Porcine Circovirus Type 2 ORF5 Induces Endoplasmic Reticulum Stress And Unfolded Protein Response In Porcine Alveolar Macrophages

Porcine circovirus type 2(PCV2)is the essential infectious agent that can cause serious diseases in pigs,and it is also one of the important viruses that severely harm the swine husbandry in swine-producing countries in the world,resulting in a series of severe diseases.PCV2,as a single-strand DNA virus without envelope,is classified in the genus Circovirus of the family Circoviridae.The genome of PCV2 is closed circular with a length of 1767 bp or 1768 bp.There are multiple open reading frames(ORFs)in PCV2 genome,of which 6 have been identified and some function studies have been reported.Among them,ORF1 encodes viral replicase protein(Rep),which is related to virus virulence and replication;ORF2 encodes viral capsid protein(Cap)and participates in the production of neutralizing antibodies;ORF3-encoded protein is associated with apoptosis which can be induced in host cells;ORF4-encoded protein has an anti-apoptotic effect;ORF6-encoded proteins may also be involved in the regulation of apoptosis.ORF5 is a protein that identified and named by our research group.In our previous study,it has been proved that the expression of its products exists in PCV2-infected porcine alveolar macrophages(PAMs),which can lead to the retardance of S-phase of cell cycle and upregulate the expression of the endoplasmic reticulum marker molecule GRP78.We speculate that ORF5 protein may be related to endoplasmic reticulum stress(ERS)in host cells,but there is still lack of in-depth research and direct evidence.Therefore,in this study,we constructed a eukaryotic expression plasmid that expressing PCV2 ORF5 protein and studied its regulatory signaling pathways that induced ERS and unfolded protein response(UPR)in PAMs.The obtained results are shown as follow:(1)PCV2 ORF5 protein localizes to the endoplasmic reticulum(ER)of PAMs and induces ERS.By transient expression method,pEGFP-ORF5,the plasmid that expressed ORF5 protein was transfected into PAMs.The subcellular localization of ORF5 protein was confirmed by laser confocal scanning microscopy(LCSM),and it was clarified that ORF5 protein was localized in the ER of PAMs.Then transmission electron microscope(TEM)was employed to determine the changes in ER morphology,it was found that ORF5 protein triggered ER's dilation and swelling,the occurrence of ERS was initially determined.Subsequently,the expression of ERS marker proteins-GRP78 and GRP94 were detected by Real-time RT-PCR and Western blot methods at respective nucleic acid level and protein level,and the results showed that ORF5 protein could significantly upregulate the expression of the two proteins.(2)PCV2 ORF5 protein induces ERS and UPR via the PERK(RNA-activated protein kinase-like endoplasmic reticulum kinase),ATF6(activating transcription factor 6)and IRE1(inositol requiring enzyme 1)signaling pathways.Plasmid pEGFP-ORF5 expressing ORF5 protein was transfected into PAMs by transient transfection technique.Real-time RT-PCR and Western blot were used to detect and analyze the key proteins in the three signaling pathways.In the identification of the PERK pathway,the phosphorylation of PERK and eIF2? was examined,and the results demonstrated that the ORF5 protein could significantly increase the phosphorylation levels of both proteins,and it was preliminarily determined that PERK pathway was activated.Subsequently,the expression of the downstream effectors ATF4 and GADD34 was detected,and the results showed that the expression of the two was also significantly up-regulated,which fully characterized the PERK/eIF2?/ATF4/GADD34 branch was activated by ORF5 protein.Additionally,in the identification of the ATF6 pathway,it was demonstrated that ORF5 protein induced the cleavage of ATF6 and activated the expression of downstream Calreticulin,indicating that the ATF6/Calreticulin signaling pathway was also activated.Finally,in the process of identification of IRE1 pathway,the phosphorylation of IRE1 was first verified,then the splicing of Xbp1 and the expression of EDEM were also activated by ORF5 protein,which proving the activation of the IRE1/Xbp1/EDEM pathway.To sum up,PCV2 ORF5 protein can lead to ERS in PAMs,and induce the occurrence of UPR by activating the three signal transduction pathways-PERK,ATF6 and IRE1.This study provided some new scientific evidence for the molecular mechanism of ORF5 in inducing endoplasmic reticulum stress in PAMs,but more experiments are still needed to verify the ORF5 protein during PCV2 infection from the virus-cell,virus-animal level.Induced endoplasmic reticulum stress.ERS is a kind of self-protection of host cells against unfavorable or harmful factors,which may also be exploited by viruses that invade cells,such as providing favorable conditions for virus replication,proliferation and persistent infection.This study provides some new scientific information for the molecular mechanism of this novel ORF5 protein in inducing ERS in PAMs,while more experiments are still needed to validate the function of the ORF5 protein that inducing ERS during PCV2 infection at viral-cell and viral-animal levels in vitro and vivo.