Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

Abstract

Porcine circovirus type 2 (PCV2) induces autophagy via the 5′ adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKβ) by increasing cytosolic Ca2+ via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca2+) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-γ). CaMKKβ then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca2+ both in PK-15 cells and PCV2-targeted primary cells from pigs.

Porcine circovirus type 2 capsid protein induces unfolded protein response with subsequent activation of apoptosis

“…First, we examined chaperon GRP78, which works as a sensor of unfolded proteins in the ER and regulates the activation of ER stress transducers. Upregulation of GRP78 in PK-15 cells with transient expression of Rep or Cap is consistent with that observed in PCV2 infection (Zhou et al, 2016). Next, we examined the PERK pathway as it is selectively activated during PCV2 infection.…”

“…To survive and propagate in host cells, viruses have evolved specific mechanisms to modulate or even subvert UPR (Li et al, 2015). In our recent report, we showed that PCV2 could deploy UPR to enhance its replication via PERK/eIF2α signaling pathway (Zhou et al, 2016). However, the mechanisms utilized by PCV2 to benefit its replication under stress conditions as well as the viral protein(s) involved in induction of UPR have yet to be elucidated.…”

“…There is a complex relationship between host cells and virus infection with regard to UPR and apoptosis. In addition to our early reports showing that PCV2 infection could induce autophagy (Zhu et al, 2012a;2012b;Gu et al, 2016), we have recently revealed that PCV2 could induce UPR by selective activation of the RNA-activated protein kinase-like ER kinase (PERK) pathway (Zhou et al, 2016). This study aimed to investigate which protein(s) of PCV2 could be responsible for activation of UPR and if apoptosis would be the eventual destiny of the cells undergoing UPR.…”

“…Small interfering RNAs (siRNAs) against PERK and control scrambled siRNA were purchased from GenePharma (Shanghai, China). Four PERKspecific siRNAs (siPERKs) were used as a pool as previously described (Zhou et al, 2016).…”

“…We have previously shown that persistent PCV2 infection could induce UPR via selective activation of the PERK pathway (Zhou et al, 2016). In order to identify the specific PCV2 protein(s) involved in UPR induction, we investigated the involvement of viral proteins in activating UPR.…”