Studies On The Mechanism Of Porcine Deltacoronavirus Antagonizes Type Iii Interferon Production

Porcine deltacoronavirus(PDCo V)is a newly emerged swine intestinal coronavirus in recent years,which belongs to the genus Deltacoronavirus,family Coronaviridae in the order Nidovirales.In 2012,PDCo V was first discovered in Hong Kong.In 2014,PDCo V broke out and became epidemic in the United States.Subsequently,many countries also reported the occurrence and prevalence of the disease.PDCo V mainly infects newborn piglets,causing diarrhea,vomiting,dehydration and even death,leading to great economic losses to the pig industry.Recent studies have confirmed that cattle,chickens,and mouse are also susceptible to PDCo V.And PDCo V also has the potential to infect humans,posing a major threat to human and animal health.Interferon(IFN)is an important cytokine for the host to resist the invasion of outside pathogens.Previous studies have reported that PDCo V infection can evade the host's antiviral immune response by antagonizing the type I interferon responses.However,as an enterovirus,PDCo V mainly infects the intestinal mucosa.Studies have shown that type III interferons(IFN-?s)play a more important antiviral effect than type I interferons in the intestinal mucosa.Intestinal mucosal epithelial cells can produce a type III interferon response by expressing a large amount of type III interferon receptors and up-regulating the production of peroxisomes.However,in order to establish an effective infection,many viruses take various strategies to antagonize the type III interferon production.At present,it is unclear whether PDCo V antagonizes type III interferon production.This study confirm that PDCo V infection can antagonize IFN-?1 production and further explore its mechanism.The main research contents are as follows: 1.PDCo V infection antagonizes IFN-?1 productionIFN-?1 promoter luciferase reporter system and real-time PCR were used to detected the expression of IFN-?1 in IPI-2I and LLC-PK1 cells after PDCo V infection.It was found that PDCo V infection failed to activate IFN-?1 promoter activity and m RNA transcription,and inhibited Sendai virus(Se V)-induced IFN-?1 promoter activity and m RNA transcription.These results confirm that PDCo V infection antagonizes type III interferon production.2.PDCo V infection inhibits Se V-induced activation of IRFs and NF-?BIRFs and NF-?B are key transcription factors that mediate IFN-?1 expression.To test whether PDCo V infection inhibit IFN-?1 production by impairing the activation of IRFs and NF-?B,the IRFs and NF-?B-mediated IFN-?1 promoter luciferase reporter system was used to detect the activation of IRFs and NF-?B after PDCo V infection.It was found that PDCo V infection did not induce the activation of IRFs or NF-?B,and inhibited Se Vinduced activation of IRFs and NF-?B.These results verify that PDCo V can antagonize IFN-?1 production by inhibiting the activation of the transcription factors IRFs and NF-?B.3.PDCo V infection decreases the amounts of intracellular peroxisomes and inhibits IRF1 nuclear tianslocationPrevious studies have confirmed that PDCo V infection can interfere with RIG-I-like receptor(RLR)-mediated interferon production,and the RLR signaling pathway is also an important way for type III interferon production.Therefore,it is speculated that PDCo V may inhibit IFN-?1 production by impairing the activity of some key signaling molecules in the RLR signaling pathway.To verify this possibility,the effect of PDCo V on the IFN-?1 promoter activity mediated by key molecules in the RLR signaling pathway was detected by dual luciferase activity analysis.The results show that PDCo V can inhibit RLRmediated IFN-?1 promoter activity by targeting the key linker protein MAVS in the RLR signaling pathway.Peroxisome is an antiviral signaling platform for type III IFN signaling pathway.MAVS localized by peroxisomes is related to type III interferon production.Through indirect immunofluorescence assays,it was found that the amounts of peroxisomes in the cells decreased significantly after PDCo V infection.These results further corroborate that PDCo V inhibits IFN-?1 production by targeting MAVS.In addition,the activation of transcription factor IRF1 plays a crucial role in inducing type III interferon production,and its form of activation is nuclear tranlocation.The results of indirect immunofluorescence assays show that PDCo V infection inhibits IRF1 nuclear translocation.This confirms that PDCo V infection antagonizes IFN-?1 production by inhibiting IRF1 nuclear translocation.4.Preliminary study of PDCo V M protein antagonizing type III interferon productionThe IFN-?1 promoter luciferase reporter system was used to screen PDCo V-encoded proteins that antagonize IFN-?1 production.It was found that structural protein M had the property of antagonizing IFN-?1 production.Therefore,PDCo V M protein was taken as a representative,and a preliminary study on the mechanism of its antagonistic production of IFN-?1 was conducted.The results of dual luciferase activity analysis show that overexpression of M protein can inhibit the IFN-?1 promoter activation mediated by the key kinase TBK1 and its upstream signaling molecules in the RLR pathway,but does not affect the IFN-?1 promoter activation mediated by downstream signaling molecules of TBK1.This suggests that TBK1 may be the target of M protein.At the same time,through co-immunoprecipitation and indirect immunofluorescence assays,it was found that M protein interacted with TBK1.The results of competitive co-immunoprecipitation further confirm that M protein interact with TBK1 to reduce the binding of TBK1 to the transcription factor IRF3,thereby inhibiting IFN-?1 production.