Porcine Circovirus Type 2 ORF5 Protein Induces Autophagy Pathway In PK-15 Cells And The Effect Of Autophagy On Viral Replication

Porcine circovirus type 2?PCV2?is the primary pathogen of porcine circovirus-associated disease?PCVAD?,which can cause damage to infected pig lymphocytes and lead to immune suppression,secondary and mixed infection of multiple pathogens.The occurrence of infections and diseases seriously damages the health of pigs and affects the development of the pig industry.PCV2 is a DNA virus,the genome consists of 1766¹768 nucleotides,and the genomic structure is a closed circular single strand negative strand DNA.Elucidation of the pathogenesis of PCV2 is the basis for better prevention and control of viral infections and diseases.Functional analysis of virally encoded proteins is an important aspect of studying the pathogenesis of the virus.There are multiple open reading frames?ORFs?in the PCV2 genome,and the encoded products play a role in viral infection and pathogenesis.Six ORFs have been identified and the role of their encoded proteins in PCV2 infection has been investigated.ORF5 is a novel gene encoding PCV2 non-structural protein identified by the research team.?t was found that ORF5 protein can affect cell cycle,induce endoplasmic reticulum stress?ERS?,autophagy and promote viral replication.Cell ERS and autophagy play an important role in viral infection,and many signal pathways for autophagy are induced.Endoplasmic reticulum stress-cell autophagy is an important regulatory pathway.Previous studies have found that ORF5 protein can induce ERS and autophagy in host cells,and autophagy is also down-regulated after blocking ERS.?t is speculated that ORF5 protein-induced autophagy is associated with ERS.The identification of the pathway of autophagy induced by ORF5 protein and the effect of autophagy on PCV2 replication will provide new information for the study of ORF5protein-induced autophagy and its role in viral infection.?n this study,PK-15 cells were used as the research object,and the main pathway of autophagy induced by ORF5 protein was identified.The effect of induced autophagy on viral replication was studied.The study obtained the following results:?1?ORF5 protein induces autophagy in PK-15 cells.PCV2 and PCV2^?ORF5?ORF5 gene mutant?virus strains were infected with PK-15 cells,respectively,and PK-15 cells were transfected with recombinant expression plasmid pEGFP-ORF5.The expression of ORF5protein was observed by laser confocal microscopy to change the autophagy marker protein LC3 from a dispersed state to a spot aggregation,suggesting that autophagy appeared in the cells.Western blot analysis showed that the content of LC3? protein increased,indicating that LC3? protein was cleaved into LC3? protein,which induced autophagy.The endoplasmic reticulum stress inhibitor?4-PBA?inhibited the endoplasmic reticulum stress and decreased the ratio of LC3?/LC3?.The decrease of LC3 protein in the cells decreased,indicating that the autophagy was down-regulated after inhibiting endoplasmic reticulum stress..The results indicate that ORF5 protein induces autophagy in PK-15 cells,and endoplasmic reticulum stress is one of the pathways that induce autophagy.?2?Autophagy promotes replication of PCV2 in PK-15 cells.By plotting the growth curves of PCV2 and PCV2^?ORF5ORF5 strains in PK-15 cells,it was found that the DNA copy number of PCV2^?ORF5ORF5 strain was significantly lower than that of PCV2 test group within 48h of virus-infected cells?P<0.01?.At the same time,Cyclin A was up-regulated in nucleic acid and protein levels after infection of PCV2^?ORF5ORF5 strain,suggesting that ORF5 protein prolongs cell cycle and facilitates viral replication.PK-15 cells infected with PCV2 were transfected with pEGFP-ORF5 recombinant plasmid,and the mRNA and protein expression of PCV2 Cap protein were increased.The mRNA and protein expression of Cap protein decreased after inhibiting endoplasmic reticulum stress.?t is indicated that autophagy induced by virus and ORF5 protein promotes PCV2 replication.?3?The ORF5 protein induced autophagy in PK-15 cells by PERK-e?F2?-ATF4 and mTOR-ERK1/2-AMPK.The PERK signaling pathway and the mTOR signaling pathway fitting protein were detected in PK-15 cells transfected with pEGFP-ORF5 recombinant plasmid.The results showed that ORF5 protein up-regulated the phosphorylation level of PERK protein,activated the expression of the downstream pathway effector e?F2?/ATF4,and promoted the cleavage of LC3? into LC3? protein.Phosphorylation of PERK protein,e?F2?and ATF4 protein levels,and LC3? content were all reduced by the addition of pathway inhibitors.The phosphorylation of mTOR protein in PK-15 cells expressing ORF5protein was inhibited,and the phosphorylation level of ERK1/2-AMPK protein was increased in the downstream of the pathway,and the content of LC3? protein was also increased.This indicates that the mTOR-ERK1/2-AMPK signaling pathway is activated and the occurrence of autophagy.After phosphorylation of ERK1/2 and AMPK proteins by U0126 and Compound C reagents respectively,phosphorylation of mTOR protein increased,LC3? protein content decreased,and autophagy was inhibited.The results of our study showed that ORF5 protein promoted the activation of PEK-e?F2?-ATF4 and mTOR-ERK1/2-AMPK signaling pathways in PK-15 cells and induced autophagy.?4?Activation of the PERK-e?F2?-ATF4 and mTOR-ERK1/2-AMPK signaling pathways promotes replication of PCV2.The recombinant plasmid pEGFP-ORF5 was transfected into PK-15 cells,and then infected with PCV2 and PCV2 ORF5 virus strains,respectively.The study found that ORF5 protein increased the mRNA and protein expression of Cap protein.After the addition of pathway inhibitors,phosphorylation of PERK,ERK1/2and AMPK was inhibited,mRNA and protein expression of Cap protein decreased,and Rapa inhibited the expression of PCV2 after inhibiting the expression of mTOR.This indicates that the virus and ORF5 proteins can promote PCV2 replication through the PERK-e?F2?-ATF4 and mTOR-ERK1/2-AMPK pathways.The results of this study indicate that PCV2 ORF5 protein induces autophagy in PK-15 cells by activating PERK-e?F2?-ATF4 and mTOR-ERK1/2-AMPK signaling pathways,and promotes PCV2 replication.