African Swine Fever Virus MGF505-7R Regulates NLRP3 Inflammatory Response

African swine fever(ASF)is an acute hemorrhagic infectious disease caused by ASF virus(ASFV)infection.Since ASF epidemic was first reported in China in Augest 2018,the disease had rapidly spread,which has brought serious economic losses to China's pig industry.Due to the lack of commercialized vaccines and antiviral drugs available for ASF disease,the prevention and control situation of ASF is extremely grim.ASFV is a large double-stranded DNA virus that encoded at least150 proteins,of which more than 70% are non-structural proteins.These non-structural proteins are related to the functions of virus invasion,replication and evasion from host immunity.Previous reports have showed that ASFV-encoded proteins could escape the innate immunity of host.Relevant studies mainly focus on the viral proteins regulating Type I interferon and apoptosis signaling pathway,and the regulation of inflammatory response is concentrated in NF-?B signaling pathway.Inflammatory response is an important part of innate immunity,which is related to the pathogenesis and pathogenicity after viral infection.At present,researches on the regulation of inflammatory response by ASFV are in the phenomenon stage.In this regard,the mechanism by which ASFV regulates the inflammatory response is still unclear.Pathogen infection activates inflammatory response and secretes proinflammatory cytokines,and IL-1? is the important one.This study explored the mechanism of ASFV infection regulating IL-1? production from the molecular level.To investigate whether ASFV HLJ/18 strain isolated from Harbin Veterinary Research Institute infection induce inflammatory response,porcine alveolar macrophages(PAMs)were infected with ASFV,and the m RNA expressions and secretion levels of IL-1?,TNF-? and IL-6 were detected.The results showed that the low levels of IL-1? were induced by ASFV,and the levels of TNF-? and IL-6were not obviously affected.PAMs were first infected with ASFV for 24 h and then treated with LPS+Nigericin or LPS+poly(d A:d T)to stimulate inflammatory responses.We found that the transcription and secretion levels of IL-1? decreased in a gradient with the increase of ASFV dose,suggesting that the ASFV-encoded proteins may inhibit the inflammatory responses in PAMs.To investigate the regulation of inflammatory response by ASFV-encoded proteins,we constructed i GLuc(pro-IL-1?-Gluc)-based NLRP3 inflammasome reconstruction system with high sensitivity.A high-throughput screening assay was performed to screen ASFV-encoded proteins that regulate the secretion of mature IL-1? by detecting sensitively the fluorescence signal in the cell supernatants were collected to assess the IL-1? production.We found that several members of Multigene family(MGF)505/360 inhibited inflammatory response.Among them,pMGF505-7R showed the most significant inhibitory effect.To further verify and explore the regulatory ability of pMGF505-7R on inflammatory response,a recombinant ASFV lacking the MGF505-7R gene(ASFV-?7R)was generated by CRISPR/Cas9 and homologous recombination.ASFV-?7R displayed a similar growth kinetic compared to its parental ASFV-WT.PAMs were infected with ASFV-WT or ASFV-?7R at the same dose and the m RNA expressions and secretion levels of IL-1? were detected.We found that ASFV-?7R induced higher levels of IL-1?,which confirmed the inhibitory effect of MGF505-7R on inflammatory response.NLRP3-mediated IL-1? production plays an important role in regulating host immunity during DNA/RNA virus infection.It is well-known that the production of IL-1? involved two separate signaling cascades.In the first cascade,nuclear factor-kappa B(NF-?B)depolymerized with I?B is transported to the nucleus through I?B kinases(IKKs)complex-mediated degradation of Ik B,which promotes the transcription of proinflammatory cytokines such as IL-1?.In second cascade,pro-IL-1? is cleaved into mature cytokines and released through NLRP3 inflammasome-mediated Caspase-1activation.Further studies have shown that pMGF505-7R binded and inhibited IKK?-mediated NF-?B activation in the first cascade.In second cascade,pMGF505-7R interacted with NLRP3.ASC oligomerization as a reporter indicates assembly of NLRP3 inflammasome.The results of laser confocal analysis,flow cytometry and Western blotting showed that pMGF505-7R inhibited ASC oligomerization and thus inhibited the production of IL-1?.Finally,ASFV-?7R was evaluated in pigs by intramuscular injection of ASFV-WT and ASFV-?7R to detect body temperature,survival rate,and viral load in various organs.The results showed that the two kinds of virus infected animals had fever symptoms,and the difference was not significant.ASFV-WT infected animals died on the 8th day,and the mortality rate reached 100% by the 10 th day.However,more than 60% of the pigs infected with ASFV-?7R survived,and the viral load in the organs of the infected pigs was also significantly reduced.These results suggested that the deletion of MGF505-7R gene resulted in the decrease of ASFV virulence in piglets.The levels of IL-1? in the serum were further measured on day 1,5,and 8 after challenged,and it was found that ASFV-?7R infection group induced higher levels of IL-1? production and significantly lower viral load in the serum on day 5 and 8 after challenge compared with ASFV-WT infection group.In this study,we demonstrated that ASFV infection induced low levels of IL-1? production in PAMs.The ASFV-encoded MGF505-7R binded to and inhibited IKK?-mediated NF-?B activation,and interacted with NLRP3 and inhibited the assembly of the NLRP3 inflammasome complex to inhibit IL-1? production.In vivo,deletion of MGF505-7R gene induced higher levels of IL-1? production,and ASFV-?7R was less virulence than ASFV-WT.Our findings improve our understanding of ASFV pathogenesis and provide a theoretical basis for prevention and control of ASF and development of vaccine.