Porcine Reproductive And Respiratory Syndrome Virus Activates NLRP3Inflammasome

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductivefailure in sows, respiratory disease in pigs, high mortality in weaned piglets,immunosuppression and persistent infection, with the causative agent is PRRS virus (PRPSV).More recently, a report demonstrated that PRRSV can induce IL-1β secretion throughinflammasome in porcine alveolar macrophage (PAM). Upon detecting pathogen-associatedmolecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), somepattern-recognition recptors (PRRs) form a kind of large multimeric protein complexestermed ‘inflammasome’by recruiting the apoptosis-associated speck-like protein containing acaspase recruitment domain (ASC) and caspase-1that is activated by autocatalytic cleavagewithin the complex. The active caspase-1catalyzes proteolytic processing of pro-IL-1β andpro-IL-18into active cytokines, the most significant inflammatory cytokines in triggeringtissue inflammation. The work on the mechanism of PRRSV-induced inflammation activationis not only useful for illustrating the inflammatory induced by PRRSV, but also could providevaluable theory evidence in preventing and treating PRRSV.In this study, cDNA from PRRSV HuN4-infected PAM was used as template to clonenucleotidebinding oligomerization domain (NOD)-like receptors containg pyrin domain3(NLRP3), ASC and caspase-1, which was the basis of studying NLRP3inflammasomeindeced by PRRSV. The results are as follows in detail:(1) The cDNA of NLRP3, ASC and caspase-1were amplified by RT-PCR and clonedinto pET-28a(+) for expression in E.coli with primers designed according to mRNAsequences in NCBI. The antibodies against NLRP3, ASC and caspase-1were prepared inBalb/c mice and rabbits immunized with the purified NLRP3(1-900)/NLRP3(2686-3111),ASC and p20recombinant proteins and were characterized by Western Blot and Indirectimmunofluorescent assay (IFA).(2) PRRSV HuN4was slected by ELISA and Real-time PCR assay as experimental virus,which can induce caspase-1activation and then processing pro-IL-1β to IL-1β. ELISA andQuantitative RT-PCR were developed to demonstrate IL-1β secretion and pro-IL-1βexpression induced by PRRSV HuN4in PAM was in a does-dependent manner. In addition,IL-1β secretion can be inhibited by the specific caspase-1inhibitor yVAD-CHO, indicating that PRRSV HuN4inducing IL-1β secretion was dependent on caspase-1activation. Proteinlysates from unifected or infected or ATP+LPS treated PAMs were incubated with anti-ASCpolyclonal antibaody or preimmune rabbit IgG and immunoprecipitated with protein A+GSepharose. The immunoblot result showed NLRP3and caspase-1were interacted with ASCin PAMs upon PRRSV infection and ATP+LPS treatement. Furthermore, the level of IL-1βwas descread in PAM transfected with siRNA for NLRP3. In conclusion, PRRSV HuN4canactivate NLRP3inflammasome in PAM. Besides, lysosomal acidification and potassiumefflux were involed in inflammasome activation induced by PRRSV HuN4.(3) IL-1β secretion cannot be induced by UV-inactivated PRRSV HuN4, however, it canbe induced by PRRSV HuN4RNA, which can also induce caspase-1activation. Like byPRRSV HuN4, IL-1β secretion induced by PRRSV HuN4in PAM was also in adoes-dependent manner.(4) DEAD (Asp-Glu-Ala-As) box polypeptide19A (DDX19A) was identified as a novelcomponent of in PRRSV-mediated NLRP3inflammasome complex by IP-MS of anti-ASCmonoclonal antibody incubating with proterin lysis from unifected or infected PAMs. Theinteraction of DDX19A and NLRP3was indentifed by Co-Immunoprecipitation assay andGST pulldown assay. In addition, the co-localization of DDX19A and NLRP3in thetransfected HEK293cell was detected under confocal microscopy. The Domain1ofDDX19A and the NOD and LRR domain of NLRP3were responsible for the interaction ofDDX19A and NLRP3. DDX19A binding to PRRSV HuN4RNA was performed by RNApulldown assay. The domain1and domain2of DDX19A and the NOD and5’UTR and3’UTR were responsible for the interaction of DDX19A and PRRSV HuN4RNA.In conclusion, this study demonstrated that PRRSV could activate NLRP3inflammasome and DDX19A took part in the inflammasome activation, which may provideuseful information for the basic and clinical research on PRRSV study.