Duck enteritis virus activates CaMKKβ-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium

DEF cells were obtained from 9 to 11 days specific pathogen-free duck embryos, as described previously [24], and cultured in Dulbecco’s modified Eagle’s medium (cat. no. 8116176; Gibco, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (cat. no. 1722658; Gibco) and antibiotics (0.1 mg/ml of streptomycin and 0.1 mg/ml penicillin) at 37 °C under an atmosphere of 5% CO₂/95% air. DEV strain CSC was kept in our laboratory.

To construct a GFP-LC3 recombination plasmid, the LC3 gene was amplified from DEF cells with the primer pair LC3F 5`-ATG CAA CCG CCT CTG-3` and LC3R 5`-TCG CGT TGG AAG GCA AAT C-3`, corresponding to the GenBank sequence for duck LC3B gene (NW_004676873.1), and cloned into the pEGFP-C1 vector, to express LC3B protein with the GFP protein.

DEF cells were infected with DEV for 2 h at 37 °C, washed three times with sterile phosphate-buffered saline (pH 7.4), then maintained in 2% in culture medium supplemented with fetal bovine serum for various time points until samples were harvested. The cells were then cultured in 2% culture medium supplemented with fetal bovine serum with or without pre-treatment with the same drug for the indicated times. The optimal concentrations of chemicals used in this experiment were 10 mM 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N-tetraacetic acid (BAPTA-AM; Abcam, Cambridge, UK), 5 μM STO-609 (Merck-Millipore, Darmstadt, Germany),4 μM ionomycin and 2.5 μM Fluo-3 AM (Beyotime Institute of Biotechnology, Haimen, China). The toxicities of both drugs and siRNAs were tested using the WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime), according to the manufacturer’s instructions. At 36, 48, and 60 h post-infection (hpi), DEF cells were collected for subsequent analysis.

Proteins from cells treated with either drugs or siRNAs, or infected with DEV were extracted using immunoprecipitation lysis buffer (Beyotime) with the protease inhibitor phenylmethylsulfonyl fluoride (Beyotime), then boiled for 10 min in 5× loading buffer, separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes (GE Healthcare Life Sciences, Little Chalfont, UK), according to manufacturers’ instructions. The membranes were blocked with 3% bovine serum albumin (Sigma-Aldrich Corporation, St. Louis, MO, USA) for 2 h at room temperature and then incubated with the following primary antibodies for 2 h at room temperature: rabbit anti-LC3B antibody (Sigma-Aldrich Corporation), mMouse anti-CaMKKβ antibody (Sigma-Aldrich Corporation), rabbit anti-p-AMPK antibody (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-AMPK antibody (Thermo Fisher Scientific), mouse anti-β-actin antibody (Sigma-Aldrich Corporation).Then, the membranes were incubated with IRDye 800 CW goat anti-mouse or goat anti-rabbit immunoglobulin IgG as secondary antibodies for 1 h at room temperature. Antibody detection was conducted using an Odyssey Infrared Fluorescence Scanning Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Quantitation from western blot image intensity was achieved by adding rectangle to the image to gain data directly using the Odyssey Infrared Fluorescence Scanning Imaging System Application Software Version3.0.

For the detection of autophagosomes, DEF cells at 70–80% confluence in culture dishes were transfected with 2.5 μg of the GFP-LC3 plasmid using the Calcium Phosphate Transfection Kit (cat. no. K2780–01; Invitrogen Corporation, Carlsbad, CA, USA). At 24 hpi, chemical-treated or virus-infected DEF cells at different time points were fixed with absolute ethanol for 30 min and the cell nuclei were stained with 4′-6-diamidino-2-phenylindole (cat. no. D1306; Beyotime). The green fluorescence of GFP-LC3 was observed by confocal laser microscopy using a Leica SP2 confocal system (Leica Microsystems, Wetzlar, Germany).

In order to further study the effects of cell autophagy on viral replication, siRNA targeting the autophagy-related gene beclin-1 was synthesized (Shanghai GenePharma Co., Ltd., Shanghai, China). The sequence of the siRNA was GCC UAC AAC GAG GAC GAU ATT (sense) and UAU CGU CCU CGU UGU AGG CTT (antisense). Six-well plates were transfected with siRNA and negative control (NC)-siRNA using transfection reagents for 24 h and then infected with DEV. Cell samples were collected to detect the effects of siRNA.

DEF cells were cultured in covered 96-well plates and then infected with DEV virus diluted to 10^− 1 to 10^− 8. At 72 hpi, the cells were observed and pathological changes were recorded. Viral titers were determined according to the Reed–Muench method.

Cytosolic free Ca²⁺ ions were detected by using Fluo-3 AM. Fluo-3 AM itself is not combined with Ca²⁺ ions, but once dye is added to the cells, it can hybridize with Fluo-3 AM, and Fluo-3 AM will fluorescence upon binding to Ca²⁺. DEF cells were infected with DEV or treated with BAPTA-AM for the indicated times, then incubated with Fluo-3 AM in the dark at 37 °C for 1 h. Afterward, the cells were suspended in phosphate-buffered saline. To observe fluorescence, as an indicator of intracellular Ca²⁺ ions, the cells were monitored using a flow cytometer (BD FACSAria™; BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 488 nm.

Autophagy of DEV was induced via the AMPK/TSC2/mTOR pathway in DEF cells. An investigation to determine whether any other underlying mechanism of AMPK activation was involved in autophagy induction showed that DEV infection significantly increased intracellular levels of CaMKKβ and its substrate molecule phosphorylated AMPK (p-AMPK) at 36, 48, and 60 hpi, as compared to mock-infected cells. Microtubule-associated protein light chain (LC3) is an autophagy marker protein on the membranes of autophagosomes. When autophagosomes form, LC3I is phosphorylated by phosphatidyl ethanolamine (PE) to LC3II. LC3II remains on autophagosome membranes until fusion with lysosomes. Therefore, to some extent, LC3II expression measures the number of autophagosomes [22]. The expression level of LC3II also was significantly increased (Fig. 1a, b). This result indicated that CaMKKβ-AMPK may be involved in DEV-induced autophagy.

Next, we further verified the role of CaMKKβ in DEV-induced autophagy. Since CaMKKβ is activated in DEV-infected DEF cells, STO-609, a known CaMKKβ inhibitor, was used to assess changes in autophagy corresponding to the inhibition of CaMKKβ. The results showed that activation of AMPK, LC3I transformation to LC3II were significantly lower in DEV-infected cells treated with STO-609, as compared to control cells. In addition, less DEV gB protein was observed in the drug-treated control cells (Fig. 2a).

To eliminate nonspecific effects of chemical drugs, siRNA was used to inhibit CaMKKβ expression. As shown in Fig. 2b, as compared to cells transfected with the NC-siRNA, DEF cells transfected with siRNA, expression levels of CaMKKβ, activated AMPK, and LC3II were significantly reduced (Fig. 2b). The number of GFP-LC3 puncta decreased dramatically in siCaMKKβ -treated DEV-infected cells, as compared to control cells (Fig. 2c).

Due to the decreased expression of viral proteins in response to CaMKKβ inhibition by chemical drugs or siRNA, we further checked whether inhibition of CaMKKβ reduces viral replication. Viral titers were significantly decreased in DEV-infected DEF cells treated with STO-609 or siCaMKKβ. The TCID₅₀, as compared to control cells, at 48 hpi is shown in Fig. 2 d, e These results indicated that autophagy is related to CaMKKβ activation in DEV-infected DEF cells, and CaMKKβ is an upstream activator of AMPK involved in DEV-induced autophagy (Fig. 2d, e).

In order to further explore mechanisms involved in autophagy induction, upstream regulators were progressively explored. Some reports showed that an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]cyto) promoted the autophagic process [14, 25]. DEV-infected DEF cells were incubated with Fluo-3 AM. Then, at 36, 48 and 60 phi, intracellular Ca²⁺ was detected by flow cytometry, which showed that cytosolic Ca²⁺ in DEV-infected cells was higher than in mock-infected control cells, respectively (Fig. 3b). The result also showed that the increase in cytosolic Ca²⁺ was dependent on the initial viral dose with a DEV multiplicity of infection (MOI) of 0.1–10 at 48 hpi (Fig. 3a).

BAPTA-AM is a well-established chelator of intracellular Ca²⁺ ions. Mock- or DEV-infected DEF cells were treated with 25 μm BAPTA-AM for 30 h, and intracellular Ca²⁺ was detected by flow cytometry. As expected, the results suggested that the addition of BAPTA-AM reduced intracellular Ca²⁺ levels (Fig. 3 c). Accordingly, CaMKKβ and AMPK activities were significantly decreased in BAPTA-AM-treated cells. After the addition of BAPTA-AM, LC3II expression and viral protein synthesis were significantly reduced (Fig. 3d). GFP-LC3 distribution was also observed by a confocal fluorescence microscopy as discrete puncta associated with autophagic vacuoles. The change in GFP-LC3 puncta number after drug treatment may response to changes in autophagic activity. As speculation, the number of GFP-LC3 puncta remarkable decreased in BAPTA-AM-treated DEV-infected cells, as compared to control cells, suggesting the inhibition of autophagy (Fig. 3e).

The expression of gB protein was reduced in BAPTA-AM-treated DEF cells, as compared with control cells. Consistent with the result, virus titers were significantly reduced in BAPTA-AM-treated DEF cells, as compared with control group cells. TCID₅₀ assay was used to measure the DEV viral titers (Fig. 3f). Together, these data reflect that Ca²⁺ is necessary for the function of autophagy in DEV-infected DEF cells in a CaMKKβ- and AMPK-dependent process.

The siRNA targeting of endogenous genes or the drugs might have influenced cell viability and affected our results. The effects on cell viability of compounds used in this study were detected by WST-1 assays. Viability of treated cells was almost equal to mock cells, so the siRNA or pharmacological treatments did not affect DEF cell viability (Fig. 4).