The Mechanism Of PRRSV Nsp11 Inhibiting NLRP3 Inflammasome Activation

Porcine reproductive and respiratory syndrome(PRRS)is an important epidemic disease that affects the global pig industry.It is characterized by reproductive disorder of sows and respiratory diseases of growing pigs.The original pathogen of this disease is Porcine reproductive and respiratory syndrome virus(PRRSV).In 2006,a PRRSV deletion mutant appeared in China,that is,highly pathogenic PRRSV(HP-PRRSV).The wide spread of the virus caused great economic losses to the pig industry in China.In the early stage of PRRSV infection,a large number of pro-inflammatory cytokines can be detected,including TNF-α,IL-1α,IL-1β,IL-6 and so on.Further study found that the virus can activate NLRP3 inflammasome after infecting cells,thus promoting the maturation and secretion of downstream inflammatory cytokine IL-1β.In this study,it was found that HP-PRRSV could up-regulate the expression of IL-1β m RNA and IL-1β secretion in a dose-dependent manner after infection with PAMs,and the up-regulation of IL-1βsecretion depends on NLRP3 inflammasome.HP-PRRSV can cause mitochondrial damage after infecting cells,and the release of contents caused by mitochondrial damage can activate inflammatory reaction.In order to explore the influence of mitochondrial damage caused by HP-PRRSV infection on HP-PRRSV-induced inflammatory reaction,we first observed the morphological changes of mitochondria in PAMs and Marc145 cells infected with HP-PRRSV by electron microscope,and found that HP-PRRSV infection would cause the swelling of mitochondria and the disappearance of contents.Secondly,we labeled mitochondrial double-stranded RNA(mtdsRNA)by RNAscope technology,and then by observing the changes of mtdsRNA and mitochondrial co-location in non-infected and infected state,we found that PRRSV infection can release mtdsRNA from mitochondria.The mtdsRNA was synthesized by transcription in vitro and transfected into PAMs.It was found that mtdsRNA can stimulate inflammatory reaction.Further research found that the stimulation of IL-1β secretion by mtdsRNA depends on NLRP3 inflammasome.The experiment of NLRP3 inflammasome reconstruction in HEK293 T cells and the screening of PRRSV nonstructural proteins regulating inflammatory response showed that Nsp11 could strongly inhibit the production of IL-1β.At present,the research on Nsp11 shows that Nsp11 can inhibit the production of TNF-α,IL-1β and IFN in host,and inhibit NF-κB signaling pathway.However,the mechanism of Nsp11 inhibiting IL-1β production has not been clarified.To explore the mechanism of Nsp11 inhibiting IL-1β production,we first examined the interaction between Nsp11 and NLRP3,ASC and Caspase-1,and found that Nsp11 interacted with NLRP3 and Caspase-1.We then constructed a HEK293 T cell line that induced expression of Nsp11 to explore whether Nsp11 could inhibit inflammatory reaction by interacting with Caspase-1.The expression of Nsp11 was induced in cell line,which proved that Nsp11 had no effect on Caspase-1 activation and p10 and p20 assembly.Further study found that Nsp11 can inhibit the activation of NLRP3 inflammasome by inhibiting the oligomerization of ASC,thus reducing the inflammatory reaction.To sum up,this study found that HP-PRRSV infection can cause mitochondrial damage and release mtdsRNA to activate NLRP3 inflammasome,thus promoting the maturation and secretion of IL-1β.The inhibitory function of Nsp11 encoded by PRRSV on NLRP3 inflammatory reaction was screened through in vitro reconstruction system.Related experiments proved that Nsp11 interacted with NLRP3 and Caspase-1,but did not inhibit inflammatory reaction through interaction with Caspase-1.Further research proves that Nsp11 can inhibit inflammation by inhibiting the oligomerization of ASC.This study enriches people’s cognition of PRRSV and inflammatory reaction,and provides an important theoretical basis for the study of inflammatory reaction.