Study On The Role Of Porcine ZC3H11A In PRRSV And PRV Proliferation

Porcine Reproductive and Respiratory Syndrome(PRRS)is one of the major diseases that threaten the global pig industry caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV).The disease primarily manifests as reproductive disorders in sows and respiratory difficulties in piglets.Pseudorabies(PR)caused by pseudorabies virus(PRV)is an acute infectious disease characterized by fever,itching,and typical neurological symptoms such as encephalomyelitis.PRRSV and PRV cause billions of dollars in economic losses to the global pig farming industry every year.In recent years,new gene editing tools have been continuously explored,and widely used in gene function research and molecular design breeding.The preparation of genetically modified pigs targeting PRRSV and PRV major genes may be an effective complementary approach for the prevention and control of PRRSV and PRV.Therefore,the mining of other key host factors during PRRSV/PRV infection of the host is a key issue that needs to be solved in the field of disease-resistant breeding,which is of great significance for the prevention and control of PRRSV and PRV.ZC3H11A as a member of the family of CCCH-type zinc finger proteins,is one of the components of the transcription export complex(TREX),which plays a role in the process of mRNA exporting from the nucleus.It was found that ZC3H11A knockout inhibited the proliferation of many intracellular replicating viruses such as HAV-5,HIV-1,and HSV-1.However,the role of ZC3H11A in porcine viruses remains to be investigated.The aim of this study was to clarify the biological role of ZC3H11A in PRRSV and PRV proliferation and to investigate its feasibility as a target for genetic modifiation for disease resistance breeding in pigs.This study contains three research aspects.At first,the role of ZC3H11A in PRRSV proliferation was investigated.It was found that the expression of ZC3H11A increased in a dose-dependent manner with PRRSV.Deletion of ZC3H11A in Marc-145 cells significantly inhibited PPRSV-JXA1 and PRRSV-GD replication.Meanwhile,re-expression of ZC3H111A in ZC3H11A knockout cells could restore PRRSV proliferation.Overexpression of ZC3H11A in Marc-145 cells significantly promoted PRRSV replication,suggesting that ZC3H11A plays an important biological role in PRRSV proliferation.Further results showed that the elimination of ZC3H11A in PK-15CD163 cells also significantly inhibited PRRSV proliferation.Mass spectrometry and Co-IP results showed that ZC3H11A indirectly regulates PRRSV replication by interacting with host protein RPL29.Secondly,the role of ZC3H11A in PRV proliferation was investigated.We found that PRV infection induced the up-regulation of ZC3H11A expression,and then we found that ZC3H11A,as the proviral factor of PRV,plays an important role in the formation of TREX complex and the nuclear transfer of viral mRNA.Knockout of ZC3H11A resulted in the accumulation of PRV mRNA in the nucleus,which could not be transferred to the cytoplasm for translation,thus inhibiting the proliferation of PRV.Meanwhile,we further found that ZC3H11A knockout also significantly inhibited the proliferation of another intranuclear replicating virus,PCV2.Finally,ZC3H11A knockout pigs were prepared,and the primary cells were isolated to preliminarily verify its ability to anti-PRRSV and PRV.At first,two ZC3H11A knockout PFFs were obtained by CRISPR/Cas9.The statistical results showed that there was no development difference between ZC3H11A knockout and wild type of reconstructed blastocysts,indicating that ZC3H11A knockout did not affect the development of reconstructed blastocysts.Subsequently,ZC3H11A knockout pigs were successfully prepared by SCNT.The results showed that the blood indexes and tissue morphology of ZC3H11A knockout pig were not significantly different from that of wild type pig.Finally,ZC3H11A knockout pig and wild-type pig derived alveolar macrophages(PAMs)and primary kidney cells were isolated for in vitro viral infection.Real-time fluorescence quantitative results showed that ZC3H11A knockout of porcine PAMs and primary renal cells could effectively inhibit PRRSV and PRV proliferation in vitro.In summary,the role of ZC3H11A in PRRSV and PRV proliferation was clarified in this study.ZC3H11A knockout pigs were prepared using CRISPR/Cas9 system and its significant broad-spectrum antiviral effect was verified,which lays a good foundation for maintaining the stability of the pig breeding industry.