The Mechanisms Of DAP12 In PRRSV Inhibition Of Host Innate Immune Responses

PRRS is a highly contagious infectious disease and causes enormous economic losses annually,which is characterized by high morbidity,high mortality and persistent infection.PRRSV,as the causative agent,has been reported to exploit diverse strategies including inhibition of type I IFN production and delay of pro-inflammatory cytokine release to antagonize host immune responses for persistent infection.DAP12,as the important immune adaptor,is usually engaged by DAP12-associated receptors.Activated DAP12 recruites and activates downstream molecules to trigger various immune responses.The project's theme is “The function of DAP12 in PRRSV modulation of host antiviral innate immune responses”,which mainly includes three parts stated below: Part I DAP12's function in PRRSV modulation of celluler innate immune responsesKnockdown of DAP12 enhanced PRRSV-triggered innate immune responses including production of type I IFNs and pro-inflammatory cytokines,as well as IRF-3 activation.In contrast,DAP12 overexpression inhibited these responses.Subsequently,I tried to explore whether DAP12-Syk pathway was involved in PRRSV inhibition of antiviral pro-inflammatory responses.PRRSV infection triggered the activation of DAP12-Syk pathway including their phorsphorylation and interaction.Both Syk knockdown and Syk inhibition promoted the pro-inflammatory cytokine production induced by PRRSV.The further study concentrated on the effects of DAP12-Syk pathway on NF-?B and MAPK activation during HP-PRRSV infection.In PAMs,DAP12 knockdown enhanced PRRSV-triggered NF-?B and MAPK activation.DAP12 overexpression suppressed the activation of NF-?B(not for MAPK)in wild-type CRL-2843-CD163 cells,but not in Syk knockdown ones.These findings expand the knowledge of PRRSV pathogenesis.Part II The mechanism of NMHC-IIA-DAP12 pathway in PRRSV inhibition of antiviral proinflammatory responsesNMHC-IIA,a subunit of NM-IIA,takes part in various important cellular physiological processes,such as cell movement,cell shape mainteinance and cell transduction.Recently,NMHC-IIA has been reported to be critical factor involved in viral entry.The first chapter has shown that DAP12-Syk pathway participated in inhibiting PRRSV-induced pro-inflammatory responses.In this chapter,it mainly reveals the underlying mechanisms.NMHC-IIA was firstly demonstrated to be the prominent protein associated with DAP12 through IP and MS analysis.Next,Co-IP and confocal microscopy showed that PRRSV infection promote the formation of NMHC-IIA-DAP12-Syk ternary complex,which is dependent on the interaction between the carboxy-terminal non-helical region of NMHC-IIA and residues 50-57 within DAP12 transmembrane region.Since NMHC-IIA might act as a receptor to exert immunoregulatory function by coupling the adaptor DAP12,I further determined the role of NMHC-IIA in PRRSV early infection.NMHC-IIA was shown to recognize the sialic acids on PRRSV GP5 and then activate DAP12-Syk pathway to inhibit NF-?B-and/or MAPKmediated pro-inflammatory responses.Moreover,VSV(another sialylated virus)engaged NMHC-IIA-DAP12-Syk pathway to suppress host antiviral pro-inflammatory responses.Taken together,these results indicated that sialylated RNA viruses exploit NMHC-IIADAP12-Syk pathway to antagonize host immune responses.More importantly,the pathway was involved in attenuating NF-?B-mediated inflammatory responses in response to LPS.All these findings suggested that NMHC-IIA-mediated negative regulation of inflammation might be a common mechanism in host immune system,which contributes to maintaining the homeostasis.It also provides the targets for anti-inflammatory drug design.Part III The mechanism of poSn-DAP12 pathway in PRRSV inhibition of antiviral IFN responsespoSn,has been shown to be a putative receptor responsible for PRRSV attachment and internalization.However,a recent report showed that poSn knockout pigs were still susceptible to PRRSV,suggesting that it might play certain unidentified roles during PRRSV infection instead of an essential receptor.In this chapter,PRRSV infection induced poSn upregulation through IFN-?-STAT1 pathway.poSn in turn inhibited PRRSV-triggered production of type I IFNs.PRRSV infection was further shown to enhance the interaction of poSn and DAP12.Moreover,poSn-DAP12 pathway was involved in inhibiting NF-?B-and IRF-3-mediated IFN resposnes.These findings identified a novel role of poSn in PRRSV inhibition of type I IFN responses.Collectively,DAP12,as an important adaptor,mediated the negative modulation of antiviral immune responses by associating with different receptors during PRRSV infection.During PRRSV early infection,NMHC-IIA recognized sialic acids on the virions to activate DAP12-Syk pathway,which attenuated antiviral pro-inflammatory responses.During PRRSV late infection,poSn attenuated antiviral IFN responses by interacting with DAP12.Furthermore,NMHC-IIA-DAP12-Syk pathway suppressed LPS-triggered pro-inflammatory responses,and poSn-DAP12 pathway antagonized poly(I:C)-triggered type I IFN production.These findings not only provide clues for PRRS prevention and treatment,but also expand the understanding of the negative regulation mechanisms in host innate immune responses,which supplies the drug targets for immune disease treatment.