Porcine Circovirus Type 2 Employs Calcium Signaling To Initiate Autophagy In PK-15 Cells

Porcine circovirus type 2 (PCV2) is the primary pathogen of porcine circovirus-associated disease (PCVAD), and mainly targets immune organs of pigs and leads to such pathological features as lymphoid depletion, lymphopenia and histiocytes infiltration, finally causing severe immunosuppression. PCV2 infection is usually accompanied with secondary infection or mixed infection, leading to severe economic losses to the swine producers. PCV2 is a strand-stranded and covalently closed circular DNA virus with about 1.7 kb in genome size. Five open reading frames (ORFs) have been reported:ORF1 encodes the replicase (Rep) that is responsible for initiation of DNA replication; ORF2 encodes the capsid protein (Cap) that is involved in virion packing and autophagy, and functions as the main immunogenic protein of PCV2; ORF3 protein is reportedly able to induce apoptosis. Recent reports suggest that ORF4 is related to apoptosis and regulation of T cells and ORF5 could induce endocytoplasmic reticulum stress. Autophagy plays important roles in viral infection, replication and pathogenicity. Our previous research has proved that PCV2 infection induces autophagy to benefit its replication via AMPK/ERK/TSC2/mTOR pathway in PK-15 cells. However, the mechanism of AMPK activation remains unknown.The present study aims to explore:(1) the activation mechanism of AMPK induced by PCV2 infection in PK-15 cells; (2) alternative autophagy pathways to AMPK signaling during PCV2 infection; (3) the key protein region of PCV2 for autophagy induction.1. CaMKKβ is the upstream activator of AMPK in PCV2-induced autophagyTo explore the mechanism of activation of AMPK by PCV2 infection in PK-15 cells, RNA interference, Western blotting, confocal microscopy and quantitative RT-PCR (q-RT-PCR) were employed. Western blotting revealed that PCV2 infection at 24 hpi upregulated CaMKKβ (1.00 vs 2.20, P< 0.05) and activated its substrate molecules AMPK (1.00 vs 2.59, P< 0.01) and CaMKI (1.00 vs 2.37, P< 0.05). CaMKKβ inhibitor STO-609 or siCaMKKβ repressed the activation of AMPK and CaMKI caused by PCV2. Both STO-609 and siCaMKKp could block the formation of autophagosome induced by PCV2as shown by confocal imaging (14.6 vs 28.5, P< 0.01; 19.2 vs 29.0, P< 0.01). By q-RT-PCR and virus titration, we found that inhibition of CaMKKβ by STO-609 or siCaMKKβ could significantly reduce viral DNA copies (106.02 vs 108.23; 106.91 vs 108.36, P< 0.01) and TCID50 (10-2.23 vs 10-4.56, P < 0.01; 10-2.85 vs 10-4.35, P< 0.01) of progeny PCV2. These results suggest that PCV2 infection activates AMPK and its downstream pathways via CaMKKβ.2. PCV2 employs CaMKI/WIPIl for autophagy induction independent of AMPKTo explore if CaMKKβ/CaMKI/WIPI1 was also activated by PCV2 infection in PK-15 cells, RNA interference, Western blotting, confocal microscopy and q-RT-PCR were employed. We found that PCV2 infection significantly upregulated both mRNA (1.00 vs 2.60, P< 0.01) and protein levels of WIPI1 (1.00 vs 1.92, P<0.05). All treatments with siWIPI1, siCaMKI or CaMKI inhibitor KN93 significantly repressed PCV2 replication and autophagy. However, siAMPK could block autophagy induced by PCV2, but did not activate CaMKI or upregulate WBPI1. PCV2 infection triggered a significant increase in WIPI1-punctate (47.0 vs 24.5, P< 0.01) and co-localizations of WIPI1 and LC3 punctate as compared with the control (16.7 vs 8.7, P<0.01), which could be alleviated by STO-609 or KN93. These results suggest that PCV2 infection activates the CaMKKβ/CaMKI/WIPI1 pathway independent of AMPK.3. PCV2 infection increases cytosolic Ca2+ via IP3R to activate CaMKKβWestern blotting, ELISA and flow cytometry were used to further explore the mechanisms of the activation of CaMKKβ and autophagy induced by Cap protein. PCV2 infection significantly increased cytosolic Ca2+ as shown by flow cytometry (128.7% vs 100%, P< 0.01) and FRET analysis (FRET efficiency:14.1% vs 2.0%, P < 0.01) at 36 hpi. However, the increased cytosolic Ca2+ could be blocked by IP3R blocker 2-APB.2-APB significantly repressed the activation of CaMKKβ/AMPK or CaMKKβ/CaMKI pathway induced by PCV2 and inhibited virus replication. PCV2 infection did not activate PLC-y. ELISA showed that PCV2 infection did not upregulate IP3 level, suggesting that PCV2 activated IP3R without involving the PLC-IP3 pathway. Expression of Cap or its truncated forms (pCap, pCapl-1OOaa, pNLS+Cap90-190aa or pNLS+Cap185-233aa) in PK-15 cells could upregulate cytosolic Ca2+ and induce autophagy to different degrees regardless of NLS. The aa42-185 fragment of Cap seemed to play a major role in activation of Ca2+signaling. In addition, Cap and its truncated forms did not induced autophagy in HEK293 cells. These results suggest that PCV2 uses its Cap to activate CaMKKβ via IP3R-Ca2+ pathway.In conclusion, the present study has elucidated the signaling pathways in PCV2-induced autophagy in PK-15 cells:(1) PCV2 infection activates AMPK and its downstream signals via CaMKKβ; (2) PCV2 activates the CaMKKp/CaMKI/WIPIl pathway independent of AMPK signaling; (3) PCV2 utilizes its Cap to activate CaMKKβ via the IP3R-Ca2+ pathway. These findings provided theoretical foundation for better understanding of PCV2 pathogenesis and for potential targets of anti-PCV2 drugs.