Screening Of Interacting Proteins Of The Replication Initiation Region DNA Of PCV2 And Preliminary Study On Its Regulatory Role In Virus Replication

Porcine circovirus-associated disease(PCVAD)encompasses porcine circovirus type2(PCV2)infection caused multiple disease syndromes including reproductive disorders,intestinal diseases and respiratory diseases.In recent years,new subtypes of PCV2 appeared constantly,and the failure of vaccine immunization in pig farms occurred frequently,which brought huge economic losses to pig industry.Due to its limited coding capacity,PCV2 replication depends on the participation of cellular proteins.There are few studies on PCV2 replication initiation.PCV2 Ori DNA is the initiation site of viral replication complex assembly and PCV2 replication,and it contains a conserved stem-loop structure(Stem-loop,SL).Whether there are cellular proteins binding to Ori sequences involved in the replication initiation of PCV2 has not been reported.In this study,the host proteins interacting with PCV2 Ori were screened and the molecular mechanism of their influence on virus replication was preliminarily explored.The specific research contents and results are as follows:1.Screening and analysis of PCV2 Ori interacting host proteinsPCV2 Ori DNA was incubated with PK-15 cell lysates using DNA-protein pull down assay.Host proteins that interacted with PCV2 genome Ori were screened out and identified by LC-MS/MS.A total of 130 host proteins that could interact with PCV2 Ori DNA were identified.GO analysis showed that about 70% of the proteins were located in the nucleus.In terms of molecular function,116 had protein binding function,65 were related to DNA binding.KEGG pathway enrichment analysis showed that the interacting proteins were mainly involved in genetic information processing,and the proteins involved in DNA replication and repair,translation and transcription were the most abundant.The protein interaction network also suggested that DNA damage repair pathway proteins might be involved in the replication initiation of PCV2 genome,Among them,PARP1 protein had the largest number of interactions.NCL,PARP1 and HNRNPU proteins were selected for further study based on LC-MS/MS protein score and functional and pathway analysis.2.PCV2 Ori DNA interacts with NCL,PARP1 and HNRNPU proteinsThe interaction between PCV2 Ori DNA and NCL and PARP1 proteins was verified by DNA-protein pull down assay.The results showed that PCV2 Ori DNA fragments could bind NCL and PARP1 proteins in vitro.DNA immunoprecipitation assay was used to detect the interaction between NCL,PARP1 and PCV2 Ori DNA,and it was found that NCL and PARP1 had extremely significant binding ability to PCV2 Ori DNA in cells.The interaction between HNRNPU protein and PCV2 Ori DNA and genome was confirmed by agarose gel migration assay.Further studies on the interaction region between cellular proteins and Ori showed that NCL and PARP1 proteins interacted with the stem-loop region of PCV2 Ori.In conclusion,NCL,PARP1,HNRNPU all interact with PCV2 genome Ori,in which NCL,PARP1 interact with the stem-loop region of PCV2 Ori.3.Effects of NCL,PARP1 and HNRNPU proteins on replication of PCV2In order to study the effect of host protein on PCV2 replication,NCL,PARP1 and HNRNPU genes were overexpressed or silenced,and the effect on PCV2 genome copy number was analyzed.The eukaryotic expression plasmid NCL,PARP1 and HNRNPU was constructed and transfected into HEK-293 T cells.The results showed that the copy number of PCV2 genome increased after overexpression of NCL and PARP1.After silencing NCL and PARP1 genes with si RNA,the copy number of PCV2 genome decreased.These results indicate that NCL and PARP1 proteins positively regulate PCV2 replication.Overexpression or silencing of HNRNPU gene had no significant effect on PCV2 replication.4.The relationship between NCL,PARP1,HNRNPU proteins and virus proteins To investigate the role of cellular proteins in viral replication and packaging,we performed Co-IP(Co-immunoprecipitation)assay on HEK-293 T cells expressing different labeled viral proteins and cellular proteins using magnetic beads labeled with two types of labeled antibodies.The results showed that PARP1 interacted with Cap and Rep proteins,while NCL and HNRNPU proteins did not.5.PARP1 protein promotes the assembly of PCV2 virus replication complexNCL or PARP1 proteins were co-expressed when Rep protein was expressed,respectively,to study whether the binding of Rep protein to Ori was affected under the condition of overexpression of NCL or PARP1.The results showed that the binding of Rep protein to PCV2 Ori was enhanced in the case of overexpression of PARP1,while NCL overexpression did not affect the binding.These results suggest that PARP1 helps Rep protein bind PCV2 Ori and promotes assembly of viral replication complex,thus accelerating replication of PCV2.In conclusion,three host proteins interacting with PCV2 replication initiation region were screened in this study.Among them,PARP1 and NCL proteins interact with stem loop structure and promote viral replication;PARP1 protein interacts with viral capsid protein and replication protein and promote viral replication complex assembly in replication initiation region.These results lay a foundation for the selection of PCV2 drug targets and the study of replication initiation of PCV2.