Mechanism Of Porcine Circovirus Type 2-induced Apoptosis Via CHOP/ERO1α/ROS Pathway

Porcine circovirus type 2（PCV2）is the primary causative agent of porcine circovirus-associated disease with severe immunosuppression and can cause apoptosis,which causes huge economic losses to the global pig industry.It was shown that PCV2 infection triggers apoptosis through activation of the PERK-e IF2α-ATF4-CHOP signaling pathway from ER in PK-15 cells with unknown mechanisms.CHOP transcriptional target ER oxidase 1α（ERO1α）could induce ER oxidation,and form cytotoxic reactive oxygen species（ROS）in ER to further induce apoptosis.In this study,gene silencing,chemical inhibition,flow cytometry,western blot and other methods were used to analyze the molecular mechanism of endoplasmic reticulum stress induced by CHOP and ERO1αin PCV2,and laying a foundation for elucidating the pathogenesis of PCV2.1.CHOP/ERO1α/ROS pathway mediates PCV2 induced apoptosisPK-15 cells were infected with PCV2 at different time points,the expressions of CHOP and ERO1αprotein,ROS level,Ca²⁺level,and apoptosis rate of the infected cells were detected by western blot and flow cytometry.The results showed that after PCV2 infection,the expression of CHOP and ERO1αprotein was significantly increased,and caspase-3 cleavage was significantly increased.PCV2 infection significantly increased intracellular ROS level（p<0.01）,apoptosis rate（5.16%vs 14%）,and Ca²⁺level（p<0.01）.The conserved sequence of CHOP gene coding region was analyzed by software,and a series of si RNAs were designed and synthesized.A si RNA with good silencing effect was determined by transient transfection and western blot.The cells infected with PCV2 silenced CHOP were identified.CHOP and ERO1αprotein expression,ROS level,Ca²⁺level,and apoptosis rate of infected cells were determined by western blot and flow cytometry.The results showed that silencing CHOP significantly down-regulated the expression of ERO1α,ROS（p<0.01）,and Ca²⁺（p<0.01）,reduced caspase-3 cleavage,and significantly reduced the apoptosis rate of PCV2 infected cells（18.2%vs9.08%,p<0.01）.The conserved sequence of ERO1αgene coding region was analyzed by software,and a series of sh RNAs were designed and synthesized.Puromycin was used to screen ERO1α-knockdown PK-15 cell lines by lentivirus packaging and screening system,and ERO1α-knockdown cell lines infected with PCV2.The expression of ERO1αprotein,ROS level,Ca²⁺level,and apoptosis rate were determined by western blot and flow cytometry.The results showed that a stable ERO1αknockdown PK-15 cell line was obtained.Knockdown ERO1αdown-regulated ROS（p<0.01）,and Ca²⁺（p<0.01）levels in PCV2 infected cells,reduced caspase-3 cleavage,and significantly reduced apoptosis rate（10.5%vs 4.73%,p<0.01）.EN460,an ERO1αinhibitor,could effectively inhibit the expression of ERO1α.PCV2 infected cells were treated with EN460,and the expression of CHOP,ERO1α,and Cap proteins and the apoptosis rate of PCV2 infected cells were detected by western blot and flow cytometry.The results showed that EN460 inhibited ERO1αexpression,Cap protein expression,caspase-3 cleavage,and apoptosis rate（11.6%vs 9.05%,p<0.01）.In this study,p CMV-ERO1αoverexpression plasmid was constructed and transfected into PK-15 cells.PCV2 cells were infected with ERO1αand Cap protein expression and apoptosis rate were detected by western blot and flow cytometry.The results showed that overexpression of ERO1αsignificantly up-regulated Cap protein expression,increased caspase-3 cleavage and significantly increased apoptosis rate（10.9%vs 13.8%,p<0.01）.N-acetylcysteine（NAC）,an antioxidant,effectively inhibits ROS production.PCV2 infected cells were treated with NAC,and the expression of CHOP and ERO1αproteins,ROS levels,Ca²⁺levels,and apoptosis rate of PCV2 infected cells were detected by western blot and flow cytometry.The results showed that NAC significantly down-regulated CHOP and ERO1αprotein expression,Ca²⁺level（p<0.01）,reduced caspase-3 cleavage,and inhibited apoptosis（12%vs 9.42%,p<0.01）.2.Overexpression of PCV2 Cap activates CHOP/ERO1αpathway to induce apoptosisIn this study,pGFP-REP,pGFP-Cap and pGFP-ORF3 eukaryotic expression plasmids were constructed and transfected into PK-15 cells.Western blot was used to detect the expression of each protein.The results showed that overexpression of PCV2 Cap significantly upregulated the expressions of CHOP,ERO1α,and c-caspase3 proteins（p<0.01）.PK-15 cells overexpressing Cap protein were treated with NAC,and the expression of each protein was detected by western blot.The results showed that NAC significantly down-regulated the expressions of CHOP,ERO1α,and c-caspase3 proteins（p<0.01）.In conclusion,this study preliminary demonstrated that PCV2 infection could activate CHOP/ERO1α/ROS pathway to induce apoptosis,and PCV2 Cap protein played a key role in this pathway,providing a theoretical basis for the exploration of endoplasmic reticulum stress targeted PCV2 antiviral therapy.