Identification And Effects Research On Virus Replication Of The Interaction Protein GPNMB For Porcine Circovirus Type 2 ORF5

Porcine circovirus disease(PCVD),induced by porcine circovirus type 2(PCV2),is the important infectious diseases that seriously endanger breeding.The infection of PCV2 can make the body produce a strong immune suppression,which causes secondary/mixed infection of variety of pathogens,and bring greater challenges to the PCVD prevention and control.The PCV2 genome is a closed single-stranded circular arranged DNA of 1767 bp or 1768 bp.The virus particles have icosahedral structure without cystic membrane.PCV2 belongs to the family Circoviridae,genus Circovirus.There are several open reading frames(ORFs)in PCV2 genome,the function elucidation of their coding proteins is the basis for revealing the pathogenic mechanism of the virus,six ORFs have been identified and studied in PCV2 genome presently.ORF1 is the largest ORF of PCV2 and encodes two replicases Rep and Rep'protein,related to the replication of PCV2.ORF2 encodes nucleocapsid protein Cap,which is the basic structure of the virus.ORF2 plays multiple roles in the process of virus infection,which can participate in the immune response of the body.ORF3 encodes ORF3 protein,which can promote the accumulation of P53,resulting in cell apoptosis.ORF4 encodes ORF4 protein,which plays an anti-apoptotic role in PCV2 infection.The ORF6-encoded protein up-regulates the expression of a variety of cytokines and is associated with cell apoptosis.The Research team found and identified a new ORF in the PCV2 genome,named it ORF5.ORF5 gene measures 180 bp in length.ORF5-encoded protein may play important role in the induction of endoplasmic reticulum stress,cell autophagy and regulation of cell cycle,but its precise function is still scarce.The function of ORF5 protein is dependent on the its interaction proteins of the host cells.Screening and identification the intracellular proteins interact with PCV2 ORF5,and the effect study of which on PCV2 infection will provide more theoretical support for the revelation of infection and pathogenesis for PCV2,the find of new host targets and the research and development for new drugs associated with PCVD prevention and control.This research screened and verified the potential interaction proteins of ORF5 in porcine alveolar macrophages 3D4/2(PAMs),and explored the effect and potential mechanism of the interaction protein GPNMB on PCV2 replication.Following results were obtained:(1)The transcriptome data for PAMs with ORF5 expression(PAM-ORF5)and PCV2 infection(PAM-PCV2)was obtained via high-throughput sequencing.104 different expression genes(DEGs)were found both in PAM-ORF5 and PAM-PCV2,in which 24 DEGs showed up-regulated and 80 DEGs showed down-regulated,mostly participate in cell signal transduction,protein translation and cell cycle.In the results of Go function classification,the up-regulated DEGs in PAM-ORF5 showed the highest correlation with protein binding transcription factor activity and binding activity,and the down-regulated DEGs in PAM-ORF5 showed the highest correlation with metabolic activity and signaling.Most of the DEGs in PAM-ORF5 are enriched in viral myocarditis,protein digestion and absorption,dilated cardiomyopathy and so on,most of the DEGs in PAM-PCV2 are enriched in cardiac muscle contraction and tyrosine metabolism pathways and so on,and cell cycle pathway is the unique showed enrichment only in PAM-ORF5.(2)GPNMB was down-regulated both in PAM-ORF5 and PAM-PCV2,We believe that GPNMB is probably an intracellular interaction protein of ORF5 protein combined with the early interaction protein screening results of ORF5 protein via yeast two-hybridization.And the expression of GPNMB can also be detected in the different tissues of PCV2 infectious swines,which showed negative correlation with nucleicacid of PCV2.(3)There was an interaction between ORF5 protein and GPNMB.The co-localization phenomenon between ORF5 protein and GPNMB was observed by confocal microscopy in cell,the interaction between ORF5 protein and GPNMB was found via the exogenous and endogenous Co-immuno precipitatition(Co-IP),and GST-Pulldown experiment confirmed the direct interaction between them furtherly.(4)GPNMB has a inhibitory effect on the replication of PCV2.Through enhanced and lowered the level of GPNMB,then detected the viral nucleic acids and proteins following PCV2 infection,GPNMB was found to inhibit PCV2 replication.GPNMB can shorten the the cell cycle S phase and up-regulated the cell cycle protein Cyclin A significantly,which shorten the cell cycle of host cell,and this may be one of the reasons to suppress PCV2 replication.In conclution,PCV2 ORF5 can cause multiple host cell proteins and pathways abnormal,and the interaction protein GPNMB can inhibit the replication of PCV2,which may be implemented by promoting cell cycle progression.