Porcine circovirus type 2 induces autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in PK-15 cells

Abstract

Porcine circovirus type 2 (PCV2) uses autophagy machinery to enhance its replication in PK-15 cells. However, the underlying mechanisms are unknown. By the use of specific inhibitors, RNA interference, and coimmunoprecipitation, we show that PCV2 induces autophagy in PK-15 cells through a pathway involving the kinases AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2), the tumor suppressor protein TSC2, and the mammalian target of rapamycin (mTOR). AMPK and ERK1/2 positively regulate autophagy through negative control of the mTOR pathway by phosphorylating TSC2 in PCV2-infected PK-15 cells. Thus, PCV2 might induce autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in the host cells, representing a pivotal mechanism for PCV2 pathogenesis.

Fig 1

PCV2 induces autophagy by inhibiting the mTOR signaling pathway. (A and B) LC3-II, PCV2 Cap expression, phosphorylated mTOR (p-mTOR), and total mTOR in PK-15 cells infected with PCV2, mock infected (Ctrl), or treated with 0.5 μM rapamycin (Rapa) for 36 h. Their ratios to β-actin were normalized to mock infection, set at 1.0. (C) PK-15 cells were first transfected with control vector or the vector expressing active (Q64L) Rheb mutant. After 24 h, the cells were further infected with PCV2 or mock infected for 36 h. Immunoblotting results are presented as in panel A. (D) Ratios of p-mTOR and LC3-II to β-actin normalized to the control vector, set at 1.0. (E) Formation of autophagosomes, shown as green punctae in PK-15/EGFP-LC3 cells that were treated as described in panel C and analyzed by fluorescence microscopy (scale bar, 10 μm). (F) Average number of punctae in each cell from 60 to 80 cells in each treatment. All data are reported as means ± the standard errors of the mean of three independent experiments (*, P < 0.05; **, P < 0.01) here and in all subsequent figures.

Fig 2

PCV2 induces autophagy by activating ERK1/2 signaling pathway. (A and B) LC3-II, PCV2 Cap expression, phosphorylated ERK1/2 (p-ERK1/2), and total ERK1/2 in PK-15 cells infected with PCV2 or mock infected (Ctrl) for 36 h. Their ratios to β-actin were normalized to mock infection, set at 1.0. (C and D) Effect of UO126 treatment on endogenous LC3-II, p-ERK1/2, and total ERK1/2 of PK-15 cells infected with PCV2 for 36 h. Their ratios to β-actin were normalized to mock infection, set at 1.0. (E) Formation of autophagosomes, shown as green punctae in PK-15/EGFP-LC3 cells that were treated as described in panel C. (F) Average number of punctae in each cell from 60 to 80 cells in each treatment. (G and H) PK-15 cells were mock treated (NS) or treated with ERK1/2-specific siRNA for 24 h. The cells were then further infected with PCV2 or mock infected for 36 h, and p-ERK and LC3-II ratios to β-actin were normalized to mock-infected cells, set at 1.0. (I) Formation of autophagosomes, shown as green punctae in PK-15/EGFP-LC3 cells that were treated as described in panel G. (J) Average number of punctae in each cell from 60 to 80 cells in each treatment.

Fig 3

Downregulation of mTOR signaling is associated with ERK1/2 pathway activation after PCV2 infection. (A) PK-15 cells were treated as described in Fig. 2C, and whole-cell extracts were subjected to immunoblot analysis for LC3-II, p-mTOR, total mTOR, p-ERK1/2, and total ERK1/2. (B) Ratios of p-mTOR, p-ERK1/2, and LC3-II to β-actin were normalized to mock infection, set at 1.0. (C and D) PK-15 cells were treated as in Fig. 2G, and their ratios to β-actin were normalized to mock infection, set at 1.0.

Fig 4

AMPK is the upstream regulator of ERK1/2 in PCV2-mediated autophagy. (A) Immunoblotting of Ras and Raf1 in PK-15 cells infected with PCV2 or mock infected for 36 h or treated with EBSS for 2 h. (B) Immunoblotting of phosphorylated AMPK (p-AMPK) and total AMPK in PK-15 cells infected with PCV2, mock infected (Ctrl), or treated with AICAR for 36 h. (C) Immunoblotting of LC3-II, p-AMPK, and total AMPK in PK-15 cells transfected with pcDNA3.1 (vector) or pcDNA3.1-Cap for 24 h. (D and E) Effect of compound C treatment on LC3-II, phosphorylation, and total levels of mTOR, ERK1/2, and AMPK of PK-15 cells infected with PCV2 for 36 h. Their ratios to β-actin were normalized to mock-infected cells, set at 1.0. (F) Effect of UO126 treatment on p-ERK1/2, total ERK1/2, p-AMPK, and total AMPK of PK-15 cells infected with PCV2 for 36 h. (G and H) PK-15 cells were infected with PCV2 or mock infected (Ctrl), and the interactions between ERK1/2 and AMPK, between AMPK and ERK1/2, or between ERK1/2 and Raf1 were determined by using the indicated phospho-specific antibodies. EBSS treatment served as a positive control for Ras/Raf1 activation in PK-15 cells. IB, immunoblot; IP, immunoprecipitation; TCL, total cell lysates.

Fig 5

ERK1/2 downregulates mTOR activity by upregulating TSC2 after PCV2 infection. (A) PK-15 cells were mock treated (NS) or treated with TSC2-specific siRNA for 24 h. The cells were then further infected with PCV2 or mock infected for 36 h, and whole-cell extracts were subjected to immunoblot analysis for LC3-II, phosphorylation, and total levels of mTOR, TSC2, ERK1/2, and AMPK. (B) Ratios of the molecules in panel A to β-actin, which was normalized to mock infection, set at 1.0. (C) Effect of compound C treatment on LC3-II, phosphorylation, and the total levels of mTOR, TSC2, ERK1/2, and AMPK of PK-15 cells infected with PCV2 for 36 h. (D) Ratios of the molecules in panel C to β-actin. (E) Effect of UO126 treatment on LC3-II, phosphorylation, and the total levels of mTOR, TSC2, ERK1/2, and AMPK of PK-15 cells infected with PCV2 for 36 h. (F) Ratios of the molecules in panel E to β-actin. (G) PK-15 cells were infected with PCV2 or mock infected for 36 h, and the interactions between ERK1/2 and TSC2 or between TSC2 and ERK1/2 were determined by using the indicated phospho-specific antibodies. IB, immunoblot; IP, immunoprecipitation; TCL, total cell lysates.

Fig 6

Pharmacological or siRNA alterations of autophagy do not affect cell viability. Cell viability was determined by MTT assay after treatment with 20 μM UO126, 1 mM AICAR, or 5 μM compound C or transfection with siERK1/2 or TSC2 for 36 h. Percent relative cell viabilities are expressed as means ± the SEM (n = 3).

Fig 7

Hypothetical model of AMPK/ERK/TSC2/mTOR signaling in PCV2-induced autophagy in PK-15 cells. PCV2 infection activated AMPK and then activated ERK1/2 and TSC2 and suppressed mTOR signaling, thereby inducing autophagy in PK-15 cells.