Mechanism Of Nuclear Factor ?-C Regulated Porcine Reproductive And Respiratory Syndrome Virus Replication

Porcine Reproductive and Respiratory Syndrome(PRRS)is a highly infectious disease caused by PRRS virus,which is difficult to prevent and control.It brings great economic losses to the global pig industry and seriously harms the pig industry in China.Therefore,screening the host protein that can inhibit PRRSV replication and discussing its mechanism will provide a basis for the prevention and control of PRRS.In view of this,based on the discovery that NFIA and NFIB can inhibit the replication of PRRSV,this study explores whether NFIC,another member of NFI family,affects the replication of PRRSV and its mechanism.1.NFIC promotes the replication of PRRSV.Firstly,the eukaryotic expression plasmids of different transcripts of NFIC were successfully constructed,namely p3×flag-CMV-NFIC5,p3×flag-CMV-NFIC6,p3×flag-CMV-NFIC7 and P3×flag-CMV-NFIC8.These plasmids were transfected into MARC-145 cells respectively,and it was found by western blot that NFIC5 and NFIC7 could express normally,but NFIC6 and NFIC8 could not detect proteins,so NFIC5 was taken as the research object.MARC-145 cells expressing NFIC5 plasmid were infected BJ-4 PRRSV with 1 MOI,and the virus titer after 24 h infection was detected by TCID₅₀.It was found that overexpression of NFIC5 could promote the replication of PRRSV.2.NFIC can not induce the promoter activity of IFN-?alone.Because NFIA and NFIB inhibit PRRSV by up-regulating type I interferon,we will discuss whether NFIC affects PRRSV replication and whether NFIC affects type I interferon transcription.MARC-145 cells were co-transfected with plasmid NFIC5,IFN-?promoter reporter plasmid p284 and ph RL-TK plasmid.The results of double luciferase showed that overexpression of NFIC5 did not induce the activity of IFN-?promoter.3.NFIC inhibits IFN-?transcription induced by key proteins of IFN-?signaling pathway.MARC-145 cells were co-transfected with plasmid NFIC5,IFN-?promoter reporter plasmid p284,ph RL-TK plasmid and different type I interferon signal proteins(such as RIG-I,MDA5,VISA,TRIF and IKK-?).The results of double luciferase showed that NFIC5 could inhibit the activity of type I interferon promoter induced by these type I interferon signal proteins.Because IFN-?promoter contains binding motif of NFIC,IFN-?promoter was truncated to construct promoter reporter plasmids with different lengths,namely p600,p500,p360,p125 and p77.The reporter plasmids were co-transfected into MARC-145 cells with VISA,NFIC5 and ph RL-TK,respectively.The results of double luciferase showed that NFIC inhibited visa-induced IFN-?promoter activity independently of its binding site.Transcription of IFN-?requires the interaction of transcription factors such as IRF3 and NF?B.MARC-145 cells were co-transfected with plasmid NFIC5,p IRF3-Luc or p NF?B-TA,ph RL-TK and type I interferon signal protein.The results of double luciferase showed that NFIC5 could inhibit the binding motif activity of IRF3and NF?B induced by type I interferon signal protein.Then,IRF3 5D was used to simulate phosphorylated IRF3,which was co-transfected with NFIC5,p284 and ph RL-TK into MARC-145 cells.The results of double luciferase showed that NFIC5 could inhibit the phosphorylation of IRF3.MARC-145 cells were co-transfected with NFIC5 and VISA.The results of laser confocal showed that the number of IRF3 nuclei VISA-induced decreased,which indicated that NFIC5 inhibited IFN-?transcription induced by IRF3phosphorylation.4.NFIC promotes the replication of PRRSV and may be related to its inhibition of IFN-?transcription.MARC-145 cells were co-transfected with NFIC5 and VISA.MARC-145 cells were infected with BJ-4 PRRSV with 1 MOI.The virus titer after 24 h infection was detected by TCID₅₀.It was found that overexpression of NFIC5 could restore the titer of PRRSV inhibited by VISA.Fluorescence quantitative PCR showed that ORF7 RNA level of PRRSV also increased significantly,while IFN-?m RNA level decreased significantly.The results indicated that NFIC5 promoted the replication of PRRSV,which may be related to its inhibition of IFN-?transcription.To sum up,NFIC promotes the replication of PRRSV and inhibits IFN-?transcription induced by key protein of IFN-?signaling pathway.This inhibition has nothing to do with its binding site,and may be related to its inhibition of IRF3 phosphorylation.Moreover,NFIC promotes the replication of PRRSV and may be related to its inhibition of type I interferon.