Studies On The Mechanism That Nonstructural Protein 4 Of PRRSV Cleaves ZAP

Porcine reproductive and respiratory syndrome?PRRS?mainly causes reproductive disorder in sows,decreased semen quality in boars and respiratory disease in piglets,causing huge economic losses to the global pig industry.The pathogen of PRRS is porcine reproductive and respiratory syndrome virus?PRRSV?.PRRSV belongs to the Arteriviridae family and is an enveloped,single positive-stranded RNA virus with a genome approximately 15 kb in length.In the long-term coexistence with the virus,the host has evolved some antiviral genes,which encode host restriction factors inhibiting the replication of the viruses.Zinc finger antiviral protein?ZAP?is an important host restriction factor.There are two ZAP isoforms arising from alternative splicing?ZAP-L and ZAP-S?.Previous studies have confirmed that both splicing variants have the ability to inhibit the replication of viruses.At present,there is little research on porcine ZAP.Whether it can inhibit the replication of PRRSV has not been reported.In view of this,our study took PRRSV,porcine ZAP and PRRSV nsp4 as the objects to investigate whether ZAP can inhibit the replication of PRRSV and its antiviral mechanism.At the same time,we explored whether PRRSV has the mechanism of antagonizing the antiviral effect of ZAP.The main research contents are as follows:1.Overexpression of ZAP inhibits the replication of PRRSVThe full-length cDNAs of ZAP-L and ZAP-S were cloned and the Flag-ZAP-L and Flag-ZAP-S eukaryotic expression plasmids were constructed.They were transfected into MARC-145 cells,respectively.Afer transfection 24h,Cells were infected with PRRSV at an MOI of 0.5.The samples were collected at 24 hpi.Real time PCR,TCID₅₀,Western blot and indirect immunofluorescence were used to detect the effect of overexpression of ZAP on PRRSV replication.The results showed that overexpression of ZAP-L and ZAP-S significantly inhibits the replication of PRRSV in a dose-dependent manner.2.ZAP does not target 3'UTR and 5'UTR of PRRSV RNAPrevious studies have reported that ZAP can inhibit the replication of viruses such as Sindbis virus from the RNA level by targeting the 5'UTR and 3'UTR To explore whether ZAP can target the 5'UTR or 3'UTR of PRRSV RNA,the 5'UTR and 3'UTR of PRRSV were cloned into the pGL3 control vector,respectively,and then Flag-ZAP-L,Flag-ZAP-S or pCAGGS-Flag empty vector were co-transfected with pGL3control-PRRSV-5'UTR or pGL3 control-PRRSV-3'UTR and the internal reference plasmid pRL TK into HEK-293T cells.The double luciferase reporter system showed that ZAP does not target 3'UTR or 5'UTR of PRRSV RNA.3.ZAP-L inhibits the expression of PRRSV nsp7?,nsp9 and nsp12,while PRRSV nsp4 is able to cleave ZAP.Previous studies reported that ZAP-L can also inhibit the replication of viruses by degrading viral proteins such as influenza virus.To investigate whether ZAP-L can inhibit the replication of PRRSV in the same way,the eukaryotic expression plasmids of PRRSV non-structural proteins and Flag-ZAP-L were co-transfected into HEK-293T cells.Western blot analysis showed that ZAP-L can inbibit the expression of PRRSV nsp7?,nsp9 and nsp12 in a dose-dependent manner.We also unexpectedly found that PRRSV nsp4 also cleaves ZAP in a dose-dependent manner.4.The cleavage of ZAP by PRRSV nsp4 depends on its 3C-like serine protease activityPRRSV nsp4 is a typical 3C-like serine protease with cleavage activity.In order to show whether the cleavage of ZAP by nsp4 depends on its 3C-like protease activity,the eukaryotic expression plasmids?H39A,D64A,S118A?were constructed by site-directed mutagenesis.The eukaryotic expression plasmids of Flag-ZAP-L and nsp4 wild-type or enzyme activity mutants were co-transfected into HEK-293T cells.Western blot analysis showed that the enzyme activity mutants of nsp4 does not cleave ZAP,whereas wild-type nsp4 can cleave ZAP,indicating that the cleavage of ZAP by nsp4 depends on its 3C-like serine protease activity.Using inhibitors of the ubiquitin-proteasome pathway,autophagy lysosome pathway and apoptosis pathway,it was found that the cleavage of ZAP by nsp4is independent of autophagy,apoptosis,and proteasome pathways.5.PRRSV nsp4 cleaves ZAP at Glu-411 and the ability of the fragments produced by cleaving ZAP to inbibit the replication of PRRSV is significantly reduced.According to the characteristics of PRSV nsp4 recognizing the amino acid of the P1substrate and the size of the fragment produced by cleaving ZAP,it is speculated that there are six potential sites for ZAP cleavage:E288,E329A,E334A,E371A,E411A,E453A.To determine the site of ZAP cleavage by PRRSV nsp4,ZAP-L mutants eukaryotic expression plasmids?E288A,E329A,E334A,E371A,E411A,E453A?were constructed by site-directed mutagenesis.ZAP-L mutants,the wild type ZAP-L eukaryotic expression plasmids and HA-PRRSV nsp4 eukaryotic expression plasmid were co-transfected into HEK-293T,respectively.Western blot analysis revealed that in addition to mutant E411A,other mutants and wild-type ZAP-L can be cleaved by nsp4,indicating that PRRSV nsp4 cleaves ZAP at Glu-411.In order to further explore the biological significance of ZAP cleavage by PRRSV nsp4,the eukaryotic expression plasmid of pZAP?1-411?and L?412-895?fragments produced by cleaving ZAP-L and the C-terminal fragment S?412-779?produced by cleaving ZAP-S were constructed,and then compared with the full-length ZAP-L or ZAP-S about anti-PRRSV activity.As a result,it was found that the ability of the cleaved fragments to inhibit replication of PRRSV are significantly reduced,indicating that PRRSV nsp4 weakens its antiviral activity by cleaving...