRP3 inflammasome, which might guide the rational design of live attenuated vaccines or therapeutic strategies against ASF in the future. IMPORTANCE African swine fever (ASF), a lethal hemorrhagic disease, is caused by African swine fever virus (ASFV). There are no commercially available vaccines or antivirals for the disease. Here, we showed that ASFV with a deletion of the H240R gene exhibits high-level expression of interleukin 1β (IL-1β), a proinflammatory cytokine, in porcine alveolar macrophages and that the H240R protein (pH240R) exhibits robust inhibitory effects on IL-1β transcription and production. More specifically, pH240R inhibited NF-κB activation via the autophagy-mediated lysosomal degradation of NEMO, leading to the decrease of pro-IL-1β transcription. In addition, pH240R interacted with NLRP3 to inhibit its oligomerization, leading to decreased IL-1β production. Our results indicate that pH240R is involved in the evasion of host innate immunity and provide a novel target for the development of a live attenuated vaccine against ASF.