The Mechanism Of Evasion Of Host NLRP3 Inflammasome Recognition By Wild-type Rabies Virus

Rabies is an infectious disease that infects the nervous system.It is caused by rabies virus(RABV).There is no effective treatment for rabies,mainly because its pathogenic mechanism has not been fully elucidated yet.As an important component of host innate immunity,NLRP3 inflammasome mainly works on the regulation of immune responses and disease development.RABV has formed a variety of mechanisms to evade host innate immune responses during long-term evolution.Previous study has reported that infection of murine bone marrow-derived dendritic cells(BMDCs)with RABV vaccine strain could stimulate the activation of NLRP3 inflammsome,resulting in active Interleukin-1?(IL-1?)production.It is still not clear whether NLRP3 inflammasome can be activated by wild-type RABV.To investigate the recognition of NLRP3 inflammasome after wild-type RABV infection,BMDCs were infected with wild-type virus DRV-Mexico(DRV for short thereafter)and lab-attenuated virus CVS-B2c(B2c for short thereafter)respectively,and the expression of IL-1? was measured.The result showed that DRV infection significantly reduced the IL-1? expression compared with B2 c infection,and this difference was not caused by different viral titers.This result indicated that DRV could evade NLRP3 inflammasome recognition.To further determine the mechanism by which DRV affects NLRP3 activation,the two stages of NLRP3 inflammasome activation were analyzed respectively.It was found that DRV could reduce the expression of Interleukin-6(IL-6)and Tumor necrosis factor-?(TNF-?),which are downstream products of the NF-?B pathway.This result suggested that DRV could evade the first stage of NLRP3 inflammasome activation by inhibiting the NF-?B pathway.Lipopolysaccharide(LPS)was incubated with BMDCs to eliminate the difference caused by DRV and B2 c infection on the first stage of NLRP3 inflammasome activation.DRV could reduce the production of IL-1? after LPS stimulation,which indicated that DRV could evade the second stage of NLRP3 inflammasome activation as well.By measuring the mitochondrial membrane potential and the production of reactive oxygen species(ROS)in BMDCs infected with DRV of B2 c,we found that DRV could reduce the damage of mitochondrial and the production of ROS.This result demonstrated that DRV could evade the second stage of NLRP3 inflammasome activation.To investigate whether the structural proteins of DRV play a role in evading NLRP3 inflammasome activation,we explored this by enzyme-linked immunosor-bent assay(ELISA),Co-Immunoprecipitation(Co-IP)and confocal assay.The result showed that the M protein of DRV could interact with NLRP3.By Co-IP,it was found that the M protein of DRV interacted with all three domains of NLRP3,N-terminal pyrin domain(PYD),NACHT-associated domain(NBD)and C-terminal leucine-rich repeat(LRR)domain.By measuring ASC oligomerization and ASC speck,it was verified that the M protein of DRV could reduce ASC oligomerization and ASC speck.These results suggested that the M protein of wild-type RABV plays a key role in evading NLRP3 inflammasome activation.Our data shows that wild-type RABV can evade the two stages of NLRP3 inflammasome activation.And the M protein of wild-type RABV evades NLRP3 inflammasome activation by interacting with NLRP3.In a word,this study reveals that the M protein of wild-type RABV plays an important role in evading NLRP3 inflammasome recognition.