Porcine Circovirus Type 2 Induces Autophagy via the AMPK/ERK/TSC2/mTOR Signaling Pathway in PK-15 Cells

Cells culture, treatments with chemicals and virus infection.

PK-15 cells were maintained in complete minimal essential medium (HyClone, South Logan, UT) supplemented with 10% heat-inactivated newborn calf serum (Gibco, Grand Island, NY), 1% l-glutamine, 1% nonessential amino acids, 100 U of penicillin G/ml, and 100 μg of streptomycin/ml. PK-15 cells stably expressing enhanced green fluorescent protein (EGFP)-LC3 (referred to here as PK-15/EGFP-LC3 cells) (68) were maintained in complete medium containing 0.2 mg of G418/ml. All cell monolayers were incubated at 37°C and 5% CO₂.

A PCV2 strain SY4 (PCV2b; GenBank accession no. GU325754) isolated from a diseased pig (68) was propagated in PK-15 cells. Cells were infected with PCV2 at a multiplicity of infection of one 50% tissue culture infective dose for autophagy induction. After a 1-h absorption, the supernatant was removed, and the cells were rinsed with sterile phosphate-buffered saline at pH 7.2 (PBS) to remove unabsorbed viruses. The infected cells were grown in fresh medium at 37°C and 5% CO₂ for the indicated times.

The chemicals used to treat or pretreat PK-15 cells included 0.5 μM rapamycin (Merck, Whitehouse Station, NJ), 20 μM UO126 (Sigma, St. Louis, MO), 5 μM compound C (Sigma), and 1 mM 5-aminoimidazole-4-carboxamide riboside (AICAR; Sigma). The cells were then infected as described above and cultured in fresh medium in the absence or presence of the same drug (at the same concentrations for pretreatments), or the corresponding level of the solvent dimethyl sulfoxide (DMSO) as a control, unless otherwise specified in the figure legends.

Plasmid construction and small interfering RNA (siRNA) transfection.

Rheb (Rheb Q64L) is reported to positively regulate mTOR (30, 63). To construct pcDNA3.1-flag-Rheb Q64L, the porcine Rheb gene was PCR amplified from porcine liver cDNA by using the primers 5′-AAGGATCCATGCCGCAGTCCAAGTCC-3′ and 5′-CCCTCGAGTCACATCACCGAGCAGGA-3′. PCR-amplified Rheb was subsequently cloned into pcDNA3.1 (Invitrogen, Eugene, OR) fused to an N-terminal flag. A mutant construct with a Q64L substitution was generated by site-directed mutagenesis using the primers 5′-ACACAGCTGGGCTGGATGAATATT-3′ and 5′-AATATTCATCCAGCCCAGCTGTGT-3′. All constructs were confirmed by DNA sequencing. The construction of pcDNA3.1-Cap has been described previously (68).

The following ERK- and TSC2-specific siRNAs were purchased from Genepharma (Shanghai, China): ERK sense (5′-CUCCAAAGCUCUGGAUUUAtt-3′) and ERK antisense (5′-UAAAUCCAGAGCUUUGGAGtt-3′) and TSC2 sense (5′-CGAGCUCG UUGAGAGAUAUtt-3′) and TSC2 antisense (5′-AUAUCUCUCAACGAGCUCGtt-3′) (deoxyribonucleotides are shown as lowercase letters). For the delivery of siRNAs into PK-15 cells, 90 nM nontargeting siRNA (NS) and siRNAs targeting ERK and TSC2 were transfected with SuperFectin II reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. Cells were infected with PCV2 at 24 h posttransfection and subjected to further incubation for 36 h. Infected cells were then harvested for further analyses. The ERK and TSC2 knockdown efficiency was evaluated by Western blotting with rabbit anti-ERK or TSC2 monoclonal antibodies (Epitomics, Burlingame, CA).

Immunoblotting.

Immunoblotting (IB) was performed as described previously (68). Briefly, cells were lysed in lysis buffer after infection or chemical treatments for indicated times. The lysates were collected and protein concentration quantified by using a bicinchoninic acid assay kit. Proteins were separated in standard SDS-PAGE gels and electrotransferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membranes were blocked for 2 h in Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat milk and then probed for 1 h with the following primary antibodies: mouse monoclonal anti-Cap IgG (produced in our laboratory), rabbit polyclonal anti-LC3B (Sigma), mouse monoclonal anti-β-actin (MultiSciences, Hangzhou, China), and rabbit monoclonal antibodies to Ras, Raf1, mTOR, ERK1/2, AMPK, and TSC2, as well as to phosphorylated proteins of mTOR, ERK1/2, AMPK, and TSC2 (Epitomics). Blots were washed, incubated for another hour with goat anti-rabbit or anti-mouse horseradish peroxidase-labeled antibodies (KPL, Gaithersburg, MD), and visualized using SuperSignal West Pico chemiluminescent substrate (Thermo, Marina, CA) under the conditions recommended by the manufacturer. Images were captured in a Gel 3100 chemiluminescent imaging system (Sagecreation, Beijing, China), and the densities of the protein bands were normalized to the β-actin signal and quantified using Quantity One software (Bio-Rad, Hercules, CA). The abundance of interested proteins in various treatments was expressed relative to those under mock conditions.

Coimmunoprecipitation.

Cells were lysed with immunoprecipitation lysis buffer (IPLB; 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) in the presence of protease cocktail inhibitor (Roche, Mannheim, Germany). After centrifugation at 12,000 × g for 15 min, supernatant samples of the lysates were pretreated with protein A/G (Beyotime, Haimen, China) for 30 min at 4°C to eliminate nonspecific binding to the agarose gel. Antibodies against phospho-ERK1/2, phospho-AMPK, or phospho-TSC2 (1 μg) were used to pull down the protein complexes (500 μg) overnight at 4°C. The complexes were then immobilized with 20 μl of protein A/G-coated beads, followed by incubation overnight at 4°C on a rocker. The beads were spun down and washed three times with IPLB, and the proteins were eluted in SDS loading buffer by heating at 100°C for 5 min. SDS-PAGE and immunoblotting were carried out as previously described. The blots were probed with antibodies against phospho-ERK1/2, phospho-AMPK, phospho-TSC2, or Raf1 and visualized as described above. The lysates of mock-infected PK-15 cells were used as controls.

Confocal microscopy.

For the detection of autophagosomes, PK-15/EGFP-LC3 cells cultured in a petri dish with a glass bottom (10 mm in diameter) were infected with PCV2 or treated with 20 μM UO126 or transfected with pcDNA3.1, pcDNA3.1-flag-Rheb Q64L, or siERK for the indicated times. The cells were washed with PBS, fixed, and permeabilized with 80% cold acetone in PBS at −20°C for 20 min and then washed again with PBS. The cell samples were then incubated with rabbit polyclonal anti-flag for 1 h at room temperature, washed with PBS, and incubated with DyLight 549-conjugated goat anti-rabbit IgG (MultiSciences) for 1 h at room temperature. Nuclei were counterstained with DAPI (4′,6′-diamidino-2-phenylindole; Invitrogen). The fluorescence of EGFP-LC3 was observed under a laser scanning confocal microscope (TCS SP5; Leica Munich, Germany). The average number of EGFP-LC3 punctae per cell from at least 60 to 80 cells per sample was counted (39). Only cells with at least five dots were scored as positive.

MTT assay.

MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] was performed as described previously (68). In brief, PK-15 cells were seeded in a 96-well plate, followed by incubation at 37°C for 24 h. The medium was replaced with fresh complete medium containing DMSO, 20 μM UO126, or 5 μM compound C or 1 mM AICAR or transfected with siERK or siTSC2, and the plates were incubated for 36 h. MTT (10 μl) was added to each well, and the cells were further incubated for 4 h. Then, 100 μl of acidified isopropanol was added to each well to dissolve the precipitates completely. The optical density was measured at 570 nm using the SpectraMax M2 spectrophotometer. The viability of treated cells was expressed as a percentage relative to untreated cells.

Statistical analysis.

All results in figures were presented, where appropriate, as means ± the standard deviations from three independent experiments and analyzed by using the Student t tests.

PCV2 induces autophagy by inhibiting mTOR signaling.

Previously, we found that PCV2 infection activated autophagy (68). However, the underlying mechanism remains elusive. Since mTOR is a major signaling pathway that inhibits autophagy (12, 21), we investigated whether PCV2 infection inhibited mTOR by infecting PK-15 cells with PCV2 or treating with rapamycin, an mTOR inhibitor. Figure 1 shows that PCV2 or rapamycin caused significant reduction in phosphor-mTOR and increased the LC3-II/β-actin ratio compared to uninfected control cells (Fig. 1A and B, P < 0.05) (no change was detected in the levels of total mTOR), suggesting that mTOR signaling is involved in PCV2-mediated autophagy in PK-15 cells.

We then investigated whether inhibition of mTOR is an essential step in autophagy induction by PCV2. For this purpose, we overexpressed a constitutive active form of Rheb (Rheb Q64L) which positively regulates mTOR (30, 63). As shown in Fig. 1C and D, the expression of Rheb Q64L restored phospho-mTOR in PCV2-infected PK-15 cells and suppressed LC3 lipidation. The expression of Rheb Q64L also reduced the number of cells containing punctae of EGFP-LC3 (i.e., autophagosome-like vesicles) in PK-15/EGFP-LC3 cells infected with PCV2 (Fig. 1E and F, P < 0.01). Collectively, these observations indicate that PCV2 induces autophagy by inhibiting mTOR signaling.

PCV2 induces autophagy by activating ERK1/2 signaling.

The ERK1/2 pathway is reported to play a key role in regulating autophagy (5, 10), and PCV2 infection activates ERK1/2 signaling (61). Thus, we examined whether ERK1/2 is involved in autophagy induction in PCV2-infected PK-15 cells. Figure 2A and B show that PCV2-infected cells had an increased ratio of LC3-II or phospho-ERK1/2 to β-actin relative to uninfected cells (P < 0.05), whereas no change was detected in the levels of total ERK1/2. Inhibition of ERK1/2 phosphorylation with UO126 suppressed PCV2-induced conversion of LC3-I to LC3-II (Fig. 2C and D, P < 0.01). In PK-15/EGFP-LC3 cells, UO126 decreased the number of cells containing EGFP-LC3 punctae (Fig. 2E and F, P < 0.01). Similar findings were seen with knockdown of ERK1/2 by siRNA. LC3-II levels were significantly decreased in PK-15 cells transfected with siRNA targeting ERK1/2 compared to control siRNA (Fig. 2G and H, P < 0.05). In PK-15/EGFP-LC3 cells, ERK1/2-targeted siRNA diminished the number of cells containing EGFP-LC3 punctae (Fig. 2I and J, P < 0.01). Therefore, it is apparent that PCV2 infection triggers autophagy in PK-15 cells through the ERK1/2 signaling pathway.

PCV2 infection activates ERK1/2 with subsequent downregulation of mTOR.

The mTOR function is tightly regulated by ERK1/2 signaling through tuberous sclerosis complex 2 (TSC2) (1, 33, 60). To characterize the molecular mechanisms underlying reduced mTOR activation and autophagy induction by PCV2 infection, UO126 was used to block the ERK1/2 pathway. As shown in Fig. 3A and B, inhibition of ERK1/2 increased phosphorylation of mTOR in PCV2-infected cells (P < 0.05), indicating that ERK1/2 was required for PCV2 to inhibit mTOR signaling. To further confirm the role of ERK1/2 in mTOR inhibition during PCV2 infection, we suppressed ERK1/2 by using siRNA. Knockdown of ERK1/2 downregulated phospho-ERK1/2 (Fig. 3C and D). The levels of mTOR phosphorylation were decreased in PCV2-infected cells with ERK1/2-specific RNA silencing compared to control siRNA (Fig. 3C and D, P < 0.05). Based on these findings, we conclude that PCV2 infection in PK-15 cells might activate the ERK1/2 pathway, which downregulates mTOR in turn with subsequent induction of autophagy.

AMPK is the upstream regulator of ERK1/2 in PCV2-mediated autophagy.

ERK1/2 activation has been shown to occur via the Ras/Raf1/ERK1/2 pathway (54). However, AMPK has recently been reported to be a positive regulator of autophagy (14, 23, 26, 48, 49). Also, AMPK could positively regulate ERK1/2 activity (7, 22, 47, 60). To determine whether ERK1/2 activation occurs downstream of the Ras/Raf1 pathway or AMPK, we analyzed whether Ras, Raf1, or AMPK was activated by PCV2 infection. Figure 4A shows that PCV2 infection did not have significant effects on expression of Ras or Raf1, suggesting that the Ras/Raf1 pathway does not seem to contribute to PCV2-induced autophagy. In contrast, the starvation medium (i.e., Earle's balanced salt solution [EBSS]; Gibco), which is known to activate the Ras/Raf1/ERK1/2 pathway (18), increased expression of both Ras and Raf1 (Fig. 4A).

We then examined whether PCV2 infection activated AMPK. PK-15 cells were infected with PCV2 or treated with AICAR (an AMPK activator) (27, 34) for 36 h. Figure 4B shows that PCV2 infection or AICAR treatment significantly increased phosphorylation of AMPK, whereas no change was detected in the levels of total AMPK. Since we found that PCV2 capsid protein induces autophagosome formation (68). We attempted to examine whether Cap is the protein that activates AMPK. By transfecting PK-15 cells with pcDNA3.1-Cap, we found that Cap expression did indeed increase phosphorylation of AMPK (Fig. 4C). Moreover, PCV2-induced activation of ERK1/2 and AMPK or LC3 lipidation was inhibitable by compound C, an agent known to inhibit AMPK (37) (Fig. 4D and E, P < 0.05), whereas the inhibition of ERK1/2 by UO126 did not inhibit AMPK activation in response to PCV2 infection (Fig. 4F), suggesting that AMPK might be an upstream regulator of ERK1/2 in PCV2-mediated autophagy. To understand how ERK1/2 is linked to AMPK, PK-15 cells were infected with PCV2 or mock-infected for 36 h, and the protein lysates were analyzed by reciprocal coimmunoprecipitation (Co-IP). Clearly, PCV2 infection activated both ERK1/2 and AMPK, and both proteins were interacted with each other (Fig. 4G). However, Raf1 did not seem to interact with ERK1/2 during PCV2 infection (Fig. 4H). Therefore, we propose that ERK1/2 interacts with its upstream regulator AMPK, but not with Raf1 in response to PCV2 infection. Since we did not see direct interaction between AMPK and Cap in Co-IP analyses (data not shown), there might be some other component upstream of AMPK that could interact with the viral protein in initiating the signaling cascade.

ERK1/2 inhibits mTOR by activating TSC2 in PCV2-induced autophagy.

ERK1/2 could activate TSC2 (60). AMPK is also involved in phosphorylation of TSC2 and enhances its activity (11, 19). We found that PCV2 infection induced activation of TSC2 and reduced mTOR activity (Fig. 5A and B). Suppression of TSC2 by siRNA recovered mTOR phosphorylation and downregulated LC3-II (Fig. 5A and B, P < 0.05), suggesting that TSC2 regulates autophagy via mTOR. To examine whether ERK1/2 inhibits mTOR via activating TSC2 after PCV2 infection or whether AMPK exerts its direct effect on TSC2 or indirectly via ERK1/2, PK-15 cells were infected with PCV2 with or without compound C or UO126. We found that inhibition of AMPK with compound C repressed ERK1/2 and TSC2, and recovered mTOR activity (Fig. 5C and 5D, P < 0.05). Inhibition of ERK1/2 also suppressed TSC2 activation (Fig. 5E and F, P < 0.05). However, TSC2 remained repressed irrespective of the high level of p-AMPK when p-ERK1/2 was inhibited by UO126. Co-IP analysis suggests that PCV2 infection activated both TSC2 and ERK1/2 and that both proteins interacted with each other (Fig. 5G). These results indicate that TSC2 is the downstream effector of ERK1/2, and AMPK seems to affect TSC2 via ERK1/2 in regulation of autophagy during PCV2 infection.

Pharmacological treatments and RNA interference do not affect cell viability.

To assess whether treatments with UO126, AICAR or compound C and siRNA targeting ERK1/2 or TSC2 transfection affected cell viability, which could ultimately affect experimental results in host cells, we used the MTT assay to analyze their effects on cell viability. There were virtually no significant effects on the viability of PK-15 cells treated with the chemicals or siRNA transfection (Fig. 6).