Mechanisms Of Autophagy In PK-15Cells Induced By Porcine Circovirus Type2Infection

Porcine circovirus type2(PCV2), a member of the genus Circovirus of the Circoviridae family, is a small DNA virus with single-stranded circular genome (1767or1768bp in length). Three major open reading frames (ORFs) have been identified for PCV2, ORF1, named the rep gene, encodes a protein involved in virus replication, ORF2, named the cap gene, encodes a immunogenic capsid protein, and the third protein encoded by ORF3is associated with cell apoptosis. PCV2can affect the immune system of pig and induce the immune suppression. The affected pigs show lymphadenectasis and lymphocytopenia, which is closely associated with postweaning multisystemic wasting syndrome (PMWS). Now, PMWS incurs great economic loss to the pig industry and restricts seriously the development of pig industry.Histopathologic lesion caused by PCV2infection is lymphoid follicles disappearance in lymphatic organization, however, recent study have showed that lymphoid tissue was replaced by histiocyte which may related to PCV2-induced apoptosis. On one hand, autophagy plays a key role in antiviral infections by degradation of viral components, presenting viral antigens, activating the immune response, on the other hand, the viruses can also escape the protective antiviral activity and maintain their own survival and replication by inducing autophagy of the host cells. A number of papers suggest that autophagy and apoptosis are co-adjust each other, and autophagy is prior to apoptosis. We speculate whether autophagy triggers apoptosis involved in PCV2infection. However, it remains largely unknown on the mechanisms of PCV2-induced lymphoid depletion in affected pigs.This study was in an attempt to clarify (1) Interactions between PCV2infection and autophagy,(2) The effect of autophagy on the viral replication,(3) The signaling pathway and key signaling molecules involved in PCV2-induced autophagy.1. We analyzed the relationship between PCV2infection and autophagy through the laser confocal microscope, immunoblotting and electron microscopy in PCV2-infected PK-15cells. Results showed that eGFP-LC3protein was able to aggregate to form puncta in autophagy-induced cells and it was found that more puncta appeared in PCV2-infected cells (4.33vs27.51, P<0.01) than in mock cells. The ratio of LC3-Ⅱ/β-actin evidently increased (24h:1.00vs2.97, P<0.05;36h:1.29vs4.11, P<0.01) in PCV2-infected cells compared to mock-treated cells. Transmission electron microscope showed that compared with mock cells, in which autophagic vacuoles were rarely seen, accrual of0.5-1.0μm double-membrane vacuoles in virus-infected cells were observed. PCV2infection caused p62protein degradation, and p62and LC3-Ⅱ protein level treated with autolysosome inhibitors chloroquine were significantly higher than that of untreated group (p62:0.83vs1.87, P<0.05; LC3-II:2.02vs6.97, P<0.05), suggesting PCV2infection accelerated the autophagy substrate degradation. Besides enhancement of colocalization of LysoTracker or LAMP1with LC3suggested induction of autolysosome formation after PCV2infection, suggesting the complete autophagy reaction. PK-15cells were transfected with vectors expressed Cap or the truncated Cap, results showed that the capsid protein could induce autophagosome formation, and amino acids90-233was the key area that induced autophagy.2. We treated PK-15cells with different autophagy inhibitors or inducers, and analyzed them effect on progeny virus yield and capsid protein expression. The results from real-time PCR and TCID50showed that autophagy inhibitors significantly weakened the PCV2titer (real-time PCR:3-methyladenine (3-MA):71.51vs8.96, P<0.01; chloroquine (CQ):71.51vs2.74, P<0.001; TCID50:3-MA:4.66vs3.69, P<0.01; CQ:4.66vs3.56, P<0.01) and Cap protein expression. Autophagy induction increased PCV2titer (real-time PCR:Rapamycin:3.52vs23.19, P<0.05; Earle’s balanced salts (EBSS):3.52vs130.65,P<0.05; TCID50:Rapa:4.66vs5.09, P<0.05; EBSS:4.66vs5.13, P<0.05) and Cap protein expression. These results indicate that autophagy could facilitate virus replication.3. A number of signaling pathways have been reported in regulation of autophagy. The mammalian target of rapamycin (mTOR) pathway is a well-known pathways involved in the regulation of autophagy. AMPK (AMP-activated protein kinase), ERK1/2(the extracellular signal-regulated kinase1/2) and TSC2(tuberous sclerosis protein2) lie on the upstream of mTOR. We analyzed the mechanism PCV2-induced autophagy by Immunoblotting, co-immunoprecipitation and confocal microscopy. Results showed that PCV2infection inhibited mTOR and activated ERK1/2. UO126or siERK1/2down-regulated ERK1/2, recovered the phosphorylation levels of mTOR and inhibited autophagy. Besides, compound C (AMPK inhibitor) reduces ERK1/2and TSC2activity, but also recovers the expression of mTOR, thereby inhibiting autophagy in PCV2-infected PK-15cells. UO126down-regulated the phosphorylation levels of TSC2, but not inhibited AMPK activation. Knockdown of TSC2didn’t inhibit AMPK and ERK1/2activation, but recovered the phosphorylation of mTOR and down-regulated LC3lipidation (LC3-Ⅱ). Co-immunoprecipitation results suggest that activated ERK1/2interacts with activated AMPK and TSC2. Taken together, these results suggest that PCV2might induce autophagy via AMPK/ERK/TSC2/mTOR signaling pathway in host cells, which may thus represent a pivotal mechanism for PCV2persistence and pathogenesis.In conclusion, this study demonstrated that (1) PCV2infection induced autophagy in PK-15cells, and the viral capsid protein induces formation of autophagosomes, and amino acids90-233is a key site that induces autophagy,(2) Autophagy could facilitate virus replication,(3) PCV2might induce autophagy via AMPK/ERK/TSC2/mTOR signaling pathway in host cells. The achieved findings will help better understanding the pathogenesis of PCV2infections and provide clues to targets for screening novel antiviral chemicals.