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. 2012 Dec 21;287(52):43651-64.
doi: 10.1074/jbc.M112.399964. Epub 2012 Oct 29.

RNase L induces autophagy via c-Jun N-terminal kinase and double-stranded RNA-dependent protein kinase signaling pathways

Mohammad Adnan Siddiqui  1 , Krishnamurthy Malathi
Affiliations

RNase L induces autophagy via c-Jun N-terminal kinase and double-stranded RNA-dependent protein kinase signaling pathways

Mohammad Adnan Siddiqui et al. J Biol Chem. .

Abstract

Autophagy is a tightly regulated mechanism that mediates sequestration, degradation, and recycling of cellular proteins, organelles, and pathogens. Several proteins associated with autophagy regulate host responses to viral infections. Ribonuclease L (RNase L) is activated during viral infections and cleaves cellular and viral single-stranded RNAs, including rRNAs in ribosomes. Here we demonstrate that direct activation of RNase L coordinates the activation of c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) to induce autophagy with hallmarks as accumulation of autophagic vacuoles, p62(SQSTM1) degradation and conversion of Microtubule-associated Protein Light Chain 3-I (LC3-I) to LC3-II. Accordingly, treatment of cells with pharmacological inhibitors of JNK or PKR and mouse embryonic fibroblasts (MEFs) lacking JNK1/2 or PKR showed reduced autophagy levels. Furthermore, RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Viral infection with Encephalomyocarditis virus (EMCV) or Sendai virus led to higher levels of autophagy in wild-type (WT) MEFs compared with RNase L knock out (KO) MEFs. Inhibition of RNase L-induced autophagy using Bafilomycin A1 or 3-methyladenine suppressed viral growth in initial stages; in later stages autophagy promoted viral replication dampening the antiviral effect. Induction of autophagy by activated RNase L is independent of the paracrine effects of interferon (IFN). Our findings suggest a novel role of RNase L in inducing autophagy affecting the outcomes of viral pathogenesis.

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Figures

FIGURE 1.
FIGURE 1.
2–5A-mediated activation of RNase L induces autophagy. A, HT1080 cells were transfected with 10 μm of 2–5A and cleavage of rRNA (shown by arrows) was analyzed on RNA chips using Agilent Bioanalyzer 2100. B, 2–5A was transfected for indicated times and conversion of unconjugated LC3-I to lipidated LC3-II and degradation of p62 was monitored on immunoblots and normalized to β-actin levels. Band intensity was calculated using Image J software and ratios of LC3-II/β-actin or p62/β-actin was determined. Similar results were observed in three independent experiments. C and D, each bar represents the ratios of LC3-II/β-actin or p62/β-actin corresponding to the time points in B from three experiments shown as mean ± S.E. E, GFP-LC3 expressing HT1080 cells were transfected with 10 μm of 2–5A and the percentage of GFP+ cells showing puncta formation compared with mock-treated cells is shown. Results shown represent mean ± S.E. for three experiments and at least 100 cells were analyzed per assay. F, effect of 3-MA or BafA1 on autophagic flux induced by 2–5A. HT1080 cells were transfected with 2–5A, during the last 4 h of the 24 h treatment, 5 mm of 3-MA or 100 nm of BafA1 was optionally added. Cell lysates were analyzed on immunoblots and quantitated as in B. G, numbers of GFP+ vesicles per cell enumerated from at least 10 cells per sample. Data represent mean ± S.E. from n = 3. Student's t test, *, p < 0.0001, **, p < 0.001. H, GFP-LC3-expressing HT1080 cells were transfected with 10 μm of 2–5A alone or in the presence of Bafilomycin A1 (100 nm), JNK inhibitor (SP600125, 25 μm) or PKR inhibitor (2-aminopurine, 5 mm) or Bafilomycin A1 alone. Cells were stained with Lysotracker Red (lysosomes) or DAPI (nucleus, blue) and images taken at ×60 magnification using confocal microscope. Representative images are shown.
FIGURE 2.
FIGURE 2.
Autophagosome formation is inhibited in RNase L-deficient cells. WT and RNase L KO MEFs were transfected with 10 μm of 2–5A for indicated times and LC3-II lipidation (A) or p62 degradation (B) was monitored on immunoblots. Cells treated with rapamycin (100 nm, R) served as control for autophagy induction. Ratios of LC3-II/β-actin or p62/β-actin were determined using Image J. Each bar shown below represents the ratios from three different experiments shown as mean ± S.E. C, WT and RNase L KO MEFs were initially transfected with GFP-LC3 and 24 h later with 10 μm of 2–5A. The percentage of GFP+ cells showing puncta formation compared with mock-treated cells is shown. Results shown represent mean ± S.E. for three experiments and at least 100 cells were analyzed per assay. D, HT1080 cells were treated with control siRNA (100 nm) or RNaseL siRNA (100 nm, pool of three siRNAs) for 36 h, followed by 2–5A transfection (10 μm) for indicated times. Cell lysates were analyzed for protein levels of RNase L (80% knockdown) and LC3-II lipidation and p62 degradation. Representative immunoblots are shown. E, cells were transfected with GFP-LC3 prior to transfection with relevant siRNA and 2–5A as in D. The percentage of GFP+ cells showing puncta formation compared with control siRNA-treated cells is shown. Results shown represent mean ± S.E. for three experiments and at least 100 cells were analyzed per assay. F, RNase L KO MEFs were reconstituted with WT RNase L, mutant RNase L R667A (nuclease-dead) or vector alone and transfected with 10 μm of 2–5A for 4 h. Cleavage of rRNA (shown by arrows) was analyzed on RNA chips using Agilent Bioanalyzer 2100. 100 μg of lysates were probed for RNase L expression on immunoblots. Conversion of unconjugated LC3-I to lipidated LC3-II and degradation of p62 was monitored on immunoblots and normalized to β-actin levels. Band intensity was calculated using Image J software and ratios of LC3-II/β-actin or p62/β-actin was determined.
FIGURE 3.
FIGURE 3.
Induction of autophagosomes in response to activation of RNase L. GFP-LC3-transfected cells (A) WT and RNase L KO MEFs, (B) HT1080 cells treated as described under “Experimental Procedures” with control siRNA, siRNase L, siBeclin1 or siAtg5, (C) WT and JNK1/2 KO MEFs, (D) WT and PKR KO MEFs were transfected with 10 μm of 2–5A. Images of cells were taken by confocal microscopy to assess formation of autophagosomes (autofluorescence of GFP-LC3 puncta). Lysosomes were visualized using Lysotracker Red, and nuclei were stained with DAPI (blue). The yellow color represents colocalization of GFP-LC3 and lysosomes. Representative images are shown (×60 magnification).
FIGURE 4.
FIGURE 4.
RNase L-induced autophagy is regulated by activity of JNK and PKR. A, HT1080 cells were transfected with 10 μm of 2–5A for indicated times. Cell lysates were immunoblotted with phospho-PKR (T451), total PKR, phospho-eIF2α, total eIF2α, phospho-JNK (T183,Y185), and total JNK antibodies. B, HT1080 cells were pretreated with PKR inhibitor (2-aminopurine, 5 mm) or JNK inhibitor (SP600125, 25 μm) for 1 h prior to 2–5A transfection (10 μm, 16 h) and then replaced. Inhibition of autophagy was evaluated on immunoblots by LC3-II lipidation and p62 degradation normalized to β-actin levels. Inhibition of PKR and JNK was confirmed using phospho-specific antibodies. C and E, HT1080 cells, WT, PKR KO, or JNK1/2 KO MEFs transfected with GFP-LC3 were treated as in B. The percentage of GFP+ cells showing puncta formation compared with control cells is shown. Results shown represent mean ± S.E. for three experiments and at least 100 cells were analyzed per assay. D, WT and PKR KO or JNK1/2 KO MEFs were transfected with 10 μm of 2–5A and optionally with PKR inhibitor (2-AP, 5 mm) or JNK inhibitor (SP, 25 μm). LC3-II lipidation and p62 degradation were immunodetected as in B and analyzed. Activation of PKR in A, B, and D were correlated with phospho-eIF2α.
FIGURE 5.
FIGURE 5.
RNase L activation induces dissociation of Beclin1-Bcl-2 complex via JNK pathway. A, HT1080 cells were transfected with 2–5A for 3 or 6 h prior to co-immunoprecipitation with control IgG or anti-Beclin1 antibody. Endogenous Beclin1 and co-immunoprecipitated proteins, Bcl-2 or Vps34, were detected using specific antibodies. Expression of the relevant proteins in the cell lysates was confirmed on immunoblots. * indicates nonspecific protein. B, cells were pretreated with JNK inhibitor, SP600125 (25 μm), for 1 h followed by 4 h transfection with 2–5A. As control, HT1080 cells were treated with 100 nm rapamycin (R) for 4 h. Endogenous Beclin1 was immunoprecipitated from cell lysates and co-immunoprecipitated proteins were subject to immunoblotting using anti-Bcl-2 and anti-Vps34 antibodies. Phosphorylation of Bcl-2 on Ser70 residue was detected in cell lysates using phospho-specific antibodies. Protein expression in lysates was normalized to β-actin levels. Results are representative of three independent experiments.
FIGURE 6.
FIGURE 6.
Beclin1 and autophagy-related genes are required for RNase L-induced autophagy. A and B, HT1080 cells were transfected with 100 nm of indicated siRNAs (control, Beclin1 (pool of three siRNAs), Atg5 (two separate siRNAs)) for 36h, followed by 2–5A transfection to induce autophagy for indicated times. Cell lysates were analyzed for knockdown protein levels on immunoblots. Induction of autophagy was determined on immunoblots by lipidation of LC3-II and p62 degradation normalized to β-actin levels. C, quantitation of autophagy in HT1080 cells co-transfected with GFP-LC3 and control siRNA or with GFP-LC3 and siBeclin1 or siAtg5. The percentage of GFP+ cells showing puncta formation in knockdown cells was compared with control siRNA-treated cells. Results shown represent mean ± S.E. for three experiments and at least 100 cells were analyzed per assay. D, WT or Atg5 KO MEFs were transfected with 10 μm of 2–5A for indicated times and induction of autophagy was determined as in A and B. E, quantitation of autophagy in WT and Atg5 KO MEFs as in C. Results are mean ± S.E., n = 3. F, WT and ATG5 KO MEFs were transfected with GFP-LC3 followed by 10 μm of 2–5A. Images of cells were taken by confocal microscopy to assess formation of autophagosomes (autofluorescence of GFP-LC3 puncta). Lysosomes were visualized using Lysotracker Red and nuclei were stained with DAPI (blue). The yellow color represents colocalization of GFP-LC3 and lysosomes. Representative images are shown. (×60 magnification).
FIGURE 7.
FIGURE 7.
Inhibition of autophagy modulates antiviral effects of RNase L. WT and RNase L KO MEFs were infected with (A) SeV (40HAU/ml) or (B) EMCV (MOI = 0.1) for indicated times, and cell lysates were analyzed for induction of autophagy. Conversion of LC3-I to LC3-II by lipidation and degradation of p62 were normalized to β-actin levels. Expression of viral antigens were detected using anti-Sendai-virus antibody or 3D Pol antibody (for EMCV). C and D, WT and RNase L KO MEFs were pretreated with autophagy inhibitors, 3-MA (5 mm) or BafA1 (100 nm) for 1 h, followed by infection with SeV (40 HAU/ml) or EMCV (MOI = 0.1) for 16 h or 8 h. Control samples were treated with DMSO (vehicle). Viral titers for EMCV were determined by plaque assay and copy numbers of SeV genomic RNA strands were determined in supernatants by real-time RT-PCR as described under “Experimental Procedures.” E and F, fold increase in viral yields in WT and RNase L KO MEFs treated with either 3-MA (5 mm) or Bafilomycin A1 (100 nm) were compared with control samples. Data represent mean ± S.E. for three independent experiments performed in triplicate. Student's t test was used to determine p values of treated cells compared with DMSO-treated cells (Ctrl). G, WT and RNase L KO MEFs were transfected with GFP-LC3 followed by infection with EMCV (MOI = 0.1) for 8 h or SeV (40 HAU/ml) for 24 h. Lysosomes were stained with lysotracker (red), and nuclei were stained with DAPI (blue). GFP-LC3 puncta formation was visualized under confocal microscope at ×60 magnification. Representative images are shown out of three experiments.
FIGURE 8.
FIGURE 8.
Effect of inhibition of autophagy on viral growth in WT and RNase L KO MEFs. Growth curve of EMCV (MOI = 0.01, A and B) or SeV (20 HAU/ml, C and D) in WT and RNase L KO MEFs. WT and RNase L KO MEFs were pretreated with autophagy inhibitors, 3-MA (5 mm) or BafA1 (100 nm) for 1 h or not, followed by infection with SeV (20 HAU/ml) or EMCV (MOI = 0.01) for indicated times. Control samples were treated with DMSO (vehicle). Viral titers for EMCV were determined by plaque assay and copy numbers of SeV genomic RNA strands were determined in supernatants by real-time RT-PCR as described under “Experimental Procedures.” Fold increase in viral yields in WT and RNase L KO MEFs treated with either 3-MA (5 mm) or Bafilomycin A1 (100 nm) were compared with control samples. Data represent mean ± S.E. for two independent experiments performed in triplicate. Student's t test was used to determine p values of WT MEFs compared with identically treated RNase L KO MEFs. #, not significant; *, p < 0.05; **, p < 0.001.
FIGURE 9.
FIGURE 9.
Autophagy induced by RNase L is independent of paracrine effects of IFN. A, STAT1-signaling defective U3A cells or type1 IFN receptor defective IFNAR KO MEFs were transfected with 10 μm of 2–5A for indicated times. Cell lysates were analyzed for autophagic markers by monitoring increased lipidation of LC3-II and p62 degradation which was normalized to β-actin levels. B and C, ratios of LC3-II/β-actin or p62/β-actin corresponding to the time points in A from three experiments shown as mean ± S.E. D, U3A cells were transfected with GFP-LC3 and 24 h later with 2–5A. Cells were pretreated optionally with PKR inhibitor (2-aminopurine, 5 mm) or JNK inhibitor (SP600125, 25 μm) for 1 h prior to 2–5A transfection (up to 30 h) and inhibitors were added back. The percentage of GFP+ cells showing puncta formation compared with mock-treated cells is shown. Results shown represent mean ± S.E. for three experiments, and at least 100 cells were analyzed per assay. E, activation of JNK and PKR in response to 2–5A transfection. Western blot analysis of phospho-JNK (T184, Y185), and phospho-PKR (T451) in response to 2–5A transfection (10 μm) for indicated times compared with levels of total JNK and total PKR. Activation of PKR was correlated with phospho-eIF2α. Protein loading was normalized to β-actin levels.
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