The p17 nonstructural protein of avian reovirus triggers autophagy enhancing virus replication via activation of phosphatase and tensin deleted on chromosome 10 (PTEN) and AMP-activated protein kinase (AMPK), as well as dsRNA-dependent protein kinase (PKR)/eIF2α signaling pathways - PubMed (original) (raw)

The p17 nonstructural protein of avian reovirus triggers autophagy enhancing virus replication via activation of phosphatase and tensin deleted on chromosome 10 (PTEN) and AMP-activated protein kinase (AMPK), as well as dsRNA-dependent protein kinase (PKR)/eIF2α signaling pathways

Pei I Chi et al. J Biol Chem. 2013.

Erratum in

Abstract

Autophagy has been shown to facilitate replication or production of avian reovirus (ARV); nevertheless, how ARV induces autophagy remains largely unknown. Here, we demonstrate that the nonstructural protein p17 of ARV functions as an activator of autophagy. ARV-infected or p17-transfected cells present a fast and strong induction of autophagy, resulting in an increased level of autophagic proteins Beclin 1 and LC3-II. Although autophagy was suppressed by 3-methyladenine or shRNAs targeting autophagic proteins (Beclin 1, ATG7, and LC3) as well as by overexpression of Bcl-2, viral transcription, σC protein synthesis, and virus yield were all significantly reduced, suggesting a key role of autophagosomes in supporting ARV replication. Furthermore, we revealed for the first time that p17 positively regulates phosphatase and tensin deleted on chromosome 10 (PTEN), AMP-activated protein kinase (AMPK), and dsRNA dependent protein kinase RNA (PKR)/eIF2α signaling pathways, accompanied by down-regulation of Akt and mammalian target of rapamycin complex 1, thereby triggering autophagy. By using p53, PTEN, PKR, AMPK, and p17 short hairpin RNA (shRNA), activation of signaling pathways and LC3-II levels was significantly suppressed, suggesting that p17 triggers autophagy through activation of p53/PTEN, AMPK, and PKR signaling pathways. Furthermore, colocalization of LC3 with viral proteins (p17 and σC), p62 with LAMP2 and LC3 with Rab7 was observed under a fluorescence microscope. The expression level of p62 was increased at 18 h postinfection and then slightly decreased 24 h postinfection compared with mock infection and thapsigargin treatment. Furthermore, disruption of autophagosome-lysosome fusion by shRNAs targeting LAMP2 or Rab7a resulted in inhibition of viral protein synthesis and virus yield, suggesting that formation of autolysosome benefits virus replication. Taken together, our results suggest that ARV induces formation of autolysosome but does not induce complete autophagic flux.

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Figures

FIGURE 1.

FIGURE 1.

ARV induces autophagy. A, Vero cells were pretreated with different concentrations of 3-MA or rapamycin for 2 h and then infected with ARV at m.o.i. of 10. Cells lysates were separated by SDS-PAGE and immunoblotted with antibodies against ARV σC and β-actin, as indicated. The supernatants of ARV-infected cells in each well were harvested at 24 hpi for viral titration. Each value represents the mean of three independent experiments ± S.D. B, Vero cells were infected with different m.o.i. or time points as indicated. Cells lysates were separated by SDS-PAGE and immunoblotted with antibodies directed against ULK1, LC3-I/II, Beclin 1, and β-actin, as indicated. In semi-quantitative RT-PCR amplification of the Beclin 1 gene, Vero cells were transfected with p17 or infected with ARV at an m.o.i. of 10. In the bottom panel, the p17-transfected or ARV-infected cells were collected at 24 hpi, and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel and staining with ethidium bromide, the expression rate of the target gene is assessed by measuring the intensity of the band corresponding to a generated amplicon. The Beclin 1 mRNA levels in ARV-infected and p17-transfected cells were compared with those in mock-treated cells. The mRNA levels were normalized to that for GAPDH. Numbers below each lane are percentages of the control level of a specific protein in the mock treatment. C, Vero cells were transfected with p17-pcDNA3.1 and pCDN3.1, respectively. Cell lysates were harvested at 6, 12, 18, and 24 hpi and immunoblotted with respective antibodies against p17, LC3, and β-actin. Similar results were obtained from three independent experiments.

FIGURE 2.

FIGURE 2.

ARV activates PTEN, p38, AMPK, and PKR signaling pathways in a time-dependent manner. DF-1 cells were infected with an m.o.i. of 5. The results of Western blot analysis of the total cellular protein isolated from ARV-infected DF-1 cells (upper panel) and from bovine ephemeral fever virus (BEFV)-infected Vero cells (lower panel) at the indicated time points are shown. A nonrelated virus, bovine ephemeral fever virus, was used as a control to examine whether activation of p38 and AMPK by ARV is specific. Phosphorylation and protein levels were determined by Western blot assay with the respective antibodies. The β-actin was included as an internal control for normalization. Similar results were obtained in three independent experiments.

FIGURE 3.

FIGURE 3.

ARV p17 protein regulates PTEN, Akt, mTOR, S6K1, p38, AMPK, PKR, and eIF2α, and Beclin 1 in a time-dependent manner. DF-1 and Vero cells were transfected with p17 (A) as well as σC and σA (B) for 24 h. To ensure that p17 is the viral protein to activate PTEN, PKR, and AMPK, cells transfected with the σC- and σA-encoding genes of ARV were used as additional controls. The results of Western blot analysis of the total cellular protein isolated from p17-transfected DF-1 and Vero cells at the indicated time points are shown. Phosphorylation and protein levels were determined by Western blot assay with the indicated antibodies. The β-actin was included as an internal control for normalization. Similar results were obtained in three independent experiments.

FIGURE 4.

FIGURE 4.

Knockdown of PTEN, AMPK, and PKR gene expression reduced ARV σC protein synthesis and viral yield. DF-1 and Vero cells transfected with respective shRNA or scramble shRNA were mocked or infected with ARV at m.o.i. of 10 for 24 h. Cell lysates were analyzed by Western blotting with antibodies against PTEN (A), AMPK (B), and PKR (C), as well as ARV σC, LC3-II or β-actin, as indicated. D, supernatants of ARV-infected and shRNA-transfected cells in each well were harvested at 24 hpi for viral titration. Each value represents the mean of three independent experiments ± S.D.

FIGURE 5.

FIGURE 5.

Knockdown of ATG7, Beclin 1, and LC3 reduced ARV σC protein synthesis and viral yield. DF-1 and Vero cells transfected with respective shRNA or scrambled shRNA were mock-infected or infected with ARV at m.o.i. of 10 for 24 h. Cell lysates were analyzed by Western blotting with antibodies against ATG7 (A), Beclin 1 (B), LC3 (D), ARV σC or β-actin, as indicated. C, BHK-21 cells stably expressing Bcl-2 were infected with ARV at an m.o.i. of 10 for 24 h. Cells lysates were analyzed by Western blot with antibodies against LC3-II, σC, or β-actin, respectively. E, supernatants of ARV-infected and shRNA-transfected cells in each well were harvested at 24 hpi for viral titration. Each value represents the mean of three independent experiments ± S.D. F, in semi-quantitative RT-PCR amplification of the σC and p17-encoding gene of ARV, DF-1 cells were transfected with specific Beclin 1 and LC3 shRNAs for 24 h and then infected with ARV at an m.o.i. of 10. The ARV-infected cells were collected at 24 hpi, and total RNAs were extracted for semi-quantitative RT-PCR. The quantitated assay was carried out as described in Fig. 1_B_. The σC and p17 mRNA levels in LC3- and Beclin 1-depleted cells were compared with those in ARV-infected and mock-treated cells. The mRNA levels were normalized to that for GAPDH. Numbers below each lane are percentages of the control level of a specific protein in shRNA-treated cells. The shRNAs used are indicated on the top of the gel. Similar results were obtained from three independent experiments.

FIGURE 6.

FIGURE 6.

Blockade of p53 by p53 shRNA-inhibited p17 up-regulated PTEN, Beclin 1, and LC3-II. Vero and DF-1 cells were transfected with p53 shRNA, pcDNA3.1, and p17 for 24 h followed by Western blot analysis. The Western blot results of different combinations of treatments in Vero and DF-1 cells are shown. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Similar results were obtained in three independent experiments.

FIGURE 7.

FIGURE 7.

Knockdown of PTEN, AMPK, PKR, and ATG7 reduced the level of LC3-II protein in p17-transfected cells. DF-1 and Vero cells were transfected with the respective shRNA, scrambled shRNAs, and mock transfection, respectively, for 24 h. Cell lysates were analyzed by Western blotting with antibodies against PTEN (A), AMPK (B), PKR (C), PKR and AMPK (D), PKR and Beclin 1 (E), ATG7 (F), and β-actin, as indicated. G, PTEN, AMPK, and PKR signalings were suppressed in p17 shRNA-treated cells. Similar results were obtained in three independent experiments.

FIGURE 8.

FIGURE 8.

ARV p17 promotes autophagosome and autolysosome formation that benefits virus replication. A, Vero cells were infected with ARV for 12 h and then fixed and processed for immunofluorescence staining of p17, σC, and LC3. The colocalization of ARV proteins (p17 and σC) was observed under a fluorescence microscope. Scale bar, 20 μm. B, quantitation results from Fig. 1 represents mean LC3 puncta per cell. n = 13. C, coimmunoprecipitation of p17, σC, and LC3 was carried out. Cells were infected with ARV at an m.o.i. of 10 and collected 24 h postinfection. About 500 μg of cellular proteins was incubated with 4 μg of anti-p17 or σC antibodies at 4 °C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot, and then proteins were detected with indicated antibodies. Similar results were obtained in three independent experiments. IB, immunoblot. D, uninfected Vero cells were pretreated with TG for 2 h or without TG treatment (mock) in complete media as well as without TG treatment but in amino acid-free media for 2 h (starvation). In parallel experiments, two sets of Vero cells were infected with ARV at m.o.i. of 10 or transfected with p17-pCDNA3.1 plasmid, respectively. Cells lysates were collected at 24 hpi or post-transfection and then analyzed by Western blot with antibodies against p62. E, Vero cells were infected with ARV at m.o.i. of 10 for 12 h and then fixed and processed for immunofluorescence staining of p62 and LAMP2. The procedures for the TG and starvation treatments are described in D. Colocalization of p62 and LAMP2 was observed under a fluorescence microscope. Scale bar, 20 μm. F, p62- and LAMP2-positive cells were quantified by counting the numbers of positive cells in an individual field under a fluorescence microscope. Each value represents the mean of 20 fields ± S.D. G, Vero cells were transfected with LC3-GFP plasmid for 24 h and then infected with ARV at m.o.i. of 10 for 12 h. The cells were then fixed and processed for immunofluorescence staining of LC3-GFP and Rab7a. Colocalization of LC3-GFP and Rab7a was observed under a fluorescence microscope. Scale bar, 20 μm. H, Vero cells were transfected with LC3-GFP plasmid for 24 h and then infected with ARV at m.o.i. of 10 for 12 h. The cells were then fixed and processed for immunofluorescence staining of LC3-GFP and lysosome. In the p17 transfection assay, cells were transfected with p17-pCDNA3.1 plasmid. The treatment conditions for TG and starvation are described as in D. I, Vero cells were transfected with shRNAs targeting LAMP2 or Rab7a as well as scrambled shRNA for 24 h and then infected with ARV at an m.o.i. of 10 for 24 h. Cells lysates were analyzed by Western blot with antibodies against LAMP2, σC, and actin, respectively. The supernatants of LAMP2 and Rab7a knockdown in ARV-infected cells were harvested at 24 hpi for viral titration. Each value represents the mean of three independent experiments ± S.D.

FIGURE 9.

FIGURE 9.

Model depicting the mechanism of ARV p17 triggers autophagy. This study established a new regulatory network of p17 linking p53/PTEN/mTOR, AMPK, and PKR/eIF2α pathways, which up-regulate ULK1, Beclin 1, and LC3. p17 increases the levels of phosphorylated p53, PTEN, and AMPK, which in turn down-regulate Akt and mTORC1. p17 also activates PKR to phosphorylate eIF2α. Taken together, PTEN, AMPK, and PKR signaling pathways triggered by p17 that induce autophagy enhance virus replication.

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