Article

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Interactome Analysis of the NS1 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, NS1- associated 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 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-mRNA- processing 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

■ INTRODUCTION influenza A virus interaction. These approaches have revealed many host proteins as interacting with polymerase subunits or Influenza A virus is an important pathogen that can cause − the nucleoprotein (NP) of influenza A virus.9 16 Immunopre- severe illness and death in mammals and avians. The ability of fl the virus to be transmitted among natural reservoirs, such as cipitation of in uenza viral proteins combined with mass aquatic birds, and to humans occasionally has resulted in spectrometry has also facilitated the search for host factors that 17,18 fi pandemics in human populations.1,2 The virus belongs to the may regulate viral replication. The host proteins identi ed Orthomyxovirus family, which contains a segmented RNA using these approaches may not only help to characterize fl genome in an enveloped viral particle. On the basis of the replication of in uenza virus and its pathogenesis, but also may be applied to develop antivirals for treating influenza A virus combination of the viral envelope proteins hemagglutinin (HA) 19 and neuraminidase (NA), the virus has been divided into infection. fl subtypes such as H1N1 and H3N2. In addition, an H7N9 Nonstructural protein 1 (NS1) of in uenza A virus is a fl fi multifunctional protein that can suppress host innate antiviral subtype of avian in uenza A virus has recently been identi ed 20 as capable of infecting humans and resulting in severe disease responses to establish virus replication in infected cells. − and death.3 5 Moreover, it has been found that the protein can also regulate Several studies have applied genome-wide RNAi library the maturation steps of cellular mRNA such as screening to systemically explore the host factors involved in − influenza A virus replication.6 8 In addition, the yeast two- Received: February 3, 2016 hybrid system has been employed to investigate the host- Published: April 20, 2016

© 2016 American Chemical Society 1639 DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648 Journal of Proteome Research Article

− and splicing.21 23 On the basis of its properties and structure, subjected to immunoprecipitation using anti-FLAG M2 affinity the NS1 protein is divided in two domains, RNA-binding and gel (Cat. No. A2220, Sigma-Aldrich) following the manufac- effector domains, each of which can impact the regulation of turer’s instructions. For immunoblotting, the cell extracts and biological processes in host cells by binding to specific cellular the cellular proteins copurifying with the 3xFLAG NS1 protein factors.24 Previously, an interactome analysis using the yeast were separated by SDS-PAGE and transferred to PVDF two-hybrid approach identified 51 cellular interactors of the membranes. The membranes were then probed with anti- NS1/NS2 proteins encoded by nine different strains of NS1, anti-FLAG-M2 (Cat. No. F1804, Sigma-Aldrich), anti-β- influenza A virus.25 Subsequently, a recombinant influenza A actin (Cat. No. A5441, Sigma-Aldrich), anti-c-Myc (Cat. No. virus expressing epitope-tagged NS1/NS2 proteins was M4439, Sigma-Aldrich), or anti-PRPF19 (Cat. No. generated for identifying host factors that interact with these GTX106952, GeneTex, USA or Cat. No. SAB4501215, viral proteins in infected cells.26 The study has also identified Sigma-Aldrich) antibodies. To examine the NS1-PRP19 interferon-induced protein kinase (PACT) as a factor interaction during influenza A virus infection, 293T cells were interacting with NS1 and involved in replication of the transfected with a plasmid expressing Myc-tagged PRP19 for 24 influenza A virus.26 However, in the approaches used in h and then infected with the PR8 virus at an MOI of 2. At 12 h previous studies, the NS2 protein and its interacting proteins postinfection, cell lysates were collected and incubated with an were copurified. Thus, the factors that specifically bound to anti-Myc antibody (Cat. No. M4439, Sigma-Aldrich) at 4 °C NS1 could not be systemically screened. for 14 h. After mixing and incubating with Protein A and Although NS1 has been reported to interact with numerous Protein G Sepharose Fast Flow beads (GE Healthcare Life cellular molecules implicated in multiple host response,25,26 its Sciences, NJ, USA) at 4 °C for 3 h, the immunoprecipitated functions and the mechanism underlying NS1-mediated products were eluted with SDS-PAGE sample buffer and regulation of cellular mRNA processing have not been fully analyzed by immunoblotting as described above. described to date. To address this, we systemically explored the SDS-PAGE and In-Gel Protein Digestion NS1 interactome in NS1-expressing cells using immunopreci- pitation followed by SDS-polyacrylamide gel electrophoresis Proteins immunoprecipitated from 3xFLAG NS1-expressing − cells were separated by 10% SDS-PAGE and stained using a (SDS-PAGE) coupled with liquid chromatography tandem fi mass spectrometry (GeLC−MS/MS). The cellular proteins Colloidal Blue Staining kit (Thermo Fisher Scienti c, NY, identified were found to be involved in the cellular processes of USA). The stained gel lanes were cut into 20 slices and subjected to in-gel tryptic digestion as described previously.28,29 RNA splicing/processing, cell cycle, and protein folding/ fl targeting. Importantly, one of the identified proteins, pre- Brie y, gel pieces were destained in 10% methanol mRNA-processing factor 19 (PRP19; encoded by the PRPF19 (Mallinckrodt Baker, NJ, USA), dehydrated in acetonitrile ), was confirmed to interact with the NS1 protein in (ACN; Mallinckrodt Baker), and dried using a SpeedVac. The fl gel pieces were treated with 25 mM NH4HCO3 containing 10 in uenza A virus-infected cells, and depletion of cellular PRP19 ° decreased influenza A viral replication. This study compre- mM dithiothreitol (Biosynth AG, Switzerland) at 60 C for 30 hensively profiled the interactome of the NS1 protein in host min, followed by alkylation with 55 mM iodoacetamide cells, and the results demonstrate a novel regulatory role for (Amersham Biosciences, UK) at room temperature for 30 PRP19 in replication of influenza A virus. min. The proteins were then digested using sequencing-grade modified porcine trypsin (20 μg/mL; Promega, WI, USA) at 37 ■ MATERIALS AND METHODS °C for 16 h. Peptides were extracted with ACN and dried using a SpeedVac. Cells, Virus, and Plasmids Reverse-Phase LC−MS/MS Analysis HEK293T (293T; ATCC CRL-3216), Madin Darby Canine Protein identification was performed as described previ- Kidney (MDCK; ATCC PTA-6500), and A549 (ATCC CCL- ously.29,30 Briefly, each peptide mixture was reconstituted in 185) cell lines were cultivated in DMEM with 10% FBS. HPLC buffer A (0.1% formic acid; Sigma-Aldrich), loaded onto Influenza A virus Puerto Rico/1934/H1N1 strain (PR8) was a trap column (Zorbax 300SB-C , 0.3 × 5 mm; Agilent amplified with 10-day-old fertilized eggs and titrated by plaque 18 Technologies, Taiwan) at a flow rate of 0.2 μL/min in HPLC formation assays using monolayers of MDCK cells. Viral buffer A, and separated on a resolving 10 cm analytical C replication was determined by infection of A549 cells at a 18 column (inner diameter, 75 μm) with a 15-μm tip (New multiplicity of infection (MOI) of 0.001 for 24 and 48 h. A Objective, MA, USA). Using a flow rate of 0.25 μL/min across plasmid-expressed NS1 protein with a 3xFLAG was constructed the analytical column, the peptides were eluted using a linear by cloning the PR8 NS1 open reading frame into p3xFLAG- gradient of 0−10% HPLC buffer B (99.9% ACN containing Myc-CMV 26 (Sigma-Aldrich, St. Louis, MO, USA). To 0.1% formic acid) for 3 min, 10−30% buffer B for 35 min, 30− generate a human PRP19-expressing plasmid, the human 35% buffer B for 4 min, 35−50% buffer B for 1 min, 50−95% PRP19 cDNA was generated by reverse transcription and buffer B for 1 min, and 95% buffer B for 8 min. PCR using total RNA from 293T cells and inserted into the The LC apparatus was coupled online with a two- pcDNA3.1-Myc-His A vector. The pSV40-CAT(In1) plasmid dimensional linear ion trap mass spectrometer (LTQ-Orbitrap was provided by Dr. Woan-Yuh Tarn (Academia Sinica, 27 Discovery, Thermo Fisher Scientific) managed using the Taiwan). Xcalibur 2.0 software package (Thermo Fisher Scientific). An Immunoprecipitation, Immunoblotting, and Antibodies electrospray voltage of 1.8 kV was applied. Intact peptides were For NS1 immunoprecipitation, 293T cells were transfected detected by the Orbitrap at a resolution of 30 000. The ion + with the plasmids expressing 3xFLAG-tagged NS1 or control signal of (Si(CH3)2O)6H at m/z 445.120025 was used as an vectors, which express a peptide of MDYKDHDGDYKD- internal standard for mass lock. For MS analysis, we used a HDIDYKDDDDKLAAANSSIDLISVPVDSREQKLISEEDL data-dependent acquisition mode that alternated between one with a 3xFLAG tag. Extracts from 293T cells were then MS scan and six MS/MS scans for the six most abundant

1640 DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648 Journal of Proteome Research Article precursor ions. For MS scans, the m/z scan range was set to RNA transcribed from the reporter plasmid, total RNA from 350−2000 Da. The m/z values selected for MS/MS scans were the transfected cells was collected and subjected to reverse dynamically excluded for 3 min, and 5 × 104 ions were transcription (RT) and quantitative polymerase chain reaction accumulated and resolved in the ion trap to generate MS/MS (qPCR). As in the previously described method,17 DNase I- spectra. Both MS and MS/MS spectra were acquired using one treated total RNA was reverse transcribed using SuperScript III microscan with maximum fill times of 1000 and 100 ms for MS reverse transcriptase (Invitrogen) with the oligo-dT primer. and MS/MS analyses, respectively. Automatic gain control was cDNA was then amplified by qPCR using SYBR Green reagent applied to prevent overfilling of the ion trap. with a reverse primer complementary to the CAT(In1) ′ ′ Protein Database Searching for Protein Identification sequence, 5 -GTATTCACTCCAGAGCGATG-3 , and a CAT- (In1)- forward primer, 5′-CCAGACCGTTCA- For database searching, the obtained MS/MS spectra were GCTGGATATT-3′, for spliced mRNA or with the reverse analyzed using the Mascot algorithm (version 2.1, Matrix primer and a CAT(In1)- forward primer, 5′- Science, MA, USA) against the Swiss-Prot human sequence ATTGGTCTATTTTCCCACCCTTAG-3′, for the unspliced database (released Apr 16, 2014, selected for Homo sapiens, transcript. Regular PCR was performed with SuperRed PCR 20 265 entries) of the European Bioinformatics Institute. The Master Mix (Biotools, Taiwan) and primers (forward: 5′- mass tolerances of the fragment and parent ions were set to 0.5 TTTTGGAGGCCTAGGCTTTT-3′; reverse: 5′- Da and 10 ppm, with trypsin as the digestion enzyme. Up to GTATTCACTCCAGAGCGATG-3′), and the products were one missed cleavage was permitted, and searches were examined by agarose gel electrophoresis and quantitated by performed with the parameters of variable oxidation on ImageJ analysis. methionine (+15.99 Da) and fixed carbamidomethylation on Inhibition of PRP19 Expression by Small Interfering RNA cysteine (+57 Da). A random sequence database was used to and Determination of Viral Replication and Cell Viability estimate false-positive rates for peptide matches. After Mascot searching, the obtained files were processed A549 cells were transfected with small interfering RNA using Scaffold software (version 3.6.5; Proteome Software, OR, (siRNA) targeting PRP19 (5′-CUAAUCUGCUCC- USA), which includes the PeptideProphet program to assist in AUCUCUA and 5′-UAGAGAUGGAGCAGAUAAG) or con- the assignment of peptide MS spectra and the ProteinProphet trol siRNA using Lipofectamine 3000 (Thermo Fisher program for assigning/grouping peptides to a unique protein/ Scientific) following the manufacturer’s instructions. At 24 h protein family when they are shared among several isoforms. posttransfection, the cells were infected with influenza A virus We used PeptideProphet and ProteinProphet probabilities ≥ at an MOI of 0.001. At the indicated time points after infection, 0.95 to ensure an overall false-discovery rate below 0.5%. Only the supernatants were collected, and a plaque formation assay proteins with two or more identified peptides were retained in using MDCK cells was performed to determine the virus titers. this study. The viability of PRP19-knocked down A549 cells was determined by the MTT assay. Briefly, MTT (thiazolyl blue Bioinformatic Analysis tetrazolium blue, Sigma-Aldrich) at a concentration of 1 mg/ To identify components of NS1-associated protein complexes, mL was added to cells and incubated for 3 h at 37 °C. After the we performed label-free comparison between immunoprecipi- MTT solution was removed, isopropanol-diluted HCl (0.04N) tation products from cells transfected with the control vector was added to the cells. The relative cell viability of control and and the NS1 vector using the spectral counting method. The knockdown cells was determined by measuring the absorbance numbers of spectra assigned to each protein were exported at 570 nm and subtracting the background at 630 nm. ff from the Sca old software in MS Excel format. The normalized Statistical Analysis spectral count (SC) of each protein was obtained by dividing − the SC of a given protein by the total SC in the experiment. The Mann Whitney U test was used for comparing protein The fold change was determined by dividing the average of levels between immunoprecipitation products from cells normalized SCs for the NS1 vector group by that for the transfected with the control vector and those transfected with the NS1 vector. P values <0.05 were considered statistically control vector group. We failed to identify all proteins in all fi experiments; unidentified proteins or missing values in a signi cant. All data were processed by SPSS software version particular sample were assigned an SC of one to avoid dividing 12.0 (SPSS Inc., IL, USA). by zero and to prevent overestimation of fold changes.28,29 Biological process classification and signaling pathway ■ RESULTS fi analysis of the proteins identi ed as coimmunoprecipitating Interactome Analysis of Influenza A Virus-Encoded NS1 with NS1 were performed using Database for Annotation, fl Visualization, and Integrated Discovery (DAVID, version 6.7, To globally explore cellular proteins that interact with in uenza http://david.abcc.ncifcrf.gov/) and the Kyoto Encyclopedia of A NS1 protein, 293T cells were transfected with a control and Genomes (KEGG) database (http://www.genome. vector or plasmid expressing 3xFLAG-tagged NS1 encoded by fl jp/kegg/pathway.html) tools.31 The STRING online software in uenza A PR8(H1N1) strain. At 24 h posttransfection, lysates (version 10) was used to search for interaction relationships of the transfected cells were collected and subjected to among the proteins identified in NS1-associated protein immunoprecipitation with anti-FLAG resin. We initially complexes, and a required confidence (combined score) > detected the FLAG-tagged NS1 protein in the precipitate by 0.8 was used as the cutoff criterion.32 immunoblotting with both anti-FLAG and anti-NS1 antibodies (Figure 1A). To identify cellular proteins coprecipitated with Measurement of Cellular Pre-mRNA Splicing Events the NS1 protein, precipitates from cells transfected with control The 293T cells were cotransfected with the splicing reporter and NS1 vectors were separated by SDS-PAGE and plasmid pSV40-CAT(In1) and a PR8 NS1-expressing plasmid sequentially stained using the Colloidal Blue Staining kit or the empty vector for 24 h. To detect spliced and unspliced (Figure 1B). Each gel lane was then divided into 20 fractions,

1641 DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648 Journal of Proteome Research Article

Spectral Counting-Based Approach for Identifying Components of NS1-Associated Complexes To identify the proteins present in NS1-associated protein complexes, the relative levels of 1489 proteins detected in all the immunoprecipitated products were determined by spectral counting-based quantification (Table S-2). The fold change for each protein was determined by dividing the average SC of the protein in the NS1 group by that in the control group. Proteins with values larger than the mean plus two SD (the ratios were above 7.194) and detected in more than two replicates of the NS1 group were considered to be candidates possibly involved in NS1-associated protein complexes. On the basis of the cutoffs, 64 proteins were discovered (Table 1). Among them, 52 proteins were exclusively detected in the NS1 vector group, and 12 molecules were relatively increased in the NS1 vector group compared to the control group (Table 1). Analysis of Biological Process Networks for Proteins Present in NS1-Associated Complexes To highlight the biological processes in which the NS1- associated complexes are potentially involved, 64 molecules identified as described above (Table 1) were analyzed using DAVID. As shown in Table 2, these proteins are highly correlated with the biological processes of RNA splicing/ processing, cellular macromolecule localization, protein trans- port, and protein folding. Moreover, these proteins were Figure 1. Systemic proteomic analysis of cellular proteins that interact fl subjected to pathway-wise analysis using the KEGG database, with the in uenza NS1 protein. The 293T cells were transfected with a with the results revealing that the proteins most likely control vector (Ctrl vector) and plasmid expressing 3xFLAG-tagged fl participate in the , neurotrophin signaling pathway, NS1 protein (NS1 vector) encoded by in uenza A virus strain − PR8(H1N1). At 24 h posttransfection, lysates of the transfected cells and cell cycle networks (Table 3). A protein protein were collected and subjected to immunoprecipitation with anti-FLAG interaction (PPI) network of these proteins was also resin. (A) The FLAG-tagged NS1 protein in the lysates and IP constructed using the STRING online database, and the PPI precipitates was detected by immunoblotting with anti-FLAG and anti- network of the 64 proteins depicted 53 interaction links NS1 antibodies. (B) Precipitates separated by SDS-PAGE were between individual nodes/proteins (Figure 2). Two modules sequentially stained with the Colloidal Blue Staining kit. (C) Venn were found, both of which involve more than 10 nodes/ diagrams show overlaps between the proteins identified in the control proteins. One module depicted interaction of PRPF19 and the NS1 groups. (D) Venn diagrams display overlaps between the fi (encoding PRP19) with proteins involved in RNA splicing/ proteins identi ed in the three replicates. The total numbers of processing, and the other depicted interaction of YWHAE identified proteins are listed in brackets. (encoding 14−3−3 ε) with proteins involved in the cell cycle or protein targeting (Figure 2). and each fraction was sliced into three parts to provide NS1 Protein Interferes with a Cellular Pre-mRNA Splicing technical replicates. After in-gel tryptic digestion, peptides were Event − analyzed by LC MS/MS. Spectral searches of the Swiss-Prot It has been reported previously that influenza virus NS1 protein database were performed with the Mascot server, and results can regulate cellular pre-mRNA splicing.21,22,33 Our global were further integrated using the Scaffold software. When ff ≥ ≥ proteomic analysis further revealed that the NS1 protein cuto s of peptide probability 0.95 and protein probability encoded by influenza A virus PR8(H1N1) strain may interact 0.95 were imposed, 1489 nonredundant proteins with ≥ 2 fi with many cellular factors involved in the cellular pre-mRNA peptide hits were identi ed (Figure 1C, Table S-1). Among the splicing process. To examine whether PR8(H1N1) NS1 is 1489 proteins, 926 (62.19%) were present in both groups, indeed involved in a cellular pre-mRNA splicing event, we whereas 145 (9.74%) and 418 (28.07%) proteins were uniquely cotransfected the NS1-expressing plasmid with a reporter detected in the control vector and NS1 vector groups, plasmid that can be transcribed into an intron-containing pre- respectively (Figure 1C and Table S-2). mRNA substrate to monitor cellular pre-mRNA splicing.27 The To evaluate the reproducibility of the proteomic analyses, fi relative amounts of unspliced and spliced mRNAs in the proteins identi ed in three replicates were further observed for cotransfected cells were determined by quantitative RT-PCR overlap. There were 618 (57.70%) and 869 (64.66%) proteins with primers specific for the RNA species. The result showed detected in all three replicates of the control and NS1 vector that unspliced pre-mRNA was accumulated in cells expressing groups, respectively (Figure 1D and Table S-2). Approximately fl fi in uenza PR8(H1N1) NS1, whereas spliced mRNA was 80% of the proteins were identi ed in more than two replicates, reduced (Figure 3). whereas approximately 20% were exclusive to one replicate (Figure 1D). In addition, the false discovery rate (FDR) of NS1 Interacts with Splicing Factor PRP19 peptide identification was estimated using a decoy database, Because one of the important functions of the NS1 protein is to with all FDRs being below 0.05%. Collectively, the results regulate the maturation of cellular mRNA, PRP19 was selected indicate that the proteome profiling was adequately performed. for further evaluation as a component of NS1-associated

1642 DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648 Journal of Proteome Research Article Table 1. Spectral Counting-Based Identification of NS1-Interacting Proteins

spectral counts (SCs) in replicates 1/2/3 control vector NS1 vector NS1/CV protein name (protein accession number, gene name) (CV) (NS1) ratioa phosphatidylinositol 3-kinase regulatory subunit beta (P85B_HUMAN, PIK3R2) 0/0/0 40/23/31 32.855 polyadenylate-binding protein 2 (PABP2_HUMAN, PABPN1) 0/0/0 27/28/29 29.566 Hsp70-binding protein 1 (HPBP1_HUMAN, HSPBP1) 0/0/0 26/26/19 24.861 inosine-5′-monophosphate dehydrogenase 2 (IMDH2_HUMAN, IMPDH2) 0/0/0 18/17/21 19.729 scaffold attachment factor B2 (SAFB2_HUMAN, SAFB2) 0/0/0 15/19/21 19.439 spliceosome RNA helicase DDX39B (DX39B_HUMAN, DDX39B) 0/0/0 22/20/12 18.833 ATP-dependent RNA helicase DHX8 (DHX8_HUMAN, DHX8) 0/0/0 13/19/19 18.040 ethylmalonyl-CoA decarboxylase (ECHD1_HUMAN, ECHDC1) 0/0/0 18/20/13 17.866 insulin-like growth factor 2 mRNA-binding protein 2 (IF2B2_HUMAN, IGF2BP2) 0/0/0 21/10/15 16.045 regulation of nuclear pre-mRNA domain-containing protein 1A (RPR1A_HUMAN, RPRD1A) 0/0/0 12/18/15 15.887 14−3−3 protein gamma (1433G_HUMAN, YWHAG) 0/0/0 14/14/16 15.499 double-stranded RNA-binding protein Staufen homologue 1 (STAU1_HUMAN, STAU1) 2/0/0 26/19/22 14.786 acidic fibroblast growth factor intracellular-binding protein (FIBP_HUMAN, FIBP) 0/0/0 14/14/12 14.034 U2 small nuclear ribonucleoprotein A′ (RU2A_HUMAN, SNRPA1) 0/0/0 15/12/12 13.657 interferon-inducible double-stranded RNA-dependent protein kinase activator A (PRKRA_HUMAN, 0/2/5 65/59/55 12.848 PRKRA) DnaJ homologue subfamily B member 6 (DNJB6_HUMAN, DNAJB6) 0/0/0 9/10/17 12.777 RNA-binding protein PNO1 (PNO1_HUMAN, PNO1) 0/0/0 15/16/5 12.516 citron Rho-interacting kinase (CTRO_HUMAN, CIT) 0/0/0 10/14/11 12.335 U5 small nuclear ribonucleoprotein 40 kDa protein (SNR40_HUMAN, SNRNP40) 0/0/0 16/11/8 12.171 26S proteasome non-ATPase regulatory subunit 6 (PSMD6_HUMAN, PSMD6) 5/0/0 32/35/35 12.099 malate dehydrogenase, mitochondrial (MDHM_HUMAN, MDH2) 0/0/0 11/10/13 11.979 RNA binding motif protein, X-linked-like-1 (RMXL1_HUMAN, RBMXL1) 0/0/0 11/13/10 11.947 tubulin alpha-1A chain (TBA1A_HUMAN, TUBA1A) 0/0/0 12/12/8 11.193 14−3−3 protein eta (1433F_HUMAN, YWHAH) 0/0/0 11/11/8 10.504 heat shock 70 kDa protein 4 (HSP74_HUMAN, HSPA4) 0/0/0 10/12/7 10.160 septin-2 (SEPT2_HUMAN, SEPT2) 0/0/0 11/9/9 10.159 probable ATP-dependent RNA helicase DDX47 (DDX47_HUMAN, DDX47) 0/0/0 14/9/6 10.061 huntingtin (HD_HUMAN, HTT) 0/0/0 11/9/8 9.793 complement component 1 Q subcomponent-binding protein, mitochondrial (C1QBP_HUMAN, C1QBP) 0/0/0 12/9/7 9.761 polypyrimidine tract-binding protein 1 (PTBP1_HUMAN, PTBP1) 7/0/2 48/51/39 9.695 protein diaphanous homologue 1 (DIAP1_HUMAN, DIAPH1) 0/0/0 6/7/14 9.613 14−3−3 protein theta (1433T_HUMAN, YWHAQ) 0/0/0 7/9/11 9.558 zinc finger RNA-binding protein (ZFR_HUMAN, ZFR) 0/0/2 13/22/21 9.434 nuclear receptor coactivator 5 (NCOA5_HUMAN, NCOA5) 0/0/0 11/9/7 9.427 probable ATP-dependent RNA helicase YTHDC2 (YTDC2_HUMAN, YTHDC2) 0/0/0 7/10/9 9.181 RNA-binding protein Musashi homologue 1 (MSI1H_HUMAN, MSI1) 0/0/0 8/8/10 9.170 ribosome biogenesis protein BRX1 homologue (BRX1_HUMAN, BRIX1) 0/0/0 10/7/9 9.115 scaffold attachment factor B1 (SAFB1_HUMAN, SAFB) 3/3/3 43/44/42 9.110 ancient ubiquitous protein 1 (AUP1_HUMAN, AUP1) 0/0/2 17/20/17 9.034 pre-mRNA-splicing factor ISY1 homologue (ISY1_HUMAN, ISY1) 0/0/0 7/9/9 8.826 14−3−3 protein beta/alpha (1433B_HUMAN, YWHAB) 0/0/0 9/8/8 8.771 lysophospholipid acyltransferase 7 (MBOA7_HUMAN, MBOAT7) 0/0/0 6/13/5 8.450 fanconi anemia group D2 protein (FACD2_HUMAN, FANCD2) 0/0/0 8/8/8 8.438 cytoplasmic dynein 2 heavy chain 1 (DYHC2_HUMAN, DYNC2H1) 0/0/0 9/7/8 8.416 DnaJ homologue subfamily B member 11 (DJB11_HUMAN, DNAJB11) 0/0/0 12/6/6 8.329 pre-mRNA-splicing factor SYF1 (SYF1_HUMAN, XAB2) 0/0/0 0/11/11 8.269 T-complex protein 1 subunit beta (TCPB_HUMAN, CCT2) 0/0/0 4/10/9 8.181 multiple myeloma tumor-associated protein 2 (MMTA2_HUMAN, MMTAG2) 0/0/0 6/8/9 8.137 60S ribosomal protein L7-like 1 (RL7L_HUMAN, RPL7L1) 0/0/0 6/9/8 8.127 casein kinase II subunit alpha (CSK21_HUMAN, CSNK2A1) 0/0/0 11/8/4 7.974 probable serine carboxypeptidase CPVL (CPVL_HUMAN, CPVL) 0/0/0 13/9/0 7.865 filaggrin-2 (FILA2_HUMAN, FLG2) 0/0/0 14/8/0 7.843 oxygen-dependent coproporphyrinogen-III oxidase, mitochondrial (HEM6_HUMAN, CPOX) 0/0/0 6/7/9 7.782 14−3−3 protein epsilon (1433E_HUMAN, YWHAE) 0/3/3 34/32/21 7.734 glutaminyl-peptide cyclotransferase (QPCT_HUMAN, QPCT) 0/0/0 8/6/8 7.727 annexin A2 (ANXA2_HUMAN, ANXA2) 0/0/0 9/7/6 7.684 nicotinamide/nicotinic acid mononucleotide adenylyltransferase 1 (NMNA1_HUMAN, NMNAT1) 0/0/0 9/10/3 7.651

1643 DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648 Journal of Proteome Research Article Table 1. continued

spectral counts (SCs) in replicates 1/2/3 control vector NS1 vector NS1/CV protein name (protein accession number, gene name) (CV) (NS1) ratioa ribosome biogenesis protein NSA2 homologue (NSA2_HUMAN, NSA2) 0/0/0 10/9/3 7.630 heterogeneous nuclear ribonucleoprotein A3 (ROA3_HUMAN, HNRNPA3) 13/13/14 164/170/148 7.594 fanconi anemia group I protein (FANCI_HUMAN, FANCI) 3/0/0 17/6/21 7.563 protein ELYS (ELYS_HUMAN, AHCTF1) 0/0/0 0/13/7 7.515 pre-mRNA-processing factor 19 (PRP19_HUMAN, PRPF19) 0/0/2 15/17/13 7.513 aldose reductase (ALDR_HUMAN, AKR1B1) 0/0/0 6/7/8 7.416 MKI67 FHA domain-interacting nucleolar phosphoprotein (MK67I_HUMAN, NIFK) 3/0/0 12/14/16 7.243 aThe value was obtained by the mean normalized SC of the NS1 vector (NS1) divided by that of the control vector (VC). Proteins with ratios > mean + 2SD (7.194) are defined as an NS1 interacting partner.

Table 2. Enrichment Analysis of Biological Processes for NS1-Interacting Proteins

biological identified proteins involved in the processa process p value RNA splicing/ DHX8, PABPN1, PRPF19, HNRNPA3, 1.01 × 10−5 processing SNRPA1, DNAJB11, PTBP1, ISY1, SNRNP40, XAB2 cellular YWHAG, YWHAH, HTT, YWHAB, 4.91 × 10−3 macromolecule YWHAQ, YWHAE, STAU1 localization protein PABPN1, YWHAG, YWHAH, HTT, 8.03 × 10−3 transport/ YWHAB, YWHAQ, YWHAE targeting protein folding HSPBP1, DNAJB11, CCT2, DNAJB6, 9.26 × 10−3 HSPA4 aDAVID (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 identified proteins involved in pathway p value spliceosome DHX8, PRPF19, HNRNPA3, 1.47 × 10−5 SNRPA1, ISY1, SNRNP40, XAB2 neurotrophin YWHAG, YWHAH, YWHAB, 1.88 × 10−4 signaling pathway YWHAQ, YWHAE, PIK3R2 cell cycle YWHAG, YWHAH, YWHAB, 1.34 × 10−3 YWHAQ, YWHAE Figure 2. fi a PPI network analysis of proteins identi ed as coimmuno- DAVID was applied to functionally annotate enriched proteins. The precipitating with NS1. A PPI network of the 64 proteins listed in knowledge base used was the KEGG pathway database. Processes with Table 1 was constructed using the STRING v10 database (http:// fi at least ve protein members and P values less than 0.01 are string-db.org/), depicting 53 interaction links between individual fi considered signi cant. nodes/proteins (solid lines). Two modules, both of which involve more than 10 nodes/proteins, are shown. One depicts interactions of protein complexes. On the basis of the bioinformatics analysis, PRPF19 with proteins involved in RNA splicing/processing, and the PRP19 is involved in the RNA splicing biological process other depicts interactions of YWHAE with proteins involved in the cell (Table 2), the spliceosome functional network (Table 3), and cycle. the PPI of proteins involved in RNA splicing/processing (Figure 2). As shown in Figure 4, panel A, in NS1-expressing fl cells, the PRP19 protein coimmunoprecipitated with 3xFLAG- in Figure 4, panel B, in uenza NS1 was coprecipitated with PRP19, demonstrating that PRP19 and NS1 coexist in protein tagged NS1, showing that PRP19 is a component of NS1- fl associated protein complexes. complexes during in uenza A virus infection. To investigate the role of splicing factor PRP19 during Cellular Splicing Factor PRP19 Is Involved in Influenza Viral influenza A virus infection, we further examined the NS1- Replication PRP19 interaction during viral infection. The 293T cells were To determine the role of the cellular PRP19 protein in transfected for 24 h with a plasmid expressing Myc-tagged influenza A viral replication, we employed a specific siRNA PRP19 and then infected with influenza A PR8(H1N1) virus. targeting PRP19 to reduce the endogenous expression of this At 12 h postinfection, cell lysates were collected and subjected protein in A549 cells. At 24 h after siRNA transfection, cells to immunoprecipitation with an anti-Myc antibody. As shown were infected with PR8(H1N1) virus at an MOI of 0.001, and

1644 DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648 Journal of Proteome Research Article

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 3xFLAG- tagged 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-β- actin 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 NS1- expressing 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 Figure 5. Decreasing endogenous PRP19 protein expression quantitated with ImageJ (B, upper-left panel). The expression of the significantly reduces influenza A virus replication. (A) A549 cells NS1 protein was examined by immunoblotting with an anti-NS1 were transfected for 24 h with either control siRNA or siRNA antibody (B, lower panel). Quantitative PCR was applied to determine targeting PRP19, followed by infection with influenza A virus the relative amounts of spliced and unspliced mRNA (C). 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 viral replication was monitored at 24 and 48 h postinfection. determination by the plaque formation assay. The experiment was We found that decreasing endogenous PRP19 expression performed in triplicate, demonstrating similar trends of differences; the fi significantly reduced replication of influenza A virus (Figure gures show one set of results. (B) The viability of A549 cells transfected with control or PRP19 siRNA was determined and 5A) and that the transient knockdown of PRP19 expression did ∗ fi not affect cell viability (Figure 5B). This finding suggested that compared using the MTT assay. , P < 0.05; n.s., not signi cant. the cellular splicing factor PRP19, which interacts with influenza NS1 protein, could be involved in influenza A viral previous reports in which NS1-interacting host proteins were replication. identified using the yeast-two-hybrid approach,25,34 three human proteins, PIK3R2, PRKRA, and STAU1, were also ■ DISCUSSION found in our research. In addition, 13 of 64 proteins detected in The NS1 protein of influenza A virus functions as a modulator this study, including PIK3R2, PABPN1, IGF2BP2, STAU1, that regulates several cellular events in infected cells such as PRKRA, RBMXL1, TUBA1A, PTBP1, HNRNPA3, and several cellular antiviral responses and mRNA maturation processes. In YWHA proteins, were also found by exploring influenza NS1- this study, we applied coimmunoprecipitation and LC−MS/MS interacting partners using tandem affinity purification (TAP) approaches to systemically analyze NS1-interacting proteins in and LC−MS/MS.35 However, our NS1 interactome profile did human cells. The interactome analysis demonstrated that the not reveal components of the nuclear pore complex, including NS1 protein may interact with host proteins involved in RNA NXF1, RAE1, NUP98, p15, and E1B-AP5, which were splicing/processing, cellular macromolecule localization, pro- previously identified by a binding assay using a GST-tagged tein transport/targeting, and protein folding. Comparing to NS1 protein and 293T cell lysates.36

1645 DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648 Journal of Proteome Research Article

We also demonstrated that expression of influenza NS1 terminal deoxynucleotidyl transferase (TdT) or other cellular protein interferes with an mRNA splicing event. Among the factors such as CDC5L, PLRG1, and SPF27 to contribute to identified proteins, the interaction between NS1 and PRP19, DNA repair response.47,48 In addition to the functions which is involved in pre-mRNA splicing and the DNA damage described above, PRP19 is also an E3 ubiquitin ligase that response, was confirmed in NS1-expressing or influenza A mediates K63 ubiquitination of spliceosomal protein PRP3 for virus-infected cells by coimmunoprecipitation. We further several cellular responses. However, as it has not been investigated the role of PRP19 in replication of influenza A determined whether the NS1 protein encoded by the influenza virus and showed that decreasing PRP19 expression in a human A virus is involved in these activities within host cells, the NS1- lung cell line could reduce propagation of the virus. This result PRP19 interaction may influence more cellular responses other indicates the importance of the cellular PRP19 protein in than pre-mRNA splicing events. influenza A viral replication. In addition to proteins that are highly correlated with RNA The PRP19 protein was originally identified as a component splicing/processing biological processes, proteins involved in in the spliceosome that serves as an E3 ubiquitin ligase and cell cycle were also identified by our NS1 interactome and mediates lysine 63 (K63) ubiquitination of the U4 spliceosomal biological process analysis. Furthermore, the present systemic protein PRP3 to stabilize the U4/U5/U6 spliceosomal analysis of NS1-interacting proteins reveals that several complex.37 Therefore, it is reasonable to propose that members of the 14−3−3 protein family may interact with the interaction between PRP19 and NS1 might modulate splicing influenza NS1 protein including 14−3−3 β/α, ε, γ, η, and θ events in host cells. As in other studies, our results showed that (encoded by genes YWHAB, YWHAE, YWHAG, YWHAH, overexpression of NS1 can suppress cellular pre-mRNA and YWHAQ). It has been found that 14−3−3 proteins are splicing.21,22 involved in regulating cell cycle, apoptosis, and protein Moreover, previous investigations have revealed that the trafficking.49 Among the identified 14−3−3 proteins, 14−3−3 PRP19 protein associates with other cellular factors in a ε, also named mitochondrial import-stimulation factor L complex named Prp19C. Different PRP19 complexes involved subunit, is an adaptor protein that binds to signaling proteins in several cellular processes are found in eukaryotic cells, and with phospho-serine residues to regulate various cellular processes such as stress-mediated transcription, apoptosis Prp19C has a well-known function in cellular pre-mRNA − splicing.38,39 ISY1, a component of PRP19 complexes that inhibition, and TNF-α-induced NF-κB activation.50 52 More function in splicing, was also identified in our systemic NS1 importantly, the 14−3−3 ε protein plays a critical role in interactome analysis. This finding demonstrates that the NS1 directing activated RIG-I to mitochondrial-associated mem- protein may interact with Prp19C and affect the pre-mRNA branes for interacting with MAVS and sequentially initiating the 53 splicing function of these complexes. In addition to PRP19, antiviral response. Consequently, the NS1 protein of several factors involved in pre-mRNA splicing, such as influenza A virus may reprogram host antiviral responses via SNRNP40 and PTBP1, were identified as components of its interaction with 14−3−3 ε. Further study is warranted to NS1-associated protein complexes, indicating that NS1 may explore the unknown mechanisms by which influenza NS1 also interact with other splicing factors to modulate host protein counteracts host antiviral responses. splicing events. Although it has been proposed that influenza NS1 protein may bind to the 30-kDa subunit of cellular ■ ASSOCIATED CONTENT cleavage and polyadenylation specific factor (CPSF30) and *S Supporting Information 23,40 interfere with cellular mRNA polyadenylation and splicing, The Supporting Information is available free of charge on the other studies have shown that NS1 encoded by PR8(H1N1) 41 ACS Publications website at DOI: 10.1021/acs.jproteo- virus does not bind to CPSF30. Therefore, the interaction of me.6b00103. splicing factors and the NS1 protein could be more relevant to fi changes in host cellular mRNA splicing events. In addition, List of proteins identi ed in immunoprecipitation product of vector control and NS1 protein; spectral NS1 possesses an RNA-binding domain and can bind to various fi RNAs including U6 snRNA.22 Such binding also plays a role in counting-based identi cation of proteins coimmunopre- suppressing cellular pre-mRNA splicing.42 Nevertheless, the cipitating with NS1 (PDF) present study focused on systemic interactome analysis of the NS1 protein and further demonstrated that NS1 may interact ■ AUTHOR INFORMATION with several host proteins involved in RNA processing. Corresponding Authors fi fl Collectively, these ndings suggest that in uenza A virus may *Phone: 886-3-2118800, ext. 5093. E-mail: [email protected]. alter cellular RNA processes via its encoded NS1 protein. fl edu.tw. also occurs for two in uenza mRNAs, *Phone: 886-3-2118800, ext. 3775. E-mail: [email protected]. M1 and NS1, to generate several spliced forms of viral RNAs edu.tw. including M2, mRNA3, M4, and NS2 mRNAs. The generation Notes of viral spliced RNAs is highly regulated and requires host splicing machinery.43 Although previous studies have raised the The authors declare no competing financial interest. question of whether influenza NS1 affects the splicing of viral mRNAs,33,44,45 the biological impact of interaction between ■ ACKNOWLEDGMENTS NS1 and host splicing factors on viral mRNA splicing requires We thank Dr. Woan-Yuh Tarn for materials and Dr. Chi-Jene further investigation. Chen for helpful discussion. This work was supported by grants On the basis of the factors identified in the PRP19 to Chih-Ching Wu from the Ministry of Science and complexes, it has been suggested that these complexes may Technology (MOST), Taiwan (102-2320-B-182-029-MY3, participate in transcription, DNA repair, and proteasomal 103-2325-B-182-007, and 103-2632-B-182-001) and the degradation.46 For example, PRP19 may form a complex with Chang Gung Memorial Hospital (CGMH), Taiwan

1646 DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648 Journal of Proteome Research Article

(CLRPD190015, CMRPD2B0053 and BMRPC77) and grants (13) Kawaguchi, A.; Nagata, K. De novo replication of the influenza to Rei-Lin Kuo from the MOST (103-2321-B-182-011 and virus RNA genome is regulated by DNA replicative helicase, MCM. 104-2321-B-182-003) and the CGMH, Taiwan EMBO J. 2007, 26 (21), 4566−75. ’ (CMRPD1E0441−3 and BMRPC09). (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 ■ REFERENCES viral RNA synthesis. Journal of virology 2001, 75 (4), 1899−908. (1) Dawood, F. S.; Jain, S.; Finelli, L.; Shaw, M. W.; Lindstrom, S.; (15) Momose, F.; Naito, T.; Yano, K.; Sugimoto, S.; Morikawa, Y.; Garten, R. J.; Gubareva, L. V.; Xu, X.; Bridges, C. B.; Uyeki, T. M. Nagata, K. Identification of Hsp90 as a stimulatory host factor involved Emergence of a novel swine-origin influenza A (H1N1) virus in in influenza virus RNA synthesis. J. Biol. 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1648 DOI: 10.1021/acs.jproteome.6b00103 J. Proteome Res. 2016, 15, 1639−1648