Nonstructural proteins 2C and 3D are involved in autophagy as induced by the encephalomyocarditis virus
© Hou et al.; licensee BioMed Central Ltd. 2014
Received: 20 April 2014
Accepted: 26 August 2014
Published: 1 September 2014
Encephalomyocarditis virus (EMCV) can infect a variety of animal species and humans. Although the EMCV infection is known to induce autophagy to promote its replication in host cells, the viral proteins that are responsible for inducing autophagy are unknown.
The recombinant plasmids that were expressing the EMCV proteins were constructed to analyze the role of each protein in the induction of autophagy. Autophagy inductions by the EMCV proteins in BHK-21 cells were investigated by confocal microscopy, Western blotting and transmission electron microscopy. ER stress in BHK-21 cells was examined by detecting the marker molecules using western blotting and luciferase assays.
This study presents the first demonstration that the nonstructural proteins 2C or 3D of EMCV were involved in inducing autophagy in BHK-21 cells that were expressing 2C or 3D, and we found that inhibiting Beclin1 expression influenced this autophagy induction process. Next, 2C and 3D were shown to be involved in inducing autophagy by activating the ER stress pathway. Finally, EMCV 2C or 3D were demonstrated to regulate the proteins associated with PERK and ATF6alpha pathway.
Our findings indicate that 2C and 3D are involved in EMCV-induced autophagy by activating ER stress molecules and regulating the proteins expression associated with UPR pathway, helping to better understand the EMCV-induced autophagy process.
KeywordsEncephalomyocarditis virus (EMCV) Nonstructural protein 2C 3D Autophagy Endoplasmic reticulum (ER) stress Unfolded protein response (UPR)
Encephalomyocarditis virus (EMCV) belongs to the Cardiovirus genus of the Picornaviridae family . This virus has a wide host-range among domestic and wild animals [2–4]. Out of all the domestic animals, pigs are considered the most commonly and severely EMCV-infected animals . EMCV is not only an important pathogen in animal husbandry, but it also has potential public health significance . Therefore, understanding the specific interaction between EMCV and hosts/cells is required for the effective treatment and control of this infection.
EMCV is a nonenveloped, single-stranded, positive-sense RNA virus. The genome is approximately 7.8-kb long with a single open reading frame (ORF) that is translated into a polyprotein precursor . This precursor is proteolytically processed into structural proteins (VP1, VP2, VP3 and VP4), primarily forming the viral nucleocapsid and nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D) along with several protein intermediates that are needed for viral replication . Although the roles of EMCV proteins have been widely investigated, a further exploration of the virus-host interaction and important cellular components in the EMCV life cycle is essential to determine a control strategy for EMCV infection.
The endoplasmic reticulum is one origin of the membranes that generate autophagosomes [9, 10]. The endoplasmic reticulum is also a multifunctional organelle in eukaryotic cells, which provides a unique compartment for posttranslational modifications, folding, and the oligomerization of newly synthesized membrane and secreted proteins. However, several endogenous imbalances in cells often contribute to ER malfunction known as ER stress . In response to ER stress, a coordinated adaptive program called the unfolded protein response (UPR) is activated and serves to minimize the accumulation and aggregation of misfolded or over-expressed proteins by increasing the capacity of the ER machinery to fold correctly and degrade aberrant proteins. To date, three ER stress sensors, namely IRE1 (inositol-requiring enzyme 1), ATF6α (activating transcription factor 6α), and PERK (PKR-like ER protein kinase), have been identified in mammals for their ability to achieve different cellular adaptations .
Autophagy is a dynamic, conserved intracellular process that involves the formation of a characteristic double- or single-membrane structure (autophagosomes and autolysosomes, respectively), which delivers misfolded or long-lived cytoplasmic proteins and damaged or obsolete organelles to lysosomes for digestion and recycling [10, 13–16]. Autophagy not only plays an important role in cellular homeostasis, but it also acts as a cellular response to stress such as pathogen infection [17–19]. Some RNA viruses may subvert the defensive function of autophagy and use the autophagic double- or single-membrane vesicles to facilitate their own replication [20–22].
In recent years, a lot of attention has been paid to the relation between autophagy and viral infection [23, 24]. Our previous study indicated that EMCV infection can induce autophagy in host cells, and it is able to facilitate viral replication . Thus, to address which protein(s) in EMCV is (are) involved in autophagy induction is necessary to understand the interaction between autophagy and viral infection. In this study, we firstly demonstrated that autophagy is induced in EMCV 2C or 3D protein-expressing cells by monitoring the presence of autophagosome-like vesicles and modifying LC3, the mammalian Atg8 known as homolog microtubule-associated protein light chain 3. Moreover, we observed the interference effect of Beclin1 on autophagy induction with target-specific siRNA. In addition, we also explored changes in the ER stress molecules and UPR pathway associated proteins in 2C- or 3D-overexpressing cells.
The recombinant plasmids expressed EMCV proteins
To identify the EMCV proteins involved in autophagy induction, we first amplified the VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C and 3D genes of the EMCV BJC3, GST or LC3 genes by RT-PCR or PCR and constructed corresponding recombinant plasmids with HA tags or GFP tags. The amplified product for each gene was consistent with the size as expected (Additional file 1: Figure S1A). The expression of each EMCV protein by the recombinant plasmids was examined in BHK-21 cells through transfection and Western blotting analysis. The EMCV proteins were expressed effectively except for the 3B gene (Additional file 1: Figure S1B). The transfected BHK-21 cells were simultaneously used to analyze subsequently the expression levels of LC3-I, LC3-II and p62.
Puncta accumulated in the BHK-21 cells that were co-transfected with the EMCV protein-expressing plasmid and pEGFP-LC3
Nonstructural proteins 2C and 3D increased the autophagic activity and the formation of autophagosome-like vesicles
To determine further whether autophagy is triggered in the BHK-21 cells that were transfected with a 2C- or 3D-expressing plasmid, TEM was performed to provide an ultrastructural analysis of the transfected cells. Previous studies have shown that the VP1 of EMCV can colocalize with LC3 during viral infection . Our present finding showed that GFP-LC3-positive puncta could be simultaneously induced and colocalized with VP1 in the BHK-21 cells that were transfected with pCMV-HA-VP1 (Figure 1B). Thus, in our study, the BHK-21 cells expressing EMCV VP1 that did not induce autophagy were used as a control to analyze the autophagosome-like vesicle formation ability as induced by 2C or 3D. As shown in Figure 2B, the BHK-21 cells that were transfected with the 2C- or 3D-expressing plasmid had significantly increased double- or single-membrane vesicles in the cytoplasm in comparison with the cells that were transfected with the pCMV-HA-VP1 plasmid (as a control of viral protein that did not induce autophagy) or pCMV-HA plasmid (as a control) and mock-transfected cells; meanwhile, the recognizable cytoplasmic contents or degraded organelles seemed to be sequestered in most of the well-defined vesicles with morphologically typical autophagic vacuoles within the cells. Further quantitative analyses indicated that there was a significant increase in the number of autophagosome-like vesicles in the cytoplasm of the cells transfected with 2C- or 3D-expressing plasmid (Figure 2C).
To verify the possibility that LC3 modification was caused by autophagic signalling instead of reflecting an EMCV 2C- or 3D-induced membrane alteration, the knockdown effect of Beclin1 (one of the crucial factors for autophagosome formation) on the induced LC3 and p62 levels was assessed . We used specific siRNA to silence the Beclin1 gene in BHK-21 cells. The cells treated with specifically targeted Beclin1 siRNA showed a dose-dependent reduction in the Beclin1 protein level (Figure 2D), and 40 pmol of siRNA was then used for further study. The increased levels of LC3-II bands and degraded p62 levels in response to 2C or 3D over-expression were significantly inhibited upon Beclin1 silencing when compared with the control groups (Figure 2E), indicating that EMCV 2C and 3D can induce autophagy signalling.
Taken together, we found that EMCV 2C or 3D protein exhibited clear abilities to induce the conversion of LC3, degrade p62 and increase the number of autophagosome-like vesicles in BHK-21 cells in comparison with control cells expressing other proteins. Thus, our further studies of autophagy mechanisms primarily focused on analyzing 2C and 3D proteins.
ER Stress was induced in EMCV-infected or EMCV 2C- or 3D-expressing BHK 21 cells
Regulating the UPR pathway in the EMCV-infected or EMCV 2C- or 3D-expressing BHK 21 cells
To assess whether EMCV and 2C or 3D protein also induces the IRE1-XBP1 pathway to trigger UPR, we analyzed XBP1 mRNA splicing in BHK-21 cells that were infected with EMCV or were overexpressing 2C or 3D proteins. The XBP1 cDNA was amplified by RT-PCR and digested by PstI, for which there is a restriction site located within the 26-nt region of XBP1 cDNA as removed by IRE1-mediated splicing, as previously described [30, 35] (Figure 4B). Tunicamycin (Tu), an inducer of XBP1 splicing (XBP1s), served as a positive control . We noticed that Tu-treated cells appeared to express high XBP1s, as evidenced by the resistance of the spliced product to PstI digestion. When compared with BHK-21 cells infected with EMCV and mock-infected cells, no obvious differences were observed in the levels of XBP1, which was subtly spliced, indicating that EMCV infection is not able to significantly activate the IRE1-XBP1 pathway substantially through splicing XBP1. Although 2C or 3D proteins could cause low levels of XBP1s in BHK-21 cells tranfected with pCMV-HA-2C and pCMV-HA-3D, no significant differences were shown between the cells expressing 2C or 3D protein and the control cells (Figure 4C), indicating that the overexpression of 2C or 3D protein cannot induce the activation of the IRE1-XBP1 pathway.
ATF6a is cleaved by trans-membrane proteases and translocated to the nucleus, where it activates the genes responsible for the ER stress response [36, 37]. ATF6a cleavage was analysed in BHK-21 cells to assess whether EMCV infection or the expression of EMCV 2C or 3D protein activate the ATF6a pathway. As shown in Figure 4D, an intact form of ATF6a (90 kDa) was detected in the control cells, and in EMCV-infected or 2C- or 3D-expressing BHK-21 cells, a lower level of its intact form was uncovered by the Western blotting assay.
Viruses have developed complex mechanisms for manipulating normal cellular pathways to facilitate viral replication and to evade host defence mechanisms. Recently, several studies have revealed that cellular autophagy is involved in various pathogenic infections and plays a crucial role in these processes [5, 7, 38]. During the infection process, viruses have been shown to employ the autophagic machinery to replicate and survive [39–41]. Further studies have shown that some viruses can induce autophagy by using their proteins, such as the NSP4 of rotavirus , the NS4B of hepatitis C virus , nonstructural protein p17 of avian reovirus  and matrix protein 2 of influenza A virus . Although our previous studies showed that EMCV infection can induce the autophagy process , more detailed evidence concerning the EMCV protein(s) involved in virus-induced autophagy remain unknown.
In the present study, we found that the BHK-21 cells that were co-expressing VP1, VP3, VP4, 2B, 2C, 3A or 3D and GFP-LC3 displayed a number of positive puncta in the cytoplasm and these EMCV proteins colocalized with a subset of GFP-LC3 puncta respectively (Figure 1B, 1D, 1E, 1G, 1H, 1I, and 1L), whereas the cells that were expressing other EMCV proteins (VP2, 2A and 3C), the irrelevant protein (GST) and the cell control transfected with the pCMV-HA plasmid exhibited no positive puncta (Figure 1C, 1F, 1K, 1M and 1N). In principle, the formation of GFP-LC3 puncta accumulations represents a component of the autophagy process. However, the formation of ubiquitinated GFP-LC3 positive protein accumulations may be triggered and does not completely imply either the induction of autophagy (or autophagosome formation), or autophagic flux through the system . Therefore, to rule out this possibility, we also detected the LC3 modification and autophagic vesicles by Western blotting analysis and transmission electron microscopy to analyze the activation of the autophagy process (Figure 2A-C). Western blotting analysis showed that the increased level of LC3-II, the reduced expression of p62, the enhanced ratio of LC3-IIto β-actin and the degradated ratio of p62 to β-actin represented a significant increase in the autophagy level of 2C- or 3D-overexpressed cells, whereas no similar results were shown in other viral proteins. The number of autophagosome-like vesicles with various sizes significantly increased in 2C- or 3D-overexpressing cells in comparison with the control cells by transmission electron microscopy. The size difference in autophagosome-like vesicles most likely implied that these vesicles contained different cytoplasmic contents, such as obsolete organelles and cytoplasmic proteins.
To verify that the LC3 modification phenomenon was caused by autophagic signalling instead of 2C- or 3D- induced membrane alterations, we employed a specific siRNA targeting the autophagy-critical gene required for autophagosome formation. Disrupting the class III phosphatidyl inositol 3-kinase (PI3K) signalling complex that was required for autophagosome formation by Beclin1 siRNA clearly reduced the induction level of LC3-II and the degradation of p62 in 2C- or 3D-transfected cells (Figure 2D-E), further demonstrating that EMCV 2C or 3D induced autophagy through the variation in autophagic vesicle formation. A previous study indicated that the 2BC and 3A proteins of poliovirus are responsible for inducing autophagy . Thus, we deduced that these differences in induction ability are most likely explained by the individual expression or overexpression level of various viral proteins in cells or the significant differences between nonstructural proteins encoded by different picornaviruses . Based on our findings, we primarily engaged in further research of autophagy mechanisms to focus on EMCV 2C and 3D proteins.
The endoplasmic reticulum (ER) system, which is a major site for the synthesis and control of the membrane or secreted protein quality, is a primary compartment of signal initiation and transduction for responding to a variety of stimuli, including viral infections [35, 47]. Although studies showed that many picornaviruses induce autophagy by activating ER stress [29, 30], a question remains as to whether the underlying mechanisms of EMCV and 2C or 3D protein-induced autophagy are related to ER stress. Thus, the activation of the ER stress pathway was analyzed in BHK-21 cells infected with EMCV, expressed 2C or 3D protein or treated with Tg (as a positive control) (Figure 3). The results showed that the marker molecules of the ER stress pathway were activated in these BHK-21 cells, indicating that EMCV 2C and 3D proteins are potent ER stress inducers in BHK-21 cells and play an important role in EMCV-activated ER stress. The two proteins could trigger the activation of ER stress marker molecules not only via up-regulation at the transcriptional level but also by activation at the translational level. Interestingly, we found that, ER stress was activated and accompanied by the up-regulation of the autophagy level through an enhanced conversion of LC3 in BHK-21 cells treated with Tg, and we therefore deduced that EMCV 2C or 3D protein also induce autophagy by activating the ER stress.
A number of viruses have been shown to induce ER stress during viral infection. Cells respond to ER stress by activation of the UPR pathway to maintain homeostasis of the ER. However, the pattern of molecular interactions that occurs within the UPR pathway differs. This finding depends on the viral identity and type of host cell. Many viruses clearly induce all the UPR pathways, and some viral infections activate the partial pathway [29, 30, 48]. A detailed analysis of the UPR pathway was undertaken in our study to understand this case (Figure 4). Our results showed that EMCV infection and 2C- or 3D-protein expression could phosphorylate the associated molecules of the PERK pathway and cleave the intact form of ATF6a (90 kDa), but they could not splice XBP1 mRNA, indicating that the activation of the PERK pathway and the ATF6a pathway can transiently block mostly protein translation and regulate the transcription of the ER chaperon against ER malfunction. These pathways are specifically induced to trigger autophagy initiation [49–51]. Moreover, many genes were regulated by activation of the UPR pathway at the transcriptional and translational levels and were involved in recovering from ER stress , which may be available for EMCV replication. Although the exact contributions of the host and virus to UPR induction remain unclear, we showed that EMCV 2C or 3D protein induced autophagy by initiating the PERK and ATF6a pathways in response to ER stress, which finally benefits this cellular event and viral replication.
Although the molecular mechanisms regulating autophagic signal pathways to facilitate viral survival and replication by viral proteins are required to be further explored, a growing number of studies have demonstrated that the interactions between viral proteins and the cellular proteins associated with autophagy play important roles in regulating autophagy process. Foot and mouth disease virus (FMDV) nonstructural protein 2C binds to Beclin1, a central regulator of the autophagy pathway, to prevent the fusion of lysosomes to autophagosomes, thereby allowing for viral survival . Additionally, a study showed that the HCV NS4B protein can induce autophagy and promote viral replication through recruiting the Rab5 and Vps34 complex . Thus, analyzing the interaction between viral proteins and autophagy-associated proteins of host cells is worthy for better evaluating the role of autophagy in relation to EMCV infection.
Taken as a whole, our findings are the first to indicate that nonstructural proteins 2C and 3D are involved in autophagy as induced by EMCV. The fact that Beclin1 knockdown inhibited the conversion of LC3 and degradation of p62 further demonstrated 2C or 3D-inducing functions. Additionally, the 2C- or 3D-induced autophagy was shown to be associated with endoplasmic reticulum (ER) stress pathway via regulating the UPR protein expression. Our present studies help to better understand the EMCV-induced autophagy process.
Materials and methods
Cells, virus and plasmids
BHK-21 cells were originally obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 100 U/ml of penicillin G, and 100 g/ml streptomycin at 37°C in a humidified 5% CO2 incubator. An EMCV strain (BJC3) that was isolated in our laboratory was used in this study . Plasmids pEGFP-C1 and pCMV-HA were purchased from Clontech (Mountain View, CA, USA), and plasmids pGL3-Basic and pRL-TK were purchased from Promega (Madison, WI, USA).
Antibodies and reagents
The following antibodies were used: anti-GRP78, anti-GRP94, anti-ATF6, anti-PERK, anti-phospho-PERK, anti-eIF2α, and rabbit anti-phospho-eIF2α antibody (Cell Signal Technology, Inc., Danvers, MA, USA). The rabbit antibodies against LC3 and p62 and a mouse monoclonal antibody against HA and β-actin, the secondary antibodies tetramethyl rhodamine isothiocyanate TRITC-conjugated goat anti-mouse and horseradish peroxidase conjugated goat anti-mouse or anti-rabbit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Beclin1 rabbit antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-VP1 mAb was prepared in our laboratory. Tunicamycin (Tu) and thapsigargin (Tg) were purchased from Sigma-Aldrich. RIPA lysis buffer was obtained from Beyotime (Jiangsu, China). Lipofectamine LTX and PLUS and RNAiMAX reagent were purchased from Invitrogen (Auckland, NY, USA).
RNA preparation and RT-PCR analysis
Total RNA from cultured cells was isolated with an RNeasy Mini Kit (Qiagen, Hilden, Germany) by following the manufacturer’s protocol. First-strand cDNAs were reverse-transcribed (RT) with 2 μg of RNA as the template, and the target genes were PCR-amplified by following the procedures described in the one-step RT-PCR kit (Qiagen) according to the manufacturer’s recommendations.
The XBP1 and β-actin genes were amplified by RT-PCR with the specific primers listed in Additional file 1: Table S1, and the primers were designed according to the XBP1 and Actin sequences available in the GenBank database (accession no. NM_001244047 and no. XM_006176094, respectively).
Constructing the expression plasmids for LC3, GST and EMCV genes
The open reading frame fragment of the Atg8 homolog known as the microtubule-associated protein light chain 3 (LC3) gene from BHK-21 cells was amplified by RT-PCR, and the specific primers were designed in accordance with the Atg8 gene sequence in the GenBank database (accession no. XM_003495104) (Additional file 1: Table S1). The amplified cDNA was then subcloned into pEGFP-C1. Each gene of the EMCV protein was amplified by RT-PCR with the primers listed in Additional file 1: Table S1 and cloned into pCMV-HA to generate the following expression plasmids: pCMV-HA-VP1, pCMV-HA-VP2, pCMV-HA-VP3, pCMV-HA-VP4, pCMV-HA-2A, pCMV-HA-2B, pCMV-HA-2C, pCMV-HA-3A, pCMV-HA-3B, pCMV-HA-3C and pCMV-HA-3D. The glutathione S-transferase (GST) gene was amplified by PCR from cloning vector pGEX-6P-1 (accession no. U78872) with the specific primers listed in Additional file 1: Table S1 and then cloned into pCMV-HA to generate recombinant plasmid pCMV-HA-GST. The pGL3-GRP78-luc, pGL3-GRP94-luc, pGL3-calreticulin-luc, pGL3-ATF4-luc, pGL3-CHOP-luc-carrying mouse GRP78, GRP94, calreticulin, ATF4 and CHOP promoters were cloned into the pGL3-basic vector . The plasmids were sequenced to confirm that each amplified product had no errors introduced as a result of PCR amplification.
In accordance with the requirements of different experiments, the BHK-21 cells were infected with either EMCV BJC3, or they were mock-infected with phosphate-buffered saline (PBS). Following a 1 h absorption period, unattached viruses were removed by aspiration. The cells were then washed thrice with PBS and cultured in complete medium at 37°C for the indicated time points until different samples had been harvested for further experiments.
A whole cell lysate of BHK-21 cells was prepared at the indicated time points after transfecting with the RIPA lysis buffer according to the manufacturer’s protocol. The supernatant was stored at −80°C for western blotting. Twenty micrograms of each extract was subjected to electrophoresis on an SDS-polyacrylamide gel and transferred onto an Immobilon-PSQ transfer membrane (Millipore, Billerica, MA, USA). The membrane was probed with the indicated primary antibodies and appropriate secondary antibodies, which were detected by using a chemiluminescence detection kit (Thermo Scientific, Inc., Waltham, MA, USA) and then exposed to a chemiluminescence apparatus (Proteinsimple, Santa Clara, CA, USA).
BHK-21 cells were grown to 70-80% confluence in 24-well culture plates (Corning Inc., NY) and transfected with firefly luciferase reporter vectors along with the internal control Renilla luciferase reporter construct, pRL-TK (firefly luciferase reporter construct and pRL-TK in a ratio of 20:1 for BHK-21 cells) and the indicated EMCV protein recombinant plasmids. Forty-eight hours after the transfection, the cells were harvested and assayed for luciferase activity by using the Dual-Luciferase assay system according to the manufacturer’s instructions from Promega (E1910).
BHK-21 cells that had grown to approximately 70-80% confluence in 24-well culture plates were co-transfected with each EMCV protein-expressing vector, GST protein-expressing plasmid (as a control) and pEGFP-LC3 for 48 h, or they were infected with EMCV after transfecting with pEGFP-LC3 plasmid for 12 h. The cells were fixed with pre-cooled 4% paraformaldehyde at the indicated times. The cells were then washed three times with PBS (pH 7.4), and a mouse monoclonal antibody against HA was allowed to incubate with the cells for 2 h at 37°C. The cells were washed three times with PBS and then incubated for 2 h at 37°C with a 1:200 dilution of goat anti-mouse secondary antibodies conjugated to TRITC. Finally, the cells were washed with PBS and directly visualized under a Nikon TE-2000E confocal immunofluorescence microscope (Nikon Instruments, Inc., Melville, NY, USA).
Transmission electron microscopy (TEM)
Sub-confluent monolayers of BHK-21 cells grown on 6-well culture plates (Corning Inc.) were transfected with pCMV-HA-2C, pCMV-HA-3D, pCMV-HA-VP1 or pCMV-HA and mock-transfected cells for 48 h. The cell samples were then processed as previously described . Ultrathin sections were prepared, examined and imaged under a Hitachi H-7500 transmission electron microscope (Hitachi Ltd; Japan).
RNA interference (RNAi) knockdown of Beclin1
siRNAs designed by the GenePharma Company (Shanghai, China) were used to knock down Beclin1 in BHK-21 cells. The siRNA sequences included siBeclin1 (sense, 5′-CGGGAAUACAGUGAAUUUATT-3′; antisense, 5′-UAAAUUCACUGUAUUCCCGTT-3′) and negative control siRNA (as a control) (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′). The BHK-21 cells were dissociated and mixed with different concentrations of siRNA by using Lipofectamine RNAiMAX reagent as recommended by the manufacturer’s protocol. The cells were harvested for further analysis after 48 h.
The data were expressed as means ± standard deviations or standard errors as indicated. All statistical analyses were performed by Student’s t-test, and a p < 0.05 was considered to be statistically significant
This work was supported by the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Ministry of Agriculture of the People’s Republic of China.
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