Porcine Circovirus Type 2 ORF5 Protein Induces Autophagy In Porcine Alveolar Macrophages And The Effect On Virus Replication

Porcine circovirus type 2(PCV2)is the major virulence factor that causes a series of diseases,which is called porcine circovirus disease(PCVD),mainly in weaned piglets with multiple system failure syndrome(PMWS).PCVD occurs throughout the world,causing serious economic losses to the pig industry.PCV2 is a non-enveloped DNA virus,a member of Circovirus Circoviridae,and the genome is a non-segmented single-stranded closed circular DNA.The length of PCV2 genome is 1766~1768 bp,and there are multiple open reading frames(ORFs)have been reported,in which ORF1 encodes viral replication enzyme-associated proteins Rep and Rep';ORF2 encodes the viral capsid protein Cap;ORF3 encodes a viral proapoptotic protein;ORF4 encodes an anti-apoptotic protein;ORF5 was identified by our team and can cause host cell reticulum stress(ERS).Studies have shown that PCV2 can induce autophagy by ERS and promote PCV2 proliferation.Does the ORF5 protein of PCV2 cause host cell autophagy and does it play a role in viral infection and proliferation? These questions are an important aspect to revealing the role of ORF5 protein in PCV2 infection.This study identified the effect of ORF5 on the autophagy pathway of porcine alveolar macrophages(PAMs),and the effects of ORF5 protein,endoplasmic reticulum stress,and autophagy on PCV2 replication.In addition,correlations between endoplasmic reticulum stress and autophagy and viral load in PCV2-infected porcine tissue were analyzed.Get the following results:(1)PCV2 ORF5 protein induces autophagy through ERS.DsRed-ORF5 eukaryotic expression plasmid has been transfected into PAMs to express ORF5 protein.Real-time RTPCR was used to quantitatively detect GRP-78 and Beclin 1,and then Western blot was used to detect the cleavage of autophagy key protein LC3,and changes in the number of autophagosomes and structure of endoplasmic reticulum has been observed by transmission electron microscope.It was found that the expression of GRP-78 and Beclin 1 in ORF5 group was significantly up-regulated at the nucleic acid level;the amount of LC3 II protein was significantly increased in the LC3 cleavage product;a large number of vacuolar structures appeared in the endoplasmic reticulum area and multiple autophagosomes were seen in the cytoplasm.After treated with endoplasmic reticulum stress inhibitor 4-PBA and transfected with recombinant plasmid expressing ORF5 protein,it was found that the expression of GRP-78 and Beclin 1 was significantly down-regulated,LC3 protein cleavage also weakened,endoplasmic reticulum vacuolation and autophagic corpuscles significantly reduced.These indicate that ORF5-induced cellular autophagy is mediated by the endoplasmic reticulum stress pathway.(2)PCV2 ORF5 protein promotes PCV2 replication by inducing autophagy.Using PCV2 to infect PAMs expressing ORF5,after 48 h of infection,PCV2 was detected by real-time PCR.The viral nucleic acid load was significantly increased.ORF5 protein was expressed after 4-PBA and 3-MA were respectively used to inhibited the ERS and cell autophagy,and then inoculated with PCV2.48 h after viral infection,PCV2 nucleic acid load has shown a significantly decrease,and there was no significant difference between the two groups.It shows that ORF5 promotes the replication of PCV2 by activating autophagy.(3)ERS and autophagy occurred in PCV2-infected porcine tissue,and the extent of occurrence was correlated with viral load.PCV2-infected pigs were obtained found to be negative for both CSFV and PRRSV.Heart,liver,kidney,lung,brain and small intestine tissue samples from pigs infected with PCV2 was collected,real-time RT-PCR was used to detect PCV2 nucleic acid load and the expression of GRP-78 and Beclin 1 in each samples.The number of autophagosomes and the structure of endoplasmic reticulum observed by transmission electron microscopy.The results showed that the PCV2 load was highest in the lungs,followed by the liver and kidneys,and small in the small intestine,heart,and brain.PCV2 was not detected in the stomach.GRP-78 and Beclin 1 expressed the highest levels in the lung,followed by the small intestine and once again in the liver and kidney.Transmission electron microscope observation showed that PCV2 particles could be observed in all tested organ tissues,with little difference except a large amount in the lungs.Autophagosomes and the changes in the structure of endoplasmic reticulum were observed in all tissues,with the largest number and most significant changes in the lungs,followed by the intestinal tract,with minor differences in the remaining tissues.This study found that PCV2 ORF5 protein causes the autophagy to occur through ERS and promotes the replication of the virus;PCV2 can also induce ERS and autophagy in infected pigs.