Interactome Analysis of the NS1 Protein Encoded by Influenza A H1N1

Apr 20, 2016 - ... Surgery, Chang Gung Memorial Hospital, Linkou 33305, Taiwan .... and titrated by plaque formation assays using monolayers of MDCK c...
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Interactome Analysis of the NS1 Protein Encoded by Influenza A H1N1 Virus Reveals a Positive Regulatory Role of Host Protein PRP19 in Viral Replication Rei-Lin Kuo,*,†,‡,§,⊥ Zong-Hua Li,† Li-Hsin Li,† Kuo-Ming Lee,† Ee-Hong Tam,† Helene M. Liu,∥ Hao-Ping Liu,□ Shin-Ru Shih,†,‡,§,¶ and Chih-Ching Wu*,†,§,# †

Department of Medical Biotechnology and Laboratory Science, College of Medicine, ‡Research Center for Emerging Viral Infections, College of Medicine, and §Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan ⊥ Department of Pediatrics, ¶Clinical Virology Laboratory, and #Department of Otolaryngology-Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou 33305, Taiwan ∥ Department of Clinical Laboratory Sciences and Medical Technology, College of Medicine, National Taiwan University, Taipei 10617, Taiwan □ Department of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan S Supporting Information *

ABSTRACT: Influenza A virus, which can cause severe respiratory illnesses in infected individuals, is responsible for worldwide human pandemics. The NS1 protein encoded by this virus plays a crucial role in regulating the host antiviral response through various mechanisms. In addition, it has been reported that NS1 can modulate cellular pre-mRNA splicing events. To investigate the biological processes potentially affected by the NS1 protein in host cells, NS1associated protein complexes in human cells were identified using coimmunoprecipitation combined with GeLC−MS/MS. By employing software to build biological process and protein−protein interaction networks, NS1-interacting cellular proteins were found to be related to RNA splicing/processing, cell cycle, and protein folding/targeting cellular processes. By monitoring spliced and unspliced RNAs of a reporter plasmid, we further validated that NS1 can interfere with cellular pre-mRNA splicing. One of the identified proteins, pre-mRNAprocessing factor 19 (PRP19), was confirmed to interact with the NS1 protein in influenza A virus-infected cells. Importantly, depletion of PRP19 in host cells reduced replication of influenza A virus. In summary, the interactome of influenza A virus NS1 in host cells was comprehensively profiled, and our findings reveal a novel regulatory role for PRP19 in viral replication. KEYWORDS: influenza A virus, NS1, interactome, PRP19, RNA splicing/processing

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INTRODUCTION Influenza A virus is an important pathogen that can cause severe illness and death in mammals and avians. The ability of the virus to be transmitted among natural reservoirs, such as aquatic birds, and to humans occasionally has resulted in pandemics in human populations.1,2 The virus belongs to the Orthomyxovirus family, which contains a segmented RNA genome in an enveloped viral particle. On the basis of the combination of the viral envelope proteins hemagglutinin (HA) and neuraminidase (NA), the virus has been divided into subtypes such as H1N1 and H3N2. In addition, an H7N9 subtype of avian influenza A virus has recently been identified as capable of infecting humans and resulting in severe disease and death.3−5 Several studies have applied genome-wide RNAi library screening to systemically explore the host factors involved in influenza A virus replication.6−8 In addition, the yeast twohybrid system has been employed to investigate the host© 2016 American Chemical Society

influenza A virus interaction. These approaches have revealed many host proteins as interacting with polymerase subunits or the nucleoprotein (NP) of influenza A virus.9−16 Immunoprecipitation of influenza viral proteins combined with mass spectrometry has also facilitated the search for host factors that may regulate viral replication.17,18 The host proteins identified using these approaches may not only help to characterize replication of influenza virus and its pathogenesis, but also may be applied to develop antivirals for treating influenza A virus infection.19 Nonstructural protein 1 (NS1) of influenza A virus is a multifunctional protein that can suppress host innate antiviral responses to establish virus replication in infected cells.20 Moreover, it has been found that the protein can also regulate the maturation steps of cellular mRNA such as polyadenylation Received: February 3, 2016 Published: April 20, 2016 1639

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Journal of Proteome Research and splicing.21−23 On the basis of its properties and structure, the NS1 protein is divided in two domains, RNA-binding and effector domains, each of which can impact the regulation of biological processes in host cells by binding to specific cellular factors.24 Previously, an interactome analysis using the yeast two-hybrid approach identified 51 cellular interactors of the NS1/NS2 proteins encoded by nine different strains of influenza A virus.25 Subsequently, a recombinant influenza A virus expressing epitope-tagged NS1/NS2 proteins was generated for identifying host factors that interact with these viral proteins in infected cells.26 The study has also identified interferon-induced protein kinase (PACT) as a factor interacting with NS1 and involved in replication of the influenza A virus.26 However, in the approaches used in previous studies, the NS2 protein and its interacting proteins were copurified. Thus, the factors that specifically bound to NS1 could not be systemically screened. Although NS1 has been reported to interact with numerous cellular molecules implicated in multiple host response,25,26 its functions and the mechanism underlying NS1-mediated regulation of cellular mRNA processing have not been fully described to date. To address this, we systemically explored the NS1 interactome in NS1-expressing cells using immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with liquid chromatography−tandem mass spectrometry (GeLC−MS/MS). The cellular proteins identified were found to be involved in the cellular processes of RNA splicing/processing, cell cycle, and protein folding/ targeting. Importantly, one of the identified proteins, premRNA-processing factor 19 (PRP19; encoded by the PRPF19 gene), was confirmed to interact with the NS1 protein in influenza A virus-infected cells, and depletion of cellular PRP19 decreased influenza A viral replication. This study comprehensively profiled the interactome of the NS1 protein in host cells, and the results demonstrate a novel regulatory role for PRP19 in replication of influenza A virus.

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subjected to immunoprecipitation using anti-FLAG M2 affinity gel (Cat. No. A2220, Sigma-Aldrich) following the manufacturer’s instructions. For immunoblotting, the cell extracts and the cellular proteins copurifying with the 3xFLAG NS1 protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were then probed with antiNS1, anti-FLAG-M2 (Cat. No. F1804, Sigma-Aldrich), anti-βactin (Cat. No. A5441, Sigma-Aldrich), anti-c-Myc (Cat. No. M4439, Sigma-Aldrich), or anti-PRPF19 (Cat. No. GTX106952, GeneTex, USA or Cat. No. SAB4501215, Sigma-Aldrich) antibodies. To examine the NS1-PRP19 interaction during influenza A virus infection, 293T cells were transfected with a plasmid expressing Myc-tagged PRP19 for 24 h and then infected with the PR8 virus at an MOI of 2. At 12 h postinfection, cell lysates were collected and incubated with an anti-Myc antibody (Cat. No. M4439, Sigma-Aldrich) at 4 °C for 14 h. After mixing and incubating with Protein A and Protein G Sepharose Fast Flow beads (GE Healthcare Life Sciences, NJ, USA) at 4 °C for 3 h, the immunoprecipitated products were eluted with SDS-PAGE sample buffer and analyzed by immunoblotting as described above. SDS-PAGE and In-Gel Protein Digestion

Proteins immunoprecipitated from 3xFLAG NS1-expressing cells were separated by 10% SDS-PAGE and stained using a Colloidal Blue Staining kit (Thermo Fisher Scientific, NY, USA). The stained gel lanes were cut into 20 slices and subjected to in-gel tryptic digestion as described previously.28,29 Briefly, gel pieces were destained in 10% methanol (Mallinckrodt Baker, NJ, USA), dehydrated in acetonitrile (ACN; Mallinckrodt Baker), and dried using a SpeedVac. The gel pieces were treated with 25 mM NH4HCO3 containing 10 mM dithiothreitol (Biosynth AG, Switzerland) at 60 °C for 30 min, followed by alkylation with 55 mM iodoacetamide (Amersham Biosciences, UK) at room temperature for 30 min. The proteins were then digested using sequencing-grade modified porcine trypsin (20 μg/mL; Promega, WI, USA) at 37 °C for 16 h. Peptides were extracted with ACN and dried using a SpeedVac.

MATERIALS AND METHODS

Cells, Virus, and Plasmids

Reverse-Phase LC−MS/MS Analysis

HEK293T (293T; ATCC CRL-3216), Madin Darby Canine Kidney (MDCK; ATCC PTA-6500), and A549 (ATCC CCL185) cell lines were cultivated in DMEM with 10% FBS. Influenza A virus Puerto Rico/1934/H1N1 strain (PR8) was amplified with 10-day-old fertilized eggs and titrated by plaque formation assays using monolayers of MDCK cells. Viral replication was determined by infection of A549 cells at a multiplicity of infection (MOI) of 0.001 for 24 and 48 h. A plasmid-expressed NS1 protein with a 3xFLAG was constructed by cloning the PR8 NS1 open reading frame into p3xFLAGMyc-CMV 26 (Sigma-Aldrich, St. Louis, MO, USA). To generate a human PRP19-expressing plasmid, the human PRP19 cDNA was generated by reverse transcription and PCR using total RNA from 293T cells and inserted into the pcDNA3.1-Myc-His A vector. The pSV40-CAT(In1) plasmid was provided by Dr. Woan-Yuh Tarn (Academia Sinica, Taiwan).27

Protein identification was performed as described previously.29,30 Briefly, each peptide mixture was reconstituted in HPLC buffer A (0.1% formic acid; Sigma-Aldrich), loaded onto a trap column (Zorbax 300SB-C18, 0.3 × 5 mm; Agilent Technologies, Taiwan) at a flow rate of 0.2 μL/min in HPLC buffer A, and separated on a resolving 10 cm analytical C18 column (inner diameter, 75 μm) with a 15-μm tip (New Objective, MA, USA). Using a flow rate of 0.25 μL/min across the analytical column, the peptides were eluted using a linear gradient of 0−10% HPLC buffer B (99.9% ACN containing 0.1% formic acid) for 3 min, 10−30% buffer B for 35 min, 30− 35% buffer B for 4 min, 35−50% buffer B for 1 min, 50−95% buffer B for 1 min, and 95% buffer B for 8 min. The LC apparatus was coupled online with a twodimensional linear ion trap mass spectrometer (LTQ-Orbitrap Discovery, Thermo Fisher Scientific) managed using the Xcalibur 2.0 software package (Thermo Fisher Scientific). An electrospray voltage of 1.8 kV was applied. Intact peptides were detected by the Orbitrap at a resolution of 30 000. The ion signal of (Si(CH3)2O)6H+ at m/z 445.120025 was used as an internal standard for mass lock. For MS analysis, we used a data-dependent acquisition mode that alternated between one MS scan and six MS/MS scans for the six most abundant

Immunoprecipitation, Immunoblotting, and Antibodies

For NS1 immunoprecipitation, 293T cells were transfected with the plasmids expressing 3xFLAG-tagged NS1 or control vectors, which express a peptide of MDYKDHDGDYKDHDIDYKDDDDKLAAANSSIDLISVPVDSREQKLISEEDL with a 3xFLAG tag. Extracts from 293T cells were then 1640

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Journal of Proteome Research precursor ions. For MS scans, the m/z scan range was set to 350−2000 Da. The m/z values selected for MS/MS scans were dynamically excluded for 3 min, and 5 × 104 ions were accumulated and resolved in the ion trap to generate MS/MS spectra. Both MS and MS/MS spectra were acquired using one microscan with maximum fill times of 1000 and 100 ms for MS and MS/MS analyses, respectively. Automatic gain control was applied to prevent overfilling of the ion trap.

RNA transcribed from the reporter plasmid, total RNA from the transfected cells was collected and subjected to reverse transcription (RT) and quantitative polymerase chain reaction (qPCR). As in the previously described method,17 DNase Itreated total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) with the oligo-dT primer. cDNA was then amplified by qPCR using SYBR Green reagent with a reverse primer complementary to the CAT(In1) sequence, 5′-GTATTCACTCCAGAGCGATG-3′, and a CAT(In1)-exon forward primer, 5′-CCAGACCGTTCAGCTGGATATT-3′, for spliced mRNA or with the reverse primer and a CAT(In1)-intron forward primer, 5′ATTGGTCTATTTTCCCACCCTTAG-3′, for the unspliced transcript. Regular PCR was performed with SuperRed PCR Master Mix (Biotools, Taiwan) and primers (forward: 5′TTTTGGAGGCCTAGGCTTTT-3′; reverse: 5′GTATTCACTCCAGAGCGATG-3′), and the products were examined by agarose gel electrophoresis and quantitated by ImageJ analysis.

Protein Database Searching for Protein Identification

For database searching, the obtained MS/MS spectra were analyzed using the Mascot algorithm (version 2.1, Matrix Science, MA, USA) against the Swiss-Prot human sequence database (released Apr 16, 2014, selected for Homo sapiens, 20 265 entries) of the European Bioinformatics Institute. The mass tolerances of the fragment and parent ions were set to 0.5 Da and 10 ppm, with trypsin as the digestion enzyme. Up to one missed cleavage was permitted, and searches were performed with the parameters of variable oxidation on methionine (+15.99 Da) and fixed carbamidomethylation on cysteine (+57 Da). A random sequence database was used to estimate false-positive rates for peptide matches. After Mascot searching, the obtained files were processed using Scaffold software (version 3.6.5; Proteome Software, OR, USA), which includes the PeptideProphet program to assist in the assignment of peptide MS spectra and the ProteinProphet program for assigning/grouping peptides to a unique protein/ protein family when they are shared among several isoforms. We used PeptideProphet and ProteinProphet probabilities ≥ 0.95 to ensure an overall false-discovery rate below 0.5%. Only proteins with two or more identified peptides were retained in this study.

Inhibition of PRP19 Expression by Small Interfering RNA and Determination of Viral Replication and Cell Viability

A549 cells were transfected with small interfering RNA (siRNA) targeting PRP19 (5′-CUAAUCUGCUCCAUCUCUA and 5′-UAGAGAUGGAGCAGAUAAG) or control siRNA using Lipofectamine 3000 (Thermo Fisher Scientific) following the manufacturer’s instructions. At 24 h posttransfection, the cells were infected with influenza A virus at an MOI of 0.001. At the indicated time points after infection, the supernatants were collected, and a plaque formation assay using MDCK cells was performed to determine the virus titers. The viability of PRP19-knocked down A549 cells was determined by the MTT assay. Briefly, MTT (thiazolyl blue tetrazolium blue, Sigma-Aldrich) at a concentration of 1 mg/ mL was added to cells and incubated for 3 h at 37 °C. After the MTT solution was removed, isopropanol-diluted HCl (0.04N) was added to the cells. The relative cell viability of control and knockdown cells was determined by measuring the absorbance at 570 nm and subtracting the background at 630 nm.

Bioinformatic Analysis

To identify components of NS1-associated protein complexes, we performed label-free comparison between immunoprecipitation products from cells transfected with the control vector and the NS1 vector using the spectral counting method. The numbers of spectra assigned to each protein were exported from the Scaffold software in MS Excel format. The normalized spectral count (SC) of each protein was obtained by dividing the SC of a given protein by the total SC in the experiment. The fold change was determined by dividing the average of normalized SCs for the NS1 vector group by that for the control vector group. We failed to identify all proteins in all experiments; unidentified proteins or missing values in a particular sample were assigned an SC of one to avoid dividing by zero and to prevent overestimation of fold changes.28,29 Biological process classification and signaling pathway analysis of the proteins identified as coimmunoprecipitating with NS1 were performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID, version 6.7, http://david.abcc.ncifcrf.gov/) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome. jp/kegg/pathway.html) tools.31 The STRING online software (version 10) was used to search for interaction relationships among the proteins identified in NS1-associated protein complexes, and a required confidence (combined score) > 0.8 was used as the cutoff criterion.32

Statistical Analysis

The Mann−Whitney U test was used for comparing protein levels between immunoprecipitation products from cells transfected with the control vector and those transfected with the NS1 vector. P values mean + 2SD (7.194) are defined as an NS1 interacting partner.

Table 2. Enrichment Analysis of Biological Processes for NS1-Interacting Proteins biological processa RNA splicing/ processing cellular macromolecule localization protein transport/ targeting protein folding

identified proteins involved in the process

p value

DHX8, PABPN1, PRPF19, HNRNPA3, SNRPA1, DNAJB11, PTBP1, ISY1, SNRNP40, XAB2 YWHAG, YWHAH, HTT, YWHAB, YWHAQ, YWHAE, STAU1

1.01 × 10−5

PABPN1, YWHAG, YWHAH, HTT, YWHAB, YWHAQ, YWHAE

8.03 × 10−3

HSPBP1, DNAJB11, CCT2, DNAJB6, HSPA4

9.26 × 10−3

4.91 × 10−3

a

DAVID (version 6.7) was applied to functionally annotate enriched proteins using the annotation category GOTERM_BP_FAT. Processes with at least five protein members and P values less than 0.01 are considered significant.

Table 3. Pathway Analysis of the Proteins Interacted with the NS1 Protein term in KEGG pathwaya spliceosome neurotrophin signaling pathway cell cycle

identified proteins involved in pathway DHX8, PRPF19, HNRNPA3, SNRPA1, ISY1, SNRNP40, XAB2 YWHAG, YWHAH, YWHAB, YWHAQ, YWHAE, PIK3R2 YWHAG, YWHAH, YWHAB, YWHAQ, YWHAE

p value 1.47 × 10−5 1.88 × 10−4 1.34 × 10−3

Figure 2. PPI network analysis of proteins identified as coimmunoprecipitating with NS1. A PPI network of the 64 proteins listed in Table 1 was constructed using the STRING v10 database (http:// string-db.org/), depicting 53 interaction links between individual nodes/proteins (solid lines). Two modules, both of which involve more than 10 nodes/proteins, are shown. One depicts interactions of PRPF19 with proteins involved in RNA splicing/processing, and the other depicts interactions of YWHAE with proteins involved in the cell cycle.

a

DAVID was applied to functionally annotate enriched proteins. The knowledge base used was the KEGG pathway database. Processes with at least five protein members and P values less than 0.01 are considered significant.

protein complexes. On the basis of the bioinformatics analysis, PRP19 is involved in the RNA splicing biological process (Table 2), the spliceosome functional network (Table 3), and the PPI of proteins involved in RNA splicing/processing (Figure 2). As shown in Figure 4, panel A, in NS1-expressing cells, the PRP19 protein coimmunoprecipitated with 3xFLAGtagged NS1, showing that PRP19 is a component of NS1associated protein complexes. To investigate the role of splicing factor PRP19 during influenza A virus infection, we further examined the NS1PRP19 interaction during viral infection. The 293T cells were transfected for 24 h with a plasmid expressing Myc-tagged PRP19 and then infected with influenza A PR8(H1N1) virus. At 12 h postinfection, cell lysates were collected and subjected to immunoprecipitation with an anti-Myc antibody. As shown

in Figure 4, panel B, influenza NS1 was coprecipitated with PRP19, demonstrating that PRP19 and NS1 coexist in protein complexes during influenza A virus infection. Cellular Splicing Factor PRP19 Is Involved in Influenza Viral Replication

To determine the role of the cellular PRP19 protein in influenza A viral replication, we employed a specific siRNA targeting PRP19 to reduce the endogenous expression of this protein in A549 cells. At 24 h after siRNA transfection, cells were infected with PR8(H1N1) virus at an MOI of 0.001, and 1644

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Figure 4. The NS1 protein encoded by the influenza A virus binds to PRP19 during infection. (A) Cell lysates of 293T cells transfected with either a control vector (Ctrl vector) or a plasmid expressing 3xFLAGtagged NS1 (NS1 vector) were subjected to immunoprecipitation using anti-FLAG-M2 resin. The input lysates and IP precipitates were analyzed by immunoblotting using anti-PRP19, anti-FLAG, and anti-βactin antibodies. (B) 293T cells were transfected for 24 h with a plasmid expressing Myc-tagged PRP19 (PRP19-Myc), followed by infection of influenza A virus PR8(H1N1) for 12 h at an MOI of 2 (FluA/PR8). Cell lysates were subjected to immunoprecipitation using an anti-Myc antibody. The lysates and IP precipitates were examined by immunoblotting using anti-Myc, anti-NS1, and anti-β-actin antibodies.

Figure 3. Cellular pre-mRNA splicing is regulated by the influenza A virus NS1 protein. 293T cells were cotransfected for 24 h with the pSV40-CAT(In1) splicing reporter plasmid (shown in A; the numbers represent the length in nucleotides) and empty vector or an NS1expressing vector. Total RNA was collected, and precursor mRNA (pre-mRNA) and mature mRNA transcribed from the reporter plasmid were then detected by RT-PCR followed by agarose gel electrophoresis and ethidium bromide staining (B, upper-right panel). The intensity of the bands representing mature mRNA was quantitated with ImageJ (B, upper-left panel). The expression of the NS1 protein was examined by immunoblotting with an anti-NS1 antibody (B, lower panel). Quantitative PCR was applied to determine the relative amounts of spliced and unspliced mRNA (C).

Figure 5. Decreasing endogenous PRP19 protein expression significantly reduces influenza A virus replication. (A) A549 cells were transfected for 24 h with either control siRNA or siRNA targeting PRP19, followed by infection with influenza A virus PR8(H1N1) strain at an MOI of 0.001. At 24 and 48 h postinfection, supernatants of the infected cells were collected for virus titer determination by the plaque formation assay. The experiment was performed in triplicate, demonstrating similar trends of differences; the figures show one set of results. (B) The viability of A549 cells transfected with control or PRP19 siRNA was determined and compared using the MTT assay. ∗, P < 0.05; n.s., not significant.

viral replication was monitored at 24 and 48 h postinfection. We found that decreasing endogenous PRP19 expression significantly reduced replication of influenza A virus (Figure 5A) and that the transient knockdown of PRP19 expression did not affect cell viability (Figure 5B). This finding suggested that the cellular splicing factor PRP19, which interacts with influenza NS1 protein, could be involved in influenza A viral replication.

previous reports in which NS1-interacting host proteins were identified using the yeast-two-hybrid approach,25,34 three human proteins, PIK3R2, PRKRA, and STAU1, were also found in our research. In addition, 13 of 64 proteins detected in this study, including PIK3R2, PABPN1, IGF2BP2, STAU1, PRKRA, RBMXL1, TUBA1A, PTBP1, HNRNPA3, and several YWHA proteins, were also found by exploring influenza NS1interacting partners using tandem affinity purification (TAP) and LC−MS/MS.35 However, our NS1 interactome profile did not reveal components of the nuclear pore complex, including NXF1, RAE1, NUP98, p15, and E1B-AP5, which were previously identified by a binding assay using a GST-tagged NS1 protein and 293T cell lysates.36

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DISCUSSION The NS1 protein of influenza A virus functions as a modulator that regulates several cellular events in infected cells such as cellular antiviral responses and mRNA maturation processes. In this study, we applied coimmunoprecipitation and LC−MS/MS approaches to systemically analyze NS1-interacting proteins in human cells. The interactome analysis demonstrated that the NS1 protein may interact with host proteins involved in RNA splicing/processing, cellular macromolecule localization, protein transport/targeting, and protein folding. Comparing to 1645

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terminal deoxynucleotidyl transferase (TdT) or other cellular factors such as CDC5L, PLRG1, and SPF27 to contribute to DNA repair response.47,48 In addition to the functions described above, PRP19 is also an E3 ubiquitin ligase that mediates K63 ubiquitination of spliceosomal protein PRP3 for several cellular responses. However, as it has not been determined whether the NS1 protein encoded by the influenza A virus is involved in these activities within host cells, the NS1PRP19 interaction may influence more cellular responses other than pre-mRNA splicing events. In addition to proteins that are highly correlated with RNA splicing/processing biological processes, proteins involved in cell cycle were also identified by our NS1 interactome and biological process analysis. Furthermore, the present systemic analysis of NS1-interacting proteins reveals that several members of the 14−3−3 protein family may interact with the influenza NS1 protein including 14−3−3 β/α, ε, γ, η, and θ (encoded by genes YWHAB, YWHAE, YWHAG, YWHAH, and YWHAQ). It has been found that 14−3−3 proteins are involved in regulating cell cycle, apoptosis, and protein trafficking.49 Among the identified 14−3−3 proteins, 14−3−3 ε, also named mitochondrial import-stimulation factor L subunit, is an adaptor protein that binds to signaling proteins with phospho-serine residues to regulate various cellular processes such as stress-mediated transcription, apoptosis inhibition, and TNF-α-induced NF-κB activation.50−52 More importantly, the 14−3−3 ε protein plays a critical role in directing activated RIG-I to mitochondrial-associated membranes for interacting with MAVS and sequentially initiating the antiviral response.53 Consequently, the NS1 protein of influenza A virus may reprogram host antiviral responses via its interaction with 14−3−3 ε. Further study is warranted to explore the unknown mechanisms by which influenza NS1 protein counteracts host antiviral responses.

We also demonstrated that expression of influenza NS1 protein interferes with an mRNA splicing event. Among the identified proteins, the interaction between NS1 and PRP19, which is involved in pre-mRNA splicing and the DNA damage response, was confirmed in NS1-expressing or influenza A virus-infected cells by coimmunoprecipitation. We further investigated the role of PRP19 in replication of influenza A virus and showed that decreasing PRP19 expression in a human lung cell line could reduce propagation of the virus. This result indicates the importance of the cellular PRP19 protein in influenza A viral replication. The PRP19 protein was originally identified as a component in the spliceosome that serves as an E3 ubiquitin ligase and mediates lysine 63 (K63) ubiquitination of the U4 spliceosomal protein PRP3 to stabilize the U4/U5/U6 spliceosomal complex.37 Therefore, it is reasonable to propose that interaction between PRP19 and NS1 might modulate splicing events in host cells. As in other studies, our results showed that overexpression of NS1 can suppress cellular pre-mRNA splicing.21,22 Moreover, previous investigations have revealed that the PRP19 protein associates with other cellular factors in a complex named Prp19C. Different PRP19 complexes involved in several cellular processes are found in eukaryotic cells, and Prp19C has a well-known function in cellular pre-mRNA splicing.38,39 ISY1, a component of PRP19 complexes that function in splicing, was also identified in our systemic NS1 interactome analysis. This finding demonstrates that the NS1 protein may interact with Prp19C and affect the pre-mRNA splicing function of these complexes. In addition to PRP19, several factors involved in pre-mRNA splicing, such as SNRNP40 and PTBP1, were identified as components of NS1-associated protein complexes, indicating that NS1 may also interact with other splicing factors to modulate host splicing events. Although it has been proposed that influenza NS1 protein may bind to the 30-kDa subunit of cellular cleavage and polyadenylation specific factor (CPSF30) and interfere with cellular mRNA polyadenylation and splicing,23,40 other studies have shown that NS1 encoded by PR8(H1N1) virus does not bind to CPSF30.41 Therefore, the interaction of splicing factors and the NS1 protein could be more relevant to changes in host cellular mRNA splicing events. In addition, NS1 possesses an RNA-binding domain and can bind to various RNAs including U6 snRNA.22 Such binding also plays a role in suppressing cellular pre-mRNA splicing.42 Nevertheless, the present study focused on systemic interactome analysis of the NS1 protein and further demonstrated that NS1 may interact with several host proteins involved in RNA processing. Collectively, these findings suggest that influenza A virus may alter cellular RNA processes via its encoded NS1 protein. Alternative splicing also occurs for two influenza mRNAs, M1 and NS1, to generate several spliced forms of viral RNAs including M2, mRNA3, M4, and NS2 mRNAs. The generation of viral spliced RNAs is highly regulated and requires host splicing machinery.43 Although previous studies have raised the question of whether influenza NS1 affects the splicing of viral mRNAs,33,44,45 the biological impact of interaction between NS1 and host splicing factors on viral mRNA splicing requires further investigation. On the basis of the factors identified in the PRP19 complexes, it has been suggested that these complexes may participate in transcription, DNA repair, and proteasomal degradation.46 For example, PRP19 may form a complex with

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00103. List of proteins identified in immunoprecipitation product of vector control and NS1 protein; spectral counting-based identification of proteins coimmunoprecipitating with NS1 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: 886-3-2118800, ext. 5093. E-mail: [email protected]. edu.tw. *Phone: 886-3-2118800, ext. 3775. E-mail: [email protected]. edu.tw. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Woan-Yuh Tarn for materials and Dr. Chi-Jene Chen for helpful discussion. This work was supported by grants to Chih-Ching Wu from the Ministry of Science and Technology (MOST), Taiwan (102-2320-B-182-029-MY3, 103-2325-B-182-007, and 103-2632-B-182-001) and the Chang Gung Memorial Hospital (CGMH), Taiwan 1646

DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648

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Journal of Proteome Research

(13) Kawaguchi, A.; Nagata, K. De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM. EMBO J. 2007, 26 (21), 4566−75. (14) Momose, F.; Basler, C. F.; O’Neill, R. E.; Iwamatsu, A.; Palese, P.; Nagata, K. Cellular splicing factor RAF-2p48/NPI-5/BAT1/ UAP56 interacts with the influenza virus nucleoprotein and enhances viral RNA synthesis. Journal of virology 2001, 75 (4), 1899−908. (15) Momose, F.; Naito, T.; Yano, K.; Sugimoto, S.; Morikawa, Y.; Nagata, K. Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. J. Biol. Chem. 2002, 277 (47), 45306−14. (16) Tafforeau, L.; Chantier, T.; Pradezynski, F.; Pellet, J.; Mangeot, P. E.; Vidalain, P. O.; Andre, P.; Rabourdin-Combe, C.; Lotteau, V. Generation and comprehensive analysis of an influenza virus polymerase cellular interaction network. Journal of virology 2011, 85 (24), 13010−8. (17) Liu, Y. C.; Kuo, R. L.; Lin, J. Y.; Huang, P. N.; Huang, Y.; Liu, H.; Arnold, J. J.; Chen, S. J.; Wang, R. Y.; Cameron, C. E.; Shih, S. R. Cytoplasmic viral RNA-dependent RNA polymerase disrupts the intracellular splicing machinery by entering the nucleus and interfering with Prp8. PLoS Pathog. 2014, 10 (6), e1004199. (18) Mayer, D.; Molawi, K.; Martinez-Sobrido, L.; Ghanem, A.; Thomas, S.; Baginsky, S.; Grossmann, J.; Garcia-Sastre, A.; Schwemmle, M. Identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches. J. Proteome Res. 2007, 6 (2), 672− 82. (19) Watanabe, T.; Kawakami, E.; Shoemaker, J. E.; Lopes, T. J.; Matsuoka, Y.; Tomita, Y.; Kozuka-Hata, H.; Gorai, T.; Kuwahara, T.; Takeda, E.; Nagata, A.; Takano, R.; Kiso, M.; Yamashita, M.; SakaiTagawa, Y.; Katsura, H.; Nonaka, N.; Fujii, H.; Fujii, K.; Sugita, Y.; Noda, T.; Goto, H.; Fukuyama, S.; Watanabe, S.; Neumann, G.; Oyama, M.; Kitano, H.; Kawaoka, Y. Influenza virus-host interactome screen as a platform for antiviral drug development. Cell Host Microbe 2014, 16 (6), 795−805. (20) Krug, R. M.; Garcia-Sastre, A. The NS1 protein: A master regulator of host and viral functions. In Textbook of Influenza, 2nd ed.; Webster, R. G., Monto, A. S., Braciale, T. J., Lamb, R. A., Eds.; John Wiley & Sons, Ltd.: New York, 2013; pp 114−132. (21) Fortes, P.; Beloso, A.; Ortin, J. Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J. 1994, 13 (3), 704−12. (22) Lu, Y.; Qian, X. Y.; Krug, R. M. The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing. Genes Dev. 1994, 8 (15), 1817−28. (23) Nemeroff, M. E.; Barabino, S. M.; Li, Y.; Keller, W.; Krug, R. M. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3′end formation of cellular pre-mRNAs. Mol. Cell 1998, 1 (7), 991−1000. (24) Zhao, C.; Kuo, R.-L.; Krug, R. M. The NS1 Protein of Influenza A Virus; Caister Academic Press: Norfolk, UK, 2010. (25) de Chassey, B.; Aublin-Gex, A.; Ruggieri, A.; Meyniel-Schicklin, L.; Pradezynski, F.; Davoust, N.; Chantier, T.; Tafforeau, L.; Mangeot, P. E.; Ciancia, C.; Perrin-Cocon, L.; Bartenschlager, R.; Andre, P.; Lotteau, V. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication. PLoS Pathog. 2013, 9 (7), e1003440. (26) Tawaratsumida, K.; Phan, V.; Hrincius, E. R.; High, A. A.; Webby, R.; Redecke, V.; Hacker, H. Quantitative proteomic analysis of the influenza A virus nonstructural proteins NS1 and NS2 during natural cell infection identifies PACT as an NS1 target protein and antiviral host factor. J. Virol. 2014, 88 (16), 9038−48. (27) Lee, K. M.; Hsu, I. W.; Tarn, W. Y. TRAP150 activates premRNA splicing and promotes nuclear mRNA degradation. Nucleic Acids Res. 2010, 38 (10), 3340−50. (28) Hsu, C. W.; Yu, J. S.; Peng, P. H.; Liu, S. C.; Chang, Y. S.; Chang, K. P.; Wu, C. C. Secretome profiling of primary cells reveals

(CLRPD190015, CMRPD2B0053 and BMRPC77) and grants to Rei-Lin Kuo from the MOST (103-2321-B-182-011 and 104-2321-B-182-003) and the CGMH, Taiwan (CMRPD1E0441−3 and BMRPC09).

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REFERENCES

(1) Dawood, F. S.; Jain, S.; Finelli, L.; Shaw, M. W.; Lindstrom, S.; Garten, R. J.; Gubareva, L. V.; Xu, X.; Bridges, C. B.; Uyeki, T. M. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N. Engl. J. Med. 2009, 360 (25), 2605−15. (2) Wright, P. F.; Neumann, G.; Kawaoka, Y. Orthomyxoviruses. In Fields Virology, 5th ed.; Fields, B. N., Knipe, D. M., Howley, P. M., Eds.; Lippincott Williams & Wilkins, 2007. (3) Chen, Y.; Liang, W.; Yang, S.; Wu, N.; Gao, H.; Sheng, J.; Yao, H.; Wo, J.; Fang, Q.; Cui, D.; Li, Y.; Yao, X.; Zhang, Y.; Wu, H.; Zheng, S.; Diao, H.; Xia, S.; Zhang, Y.; Chan, K. H.; Tsoi, H. W.; Teng, J. L.; Song, W.; Wang, P.; Lau, S. Y.; Zheng, M.; Chan, J. F.; To, K. K.; Chen, H.; Li, L.; Yuen, K. Y. Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet 2013, 381 (9881), 1916−25. (4) Gao, R.; Cao, B.; Hu, Y.; Feng, Z.; Wang, D.; Hu, W.; Chen, J.; Jie, Z.; Qiu, H.; Xu, K.; Xu, X.; Lu, H.; Zhu, W.; Gao, Z.; Xiang, N.; Shen, Y.; He, Z.; Gu, Y.; Zhang, Z.; Yang, Y.; Zhao, X.; Zhou, L.; Li, X.; Zou, S.; Zhang, Y.; Yang, L.; Guo, J.; Dong, J.; Li, Q.; Dong, L.; Zhu, Y.; Bai, T.; Wang, S.; Hao, P.; Yang, W.; Han, J.; Yu, H.; Li, D.; Gao, G. F.; Wu, G.; Wang, Y.; Yuan, Z.; Shu, Y.; et al. Human infection with a novel avian-origin influenza A (H7N9) virus. N. Engl. J. Med. 2013, 368 (20), 1888−97. (5) Lam, T. T.; Wang, J.; Shen, Y.; Zhou, B.; Duan, L.; Cheung, C. L.; Ma, C.; Lycett, S. J.; Leung, C. Y.; Chen, X.; Li, L.; Hong, W.; Chai, Y.; Zhou, L.; Liang, H.; Ou, Z.; Liu, Y.; Farooqui, A.; Kelvin, D. J.; Poon, L. L.; Smith, D. K.; Pybus, O. G.; Leung, G. M.; Shu, Y.; Webster, R. G.; Webby, R. J.; Peiris, J. S.; Rambaut, A.; Zhu, H.; Guan, Y. The genesis and source of the H7N9 influenza viruses causing human infections in China. Nature 2013, 502 (7470), 241−4. (6) Brass, A. L.; Huang, I. C.; Benita, Y.; John, S. P.; Krishnan, M. N.; Feeley, E. M.; Ryan, B. J.; Weyer, J. L.; van der Weyden, L.; Fikrig, E.; Adams, D. J.; Xavier, R. J.; Farzan, M.; Elledge, S. J. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 2009, 139 (7), 1243−54. (7) Karlas, A.; Machuy, N.; Shin, Y.; Pleissner, K. P.; Artarini, A.; Heuer, D.; Becker, D.; Khalil, H.; Ogilvie, L. A.; Hess, S.; Maurer, A. P.; Muller, E.; Wolff, T.; Rudel, T.; Meyer, T. F. Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 2010, 463 (7282), 818−22. (8) Konig, R.; Stertz, S.; Zhou, Y.; Inoue, A.; Hoffmann, H. H.; Bhattacharyya, S.; Alamares, J. G.; Tscherne, D. M.; Ortigoza, M. B.; Liang, Y.; Gao, Q.; Andrews, S. E.; Bandyopadhyay, S.; De Jesus, P.; Tu, B. P.; Pache, L.; Shih, C.; Orth, A.; Bonamy, G.; Miraglia, L.; Ideker, T.; Garcia-Sastre, A.; Young, J. A.; Palese, P.; Shaw, M. L.; Chanda, S. K. Human host factors required for influenza virus replication. Nature 2010, 463 (7282), 813−7. (9) Deng, T.; Engelhardt, O. G.; Thomas, B.; Akoulitchev, A. V.; Brownlee, G. G.; Fodor, E. Role of ran binding protein 5 in nuclear import and assembly of the influenza virus RNA polymerase complex. Journal of virology 2006, 80 (24), 11911−9. (10) Engelhardt, O. G.; Smith, M.; Fodor, E. Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II. Journal of virology 2005, 79 (9), 5812−8. (11) Huarte, M.; Sanz-Ezquerro, J. J.; Roncal, F.; Ortin, J.; Nieto, A. PA subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators. Journal of virology 2001, 75 (18), 8597−604. (12) Jorba, N.; Juarez, S.; Torreira, E.; Gastaminza, P.; Zamarreno, N.; Albar, J. P.; Ortin, J. Analysis of the interaction of influenza virus polymerase complex with human cell factors. Proteomics 2008, 8 (10), 2077−88. 1647

DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648

Article

Journal of Proteome Research that THBS2 is a salivary biomarker of oral cavity squamous cell carcinoma. J. Proteome Res. 2014, 13 (11), 4796−807. (29) Wu, C. C.; Chu, H. W.; Hsu, C. W.; Chang, K. P.; Liu, H. P. Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma. Proteomics 2015, 15 (19), 3394−404. (30) Yu, C. J.; Chang, K. P.; Chang, Y. J.; Hsu, C. W.; Liang, Y.; Yu, J. S.; Chi, L. M.; Chang, Y. S.; Wu, C. C. Identification of guanylatebinding protein 1 as a potential oral cancer marker involved in cell invasion using omics-based analysis. J. Proteome Res. 2011, 10 (8), 3778−88. (31) Huang; da, W.; Sherman, B. T.; Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2008, 4 (1), 44−57. (32) Szklarczyk, D.; Franceschini, A.; Wyder, S.; Forslund, K.; Heller, D.; Huerta-Cepas, J.; Simonovic, M.; Roth, A.; Santos, A.; Tsafou, K. P.; Kuhn, M.; Bork, P.; Jensen, L. J.; von Mering, C. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015, 43 (D1), D447−52. (33) Robb, N. C.; Jackson, D.; Vreede, F. T.; Fodor, E. Splicing of influenza A virus NS1 mRNA is independent of the viral NS1 protein. J. Gen. Virol. 2010, 91 (9), 2331−40. (34) Shapira, S. D.; Gat-Viks, I.; Shum, B. O.; Dricot, A.; de Grace, M. M.; Wu, L.; Gupta, P. B.; Hao, T.; Silver, S. J.; Root, D. E.; Hill, D. E.; Regev, A.; Hacohen, N. A physical and regulatory map of hostinfluenza interactions reveals pathways in H1N1 infection. Cell 2009, 139 (7), 1255−67. (35) Pichlmair, A.; Kandasamy, K.; Alvisi, G.; Mulhern, O.; Sacco, R.; Habjan, M.; Binder, M.; Stefanovic, A.; Eberle, C. A.; Goncalves, A.; Burckstummer, T.; Muller, A. C.; Fauster, A.; Holze, C.; Lindsten, K.; Goodbourn, S.; Kochs, G.; Weber, F.; Bartenschlager, R.; Bowie, A. G.; Bennett, K. L.; Colinge, J.; Superti-Furga, G. Viral immune modulators perturb the human molecular network by common and unique strategies. Nature 2012, 487 (7408), 486−90. (36) Satterly, N.; Tsai, P. L.; van Deursen, J.; Nussenzveig, D. R.; Wang, Y.; Faria, P. A.; Levay, A.; Levy, D. E.; Fontoura, B. M. Influenza virus targets the mRNA export machinery and the nuclear pore complex. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (6), 1853−8. (37) Cheng, S. C.; Tarn, W. Y.; Tsao, T. Y.; Abelson, J. PRP19: a novel spliceosomal component. Mol. Cell. Biol. 1993, 13 (3), 1876−82. (38) Chan, S. P.; Kao, D. I.; Tsai, W. Y.; Cheng, S. C. The Prp19passociated complex in spliceosome activation. Science 2003, 302 (5643), 279−82. (39) Chan, S. P.; Cheng, S. C. The Prp19-associated complex is required for specifying interactions of U5 and U6 with pre-mRNA during spliceosome activation. J. Biol. Chem. 2005, 280 (35), 31190−9. (40) Li, Y.; Chen, Z. Y.; Wang, W.; Baker, C. C.; Krug, R. M. The 3′end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo. RNA 2001, 7 (6), 920−31. (41) Kochs, G.; Garcia-Sastre, A.; Martinez-Sobrido, L. Multiple antiinterferon actions of the influenza A virus NS1 protein. Journal of virology 2007, 81 (13), 7011−21. (42) Qiu, Y.; Nemeroff, M.; Krug, R. M. The influenza virus NS1 protein binds to a specific region in human U6 snRNA and inhibits U6-U2 and U6-U4 snRNA interactions during splicing. RNA 1995, 1 (3), 304−16. (43) Dubois, J.; Terrier, O.; Rosa-Calatrava, M. Influenza viruses and mRNA splicing: doing more with less. mBio 2014, 5 (3), e00070−14. (44) Garaigorta, U.; Ortin, J. Mutation analysis of a recombinant NS replicon shows that influenza virus NS1 protein blocks the splicing and nucleo-cytoplasmic transport of its own viral mRNA. Nucleic Acids Res. 2007, 35 (14), 4573−82. (45) Robb, N. C.; Fodor, E. The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein. J. Gen. Virol. 2012, 93 (1), 113−8. (46) Chanarat, S.; Strasser, K. Splicing and beyond: the many faces of the Prp19 complex. Biochim. Biophys. Acta, Mol. Cell Res. 2013, 1833 (10), 2126−34.

(47) Mahajan, K. N.; Mitchell, B. S. Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (19), 10746− 51. (48) Zhang, N.; Kaur, R.; Lu, X.; Shen, X.; Li, L.; Legerski, R. J. The Pso4 mRNA splicing and DNA repair complex interacts with WRN for processing of DNA interstrand cross-links. J. Biol. Chem. 2005, 280 (49), 40559−67. (49) Aghazadeh, Y.; Papadopoulos, V. The role of the 14−3-3 protein family in health, disease, and drug development. Drug Discovery Today 2016, 21 (2), 278−87. (50) Kim, J.; Parrish, A. B.; Kurokawa, M.; Matsuura, K.; Freel, C. D.; Andersen, J. L.; Johnson, C. E.; Kornbluth, S. Rsk-mediated phosphorylation and 14−3-3varepsilon binding of Apaf-1 suppresses cytochrome c-induced apoptosis. EMBO J. 2012, 31 (5), 1279−92. (51) Wang, X.; Grammatikakis, N.; Siganou, A.; Calderwood, S. K. Regulation of molecular chaperone gene transcription involves the serine phosphorylation, 14−3-3 epsilon binding, and cytoplasmic sequestration of heat shock factor 1. Molecular and cellular biology 2003, 23 (17), 6013−26. (52) Zuo, S.; Xue, Y.; Tang, S.; Yao, J.; Du, R.; Yang, P.; Chen, X. 14−3-3 epsilon dynamically interacts with key components of mitogen-activated protein kinase signal module for selective modulation of the TNF-alpha-induced time course-dependent NFkappaB activity. J. Proteome Res. 2010, 9 (7), 3465−78. (53) Liu, H. M.; Loo, Y. M.; Horner, S. M.; Zornetzer, G. A.; Katze, M. G.; Gale, M., Jr. The mitochondrial targeting chaperone 14−33epsilon regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity. Cell Host Microbe 2012, 11 (5), 528−37.

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