interactome analysis of the ev71 5′ untranslated region in...

Proteomics 2016, 16, 2351–2362 2351DOI 10.1002/pmic.201600098

RESEARCH ARTICLE

Interactome analysis of the EV71 5′ untranslated region

in differentiated neuronal cells SH-SY5Y and regulatory

role of FBP3 in viral replication

Hsing-I Huang1,2,3,4∗, Ying-Ying Chang3, Jhao-Yin Lin3, Rei-Lin Kuo1,2,3,4, Hao-Ping Liu5,Shin-Ru Shih1,2,3,6 and Chih-Ching Wu2,3,7,8

1 Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Kwei-Shan,Tao-Yuan, Taiwan

2 Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University,Kwei-Shan, Tao-Yuan, Taiwan

3 Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan,Taiwan

4 Department of Pediatrics, Chang Gung Memorial Hospital, Linkou, Taiwan5 Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan6 Clinical Virology Lab, Department of Medical Technology, Chang Gung Memorial Hospital, Linkou, Taiwan7 Molecular Medicine Research Center, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan8 Department of Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan

Received: February 14, 2016Revised: April 13, 2016Accepted: June 8, 2016

Enterovirus 71 (EV71), a single-stranded RNA virus, is one of the most serious neurotropicpathogens in the Asia-Pacific region. Through interactions with host proteins, the 5′ untrans-lated region (5′UTR) of EV71 is important for viral replication. To gain a protein profile thatinteract with the EV71 5′UTR in neuronal cells, we performed a biotinylated RNA-protein pull-down assay in conjunction with LC–MS/MS analysis. A total of 109 proteins were detected andsubjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analy-ses. These proteins were found to be highly correlated with biological processes including RNAprocessing/splicing, epidermal cell differentiation, and protein folding. A protein–protein inter-action network was constructed using the STRING online database to illustrate the interactionsof those proteins that are mainly involved in RNA processing/splicing or protein folding. More-over, we confirmed that the far-upstream element binding protein 3 (FBP3) was able to bind tothe EV71 5′UTR. The redistribution of FBP3 in subcellular compartments was observed afterEV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibitedviral replication. Our results reveal various host proteins that potentially interact with the EV715′UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells.

Keywords:

Differentiated neuronal cells / EV71 / FBP3 / Internal ribosomal entry site /Microbiology

� Additional supporting information may be found in the online version of this article atthe publisher’s web-site

Correspondence: Dr. Chih-Ching Wu, Department of MedicalBiotechnology and Laboratory Science, College of Medicine,Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan Tao-Yuan 33302, TaiwanE-mail: [email protected]

Abbreviations: EV71, Enterovirus 71; FBP, far upstreamelement-binding protein; FMDV, foot-and-mouth disease virus;hnRNP, heterogeneous nuclear ribonucleoprotein; hr p.i., hours

postinfection; IRES, internal ribosomal entry site; ITAFs, IREStransacting factors; KEGG, Kyoto Encyclopedia of Genes andGenomes; MOI, multiplicity of infection; PCBP, poly(rC) bind-ing protein; PPI, protein–protein interaction; PTB, polypyrimidinetract binding protein; RNP, ribonucleoprotein; UTR, untranslatedregion∗Additional corresponding author: Dr. Hsing-I HuangE-mail: [email protected].

Colour Online: See the article online to view Figs. 1–4 in colour.

C© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

2352 H.-I Huang et al. Proteomics 2016, 16, 2351–2362

Significance of the study

Enterovirus 71 (EV71) is one of the most serious neurotropicpathogens in the Asia-Pacific region. As a single-strandedRNA virus, EV71 relies on its 5′ untranslated region (5′UTR)to interact with host proteins that are crucial for the viralreplication. To advance the understanding of the replica-tion of EV71 in neuronal cells, we intent to identify cellu-lar proteins that interact with the EV71 5′ UTR by usinga biotinylated RNA-protein pull-down assay in conjunc-tion with LC–MS/MS analysis. Among the identified 109

proteins, some were found to be highly associated with thebiological processes of RNA processing/splicing includingthe far-upstream element binding protein 3 (FBP3). We fur-ther validated the interaction of the EV71 5′UTR with FBP3,and confirmed that knocking down the expression of FBP3in host neuronal cells indeed significantly inhibited the viralreplication. Our results reveal, for the first time, that FBP3may serve as a positive regulator for EV71 replication in hostcells.

1 Introduction

Enterovirus 71 (EV71) is a positive-sense, single-strandedRNA virus that belongs to the family Picornaviridae. Invasionof the CNS by EV71 can result in serious neurologicaldiseases such as encephalitis, aseptic meningitis, acuteflaccid paralysis, and EV71 can also cause deadly pulmonaryedema [1]. Since 1998, there have been several outbreaksof EV71 across the Asia-Pacific region [2–4]. Due to itsstrong ability to cause neurological complications in youngchildren, several efforts have been made to elucidate thepathogenesis of EV71 and develop antiviral strategies.

The RNA genome of EV71 comprises a single ORF flankedby a 5′ untranslated region (5′UTR) and a 3′UTR that is fol-lowed by a poly (A) tail. The 5′UTR contains a type I internalribosomal entry site (IRES) responsible for translation of theviral genome [5]. The activity of the IRES can be modulatedby host factors, including IRES transacting factors (ITAFs),which determine the efficiency of viral RNA translation. MostITAFs are ribonucleotide-binding proteins involved in RNAtransport, processing, localization, and stability [6]. And aprevious study has shown that ITAFs might function in a celltype specific manner. For example, ITAF45 is only expressedin certain cell types and binds to the IRES of foot-and-mouthdisease virus (FMDV), to promote the binding of eIF4G/4Ato the IRES [7].

Similar to other neurotropic viruses, EV71 is capable ofinvading the CNS and infecting neuronal cells [8]. It has beendemonstrated that the damage to neurons induced by viralinfection can attribute to disease progression and the severityof the pathological condition. For example, varicella zostervirus is able to establish a persistent infection in sensory andautonomic neurons, a situation that can be used to under-stand the latency of this virus [9, 10]. In addition, Japaneseencephalitis virus can infect neuronal cells and subsequentlyevoke the production of proinflammatory mediators, whichmay attribute to the neuroinflammation process [11,12]. How-ever, the manner in which viral replication is regulated andthe interactions between neuronal cells and the virus remainunclear.

In this study, we applied a proteomics approach to identifyhost factors that interact with the EV71 5′UTR, specifically theIRES, in neuronal cells. Most of the identified proteins arenucleotide-binding proteins involved in spliceosome assem-bly and processing, including far upstream element bindingprotein (FBP) 1, heterogeneous nuclear ribonucleoprotein(hnRNP) L, FBP2, hnRNP K, hnRNP M, FBP3, and poly(rC)binding protein 1 (PCBP1). Recent studies have shown thatmany splicing-associated proteins are involved in regulatingviral replication. For example, cleaved hnRNP M has beenreported to facilitate enteroviral infection, and hnRNP C canbind to the negative RNA strand of poliovirus to stabilize thecomplex and promote the synthesis of positive RNA strands[13, 14]. A portion of the proteins identified by our analy-sis does not appear to possess nucleic acid binding capacity.These proteins may have been pulled down through interac-tions with proteins bound to the viral RNA, and the functionsof these proteins are mainly related to protein folding andglycolysis.

Among the identified proteins, FBP3 was selected for fur-ther confirmation. FBP1, FBP2, and FBP3 comprise a familyof single-stranded DNA-binding proteins that has recentlybeen shown to function as RNA-binding proteins and to reg-ulate RNA translation and gene stability [15, 16]. Previousstudies have demonstrated that FBP1 and FBP2 are able tomodulate the replication of EV71 through interactions withthe IRES region [17,18]. In this study, we showed for the firsttime that FBP3 interacts with the EV71 IRES and may act asa positive regulator of EV71 replication.

2 Materials and methods

2.1 Cells culture and virus

SH-SY5Y cells were cultured in DMEM supplemented with10% FBS and 1× penicillin/streptomycin (P/S) (all from In-vitrogen, CA, USA). The cells were maintained at 37�C in anincubator with 5% CO2. Cells were passaged at 80% conflu-ence. The clinically isolated strain EV71/Tainan/4643/98 was


Proteomics 2016, 16, 2351–2362 2353

amplified in RD cells cultured in DMEM supplemented with2% FBS and 1× P/S. The titer of the virus was measuredusing a plaque-forming assay.

2.2 Differentiation of SH-SY5Y cells and virus

infection

SH-SY5Y cells (2 × 105/well) were seeded in 12-well platesand incubated for 16 h. The DMEM (10% FBS, 1% P/S)was removed and the plate was subsequently washed using1 mL PBS (Invitrogen). One millimeter DMEM containing1% FBS, 1% P/S, and 10 �M retinoic acid (Sigma-Aldrich,CA, USA) were added and the cells were incubated for 7 days.After the wells were washed with PBS, virus was added ata multiplicity of infection (MOI) of 2 in serum-free culturemedium for 1 h at 37�C. The virus was allowed to attach forapproximately 1 h; the cells were washed with PBS to re-move unbound viruses, and 1 mL of fresh DMEM containing1% FBS and 1% P/S was added.

2.3 In vitro transcription

The construction of plasmid pT7-EV71-5′UTR was previouslydescribed [19]. A T7 promoter EV71-5′UTR IRES fragmentflanked by EcoR I sites was excised from the pCRII-TOPOvector, and in vitro RNA transcripts were obtained using theMEGAscript T7 kit (Ambion, TX, USA). Biotinylated RNAwas synthesized in a mixture containing NTP, biotin-labeledUTP (Roche, Penzberg, Germany), reaction buffer, T7 RNApolymerase, and EV71 IRES DNA. Nonbiotinylated RNA wassynthesized in the same mixture without biotin-labeled UTP.The two mixtures were incubated at 37�C for 4 h. An RNeasyProtect Mini Kit (Nobel, Hilden, Germany) was used to purifythe newly synthesized RNAs.

2.4 SDS-PAGE and in-gel protein digestion

Proteins pulled down from differentiated SH-SY5Y cells wereseparated using 10% SDS-PAGE and silver stained [20].Selected gel bands were subjected to in-gel tryptic diges-tion, as described previously [21, 22]. Briefly, the gel pieceswere destained in 10% methanol (Mallinckrodt Baker, NJ,USA), dehydrated in ACN (Mallinckrodt Baker) and driedin a SpeedVac. The proteins were reduced with 25 mMNH4HCO3 containing 10 mM dithiothreitol (Biosynth AG,Switzerland) at 60�C for 30 min and alkylated with 55 mMiodoacetamide (Amersham Biosciences, UK) at room tem-perature for 30 min. After reduction and alkylation, the pro-teins were digested with sequencing-grade modified porcinetrypsin (20 �g/mL; Promega, Madison, WI, USA) overnightat 37�C. The peptides were extracted with ACN and dried in aSpeedVac.

2.5 Reverse-phase LC–MS/MS analysis

Proteins were identified as described previously [23, 24].Briefly, each tryptically digested peptide mixture wasreconstituted in HPLC buffer A (0.1% formic acid; Sigma-Aldrich) and loaded onto a trap column (Zorbax 300SB-C18,0.3 × 5 mm; Agilent Technologies, Taiwan) at a flow rateof 0.2 �L/min in HPLC buffer A; the mixture was separatedon a resolving 10 cm analytical C18 column (inner diameter,75 �m) using a 15 �m tip (New Objective, Woburn, MA,USA). The peptides were eluted using a linear gradient of0–10% HPLC buffer B (99.9% ACN containing 0.1% formicacid) for 3 min, 10–30% buffer B for 35 min, 30–35% bufferB for 4 min, 35–50% buffer B for 1 min, 50–95% buffer Bfor 1 min, and 95% buffer B for 8 min, all at a flow rate of0.25 �L/min.

The LC apparatus was coupled to a 2D linear ion trapmass spectrometer (LTQ-Orbitrap, Thermo Fisher, CA, USA)that was operated using the Xcalibur 2.0 software package(Thermo Fisher). Intact peptides were detected in the Or-bitrap at a resolution of 30 000. Internal calibration wasperformed using the ion signal of (Si(CH3)2O)6H+ at m/z445.120025 as a lock mass. For the MS analysis, we used adata-dependent procedure that alternated between one MSscan and six MS/MS scans for the six most abundant pre-cursor ions. The m/z values selected for the MS/MS analyseswere dynamically excluded for 180 s. The electrospray voltagewas applied at 1.8 kV. Both MS and MS/MS spectra wereacquired using one microscan with maximum fill times of1000 and 100 ms for the MS and MS/MS analyses, respec-tively. Automatic gain control was used to prevent overfillingof the ion trap. For the generation of MS/MS spectra, 5 × 104

ions were accumulated and resolved in the ion trap. The m/zscan range was set at 350–2000 Da for the MS scans.

2.6 Protein database searching for protein

identification

For database searching, the obtained MS/MS spectra wereanalyzed using the Mascot algorithm (Version 2.1, MatrixScience, Boston, MA, USA) against the Swiss-Prot humansequence database (released Jun 15, 2010, selected for Homosapiens, 20 367 entries) of the European Bioinformatics Insti-tute. The fragment ion mass tolerance was set to 0.5 Da, andthe parent ion mass tolerance was set to 10 ppm, with trypsinas the digestion enzyme. Up to one missed cleavage wasallowed, and searches were performed with the following pa-rameters: variable oxidation on methionine (+15.99 Da) andfixed carbamidomethylation on cysteine (+57 Da). A randomsequence database was used to estimate false-positive ratesfor peptide matches.

After Mascot searching, the obtained files were processedusing the Scaffold software (Version 3.6.5; Proteome Soft-ware, Portland, OR, USA); this software includes the Peptide-Prophet program, which aids in the assignment of peptide MS



spectra, and the ProteinProphet program, which assigns andgroups peptides into a unique protein/protein family whenthey are shared among several isoforms. We used Peptide-Prophet and ProteinProphet probabilities of �0.95 to ensurean overall FDR of <0.5%. Only proteins with two or moreidentified peptides were retained in this study.

2.7 Bioinformatics analysis

Biological process classification and signaling pathway anal-ysis for the proteins identified as interacting with the EV715′UTR were performed with the tools at Database for An-notation, Visualization and Integrated Discovery (DAVID,v6.7, http://david.abcc.ncifcrf.gov/) and Kyoto Encyclopediaof Genes and Genomes (KEGG) (http://www.genome.jp/kegg/pathway.html), respectively [25]. The STRING onlinesoftware (version 10) was used to search for interaction rela-tionships of the proteins identified, and a required confidence(combined score) of >0.9 was used as the cut-off criterion [26].

2.8 Knockdown and overexpression of FBP3

SH-SY5Y cells were seeded in 12-well plates and differenti-ated for 7 days. The differentiated cells were washed with PBS,and fresh Opti-MEM was added; the cells were then incubatedat 37�C. The following procedure was performed in a laminar-flow cabinet. Two tubes were prepared and labeled with Aand B: Tube A contained 150 nM siRNA (Sigma-Aldrich) in100 �L Opti-MEM, and Tube B contained 6 �L LipofectamineRNAiMAX in 100 �L Opti-MEM. The content of Tube B andTube A were mixed and incubated for 30 min. The A + B mix-ture (200 �L) was added to the cells. The cells were incubatedat 37�C for 6 h; the medium was removed, and the cells werewashed. Finally, fresh DMEM (1% FBS, 1% P/S) was addedto the wells, followed by incubation at 37�C for 2 days. ForFBP3 overexpression, the full length FBP3 (NM_003934.1)was amplified by PCR and cloned into the XBaI and BamH1restriction sites of p3xFLAG-myc-CMV-26 expression vector(Sigma-Aldrich). The constructed plasmid was verified by se-quencing. For transfection, cells were transfected with 2 �gof plasmid DNA using lipofectamin 2000 (Invitrogen).

2.9 Immunofluorescence staining

Differentiated SH-SY5Y cells were infected with EV71/Tainan/4643/98 at an MOI of 2, and the cells were har-vested at different time points postinfection. The cells werewashed once with PBS and then fixed with ice-cold fixativesolution (ethanol/methanol, 1:1) for 1 min, followed by block-ing with 0.5% FBS in PBS for 30 min at room temperature.The cells were incubated at 4�C overnight with primary an-tibodies: rabbit anti-doublecortin (Cell Signaling, CA, USA,1:50), rabbit anti-MAP2 (Millipore, Taipei, Taiwan, 1:100),

rabbit anti-FBP3 (Santa Cruz, CA, USA, 1:50), and mouseanti-EV71 3D (1:800). The cells were then washed with PBStwice and then incubated with DyLight 488 conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch Lab-oratories, PA, USA, 1:800) or Dylight594 conjugated donkeyanti-mouse antibodies (Jackson ImmunoResearch Laborato-ries, 1:800). After incubation for 1 h at room temperature,the cells were then washed twice with PBS. The cell nucleiwere counterstained with DAPI (KPL, MD, USA, 1:10 000)for 5 min. The cells were examined under a fluorescencemicroscope system (Olympus, Tokyo, Japan).

2.10 Western blot analysis

To prepare total cell lysates, cells were washed with PBS,and ice-cold protein lysis buffer (50 mM Tris, 1% NP-40, and150 mM NaCl) supplemented with a protease inhibitor cock-tail was added. The cells were incubated on ice for 30 min,and the lysate was centrifuged to collect supernatant. Theprotein concentration of the supernatant was measured us-ing the Bradford method (Bio-Rad Laboratories, CA, USA).Protein sample (30 �g) was separated by 10% SDS-PAGE,and the proteins were transferred onto a polyvinylidene flu-oride membrane (Millipore). The membrane was blockedwith 5% skim milk in TBS Tween-20 buffer at room tem-perature (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl,and 0.1% Tween 20) and then incubated with an anti-EV71 3D (1:5000), anti-FBP3 (Santa Cruz, 1:2000) or anti-�-actin (Sigma-Aldrich, 1:20 000) antibody. Subsequently,the membranes were probed with a HRP-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Lab-oratories, 1:5000). The proteins were then observed usingchemiluminescence (Thermo Scientific, IL).

2.11 Plaque assay

RD cells were seeded in 6-well plates (5.5 × 105 cells per well)and incubated at 37�C in 5% CO2 for 20–24 h. The cells weresubsequently washed once with PBS and infected with 500 �Lof serially diluted virus suspension in serum-free medium.After 1 h of adsorption at 37�C in 5% CO2, the culture mediumwas removed, and the cells were washed twice with PBS toremove unbound viruses. Next, 3 mL of MEM containing 2%FBS and 0.3% agarose was added to each well. Four dayslater, the cells were fixed with 10% formaldehyde for 2 h andstained with crystal violet solution. The virus titer is expressedas pfu/mL.

3 Results

3.1 Differentiated SH-SY5Y cells are susceptible to

EV71 infection

To induce differentiation, undifferentiated SH-SY5Y cellswere treated with retinoic acid [27]. After treatment, the


http://david.abcc.ncifcrf.gov/

http://www.genome.jp/kegg/pathway.html

http://www.genome.jp/kegg/pathway.html

Proteomics 2016, 16, 2351–2362 2355

Figure 1. Differentiated SH-SY5Y cellsare permissive to EV71 infection. (A)Differentiated SH-SY5Y cells were in-fected with EV71 at an MOI of 2, andcytopathic effects were observed at dif-ferent time points. RD cells were usedfor comparison. (B) Total protein wasextracted, and Western blot analysiswas performed to detect the expres-sion of viral proteins in mock- andEV71-infected cells.

formation of long neurites was observed by microscopy (Sup-porting Information Fig. 1A and B), and differentiation wasconfirmed by immunofluorescence staining. The expressionof doublecortin, a protein marker for neural progenitor cells,was downregulated in the differentiated cells (Supporting In-formation Fig. 1C and D), whereas the expression of MAP2was enhanced in the treated cells (Supporting InformationFig. 1E and F). To assess whether the differentiated SH-SY5Y cells are susceptible to EV71 infection, the cells weretreated with EV71 at an MOI of 2. As shown in Fig. 1, theinfected differentiated SH-SY5Y cells exhibited less dramaticcytopathic effects than the RD cells. Most of the differen-tiated cells were still attached to the culture plates, even at24 h postinfection (hr p.i.). Infection was evidenced by vi-ral protein expression, and total proteins were extracted andanalyzed by Western blotting. The expression of EV71 vi-ral protein 3D was detected and first appeared at 16 hr p.i.(Fig. 1B).

3.2 Identification of proteins interacting with the

EV71 5′UTR

To identify proteins that can bind to the EV71 5′UTR, thisRNA fragment was labeled with biotin, and after incubatingwith differentiated SH-SY5Y cell lysates, streptavidin beadswere applied to capture the proteins associated with the bi-otinylated EV71 5′UTR. Pulled down proteins were sepa-rated by SDS-PAGE and silver stained (Fig. 2A). Six bands

were readily observed using the biotinylated EV71 5′UTR incomparison to the nonbiotinylated EV71 5′UTR (lane 4,Fig. 2A). Proteins in these six bands were in-gel digested withtrypsin and analyzed by LTQ-Orbitrap hybrid MS. To elimi-nate nonspecific interaction of proteins, the six correspondingbands in the nonbiotinylated 5′UTR group (lane 3, Fig. 2A)were simultaneously analyzed. Spectral searches were per-formed with the Mascot Server and Swiss-Prot database, andthe results were further integrated using the Scaffold soft-ware. Peptide probability of �0.95 and protein probability of�0.95 cutoffs were used, resulting in the detection of 157and 115 nonredundant proteins with �2 peptide hits in thebiotinylated and nonbiotinylated 5′UTR groups, respectively.Among the 157 proteins, 109 were found in the biotinylated5′UTR group and not identified in the nonbiotinylated 5′UTRgroup. These unique proteins are listed in Supporting Infor-mation Table 3, and the peptide information is shown inSupporting Information Tables 1 and 2.

3.3 Functional analysis of identified proteins

GO and KEGG pathway enrichment analyses were per-formed, and the identified 109 proteins were found to relate toglycolysis/gluconeogenesis, pyruvate metabolism, RNA splic-ing, gap junctions, and pathogenic Escherichia coli infection(Table 1). GO analysis revealed that the identified proteinsare involved in RNA splicing/processing, glycolysis, proteinfolding, cellular protein complex assembly, and negative



Figure 2. Identification of host proteinFBP3 interacting with the EV71 5′UTR. (A)A biotin-labeled EV71 5′UTR was incu-bated with lysates extracted from differ-entiated SH-SY5Y cells. Streptavidin wasapplied to pull down the labeled RNAand associated host proteins. The pro-teins were then eluted and subsequentlysubjected to SDS-PAGE analysis. Lane 1:marker; lane 2: input of differentiated SH-SY5Y cell lysates; lane 3: differentiatedSH-SY5Y cell lysates incubated with thenon-biotinylated EV71 5′UTR; lane 4: dif-ferentiated SH-SY5Y cell lysates incubatedwith the biotinylated EV71 5′UTR. (B) TheEV71 5′UTR was purified, labeled with bi-otin and incubated with lysate extractedfrom differentiated SH-SY5Y cells. 5′UTR-associated proteins were then pulled downwith biotinylated RNA via interaction withstreptavidin. The proteins were washedand eluted for SDS-PAGE electrophoresis.Western blotting was performed, and ananti-FBP3 Ab was used to detect the pres-ence of FBP3. Anti-FBP1 and anti-FBP2 Abswere also used to confirm the presence ofthese two proteins. (C) The expression ofFBP3 was examined in differentiated SH-SY5Y cells using anti-FBP3 antibodies. Cellnuclei were counterstained with DAPI.

regulation of cellular protein metabolic process (Table 2). Asexpected by the fact that we used 5′ UTR RNA to pull downhost proteins, several RNA-binding proteins were identified.However, we noticed that some proteins without nucleotide-binding activity were also identified; these proteins may havebeen pulled down through interactions with RNA-bindingproteins. A protein–protein interaction network of theidentified proteins (Supporting Information Table 3) wasconstructed using the STRING online database. As shown inFig. 3, 154 interaction links between the proteins are present,of which three modules with more than ten protein–proteininteractions are represented. The first module showsinteractions of proteins involved in RNA splicing andmRNA processing, the second module shows interactionsof proteins functionally associated with protein folding andprotein metabolic process, and the third module shows

interactions between proteins associated with glycolysis(Fig. 3).

Many proteins in module 1 (Fig. 3) have been studiedfor their effects in regulating viral expression and propa-gation. Several tubulin genes, such as TUBB3, TUBB4B,TUBA1B, and TUBA1C, are depicted in Fig. 3. Althoughthe role of tubulin in EV71 infection has not yet beendemonstrated, a previous study has shown that FMDVinfection alters the distribution of cytoskeleton [28]. Fur-thermore, RNA-binding proteins have been demonstratedto be able to bind to microtubules and/or microfilaments[29], it is not surprising that our results showed severalmicrotubule proteins. Several glycolysis-related enzymeswere also identified, the presence of which might be ex-plained by their interactions with RNA or RNA-bindingproteins [30].

Table 1. Pathway analysis of EV71 5′UTR-associated proteins

KEGG pathway termsa) Identified proteins p value

Glycolysis/gluconeogenesis PGM2, ALDOA, GPI, LDHB, ALDOC, DLD, PDHB 3.48 × 10−5

Pyruvate metabolism LDHB, AKR1B1, DLD, MDH2, PDHB, MDH1 5.55 × 10−5

RNA splicing/spliceosome PRPF19, HNRNPA3, HNRNPM, HSPA1A, HNRNPC, HNRNPA1 1.03 × 10−3

Gap junctions GNAI3, GNAI2, TUBB2C, TUBA1B, TUBA1C, TUBB3 2.36 × 10−3

Pathogenic E. coli infection ACTG1, TUBB2C, TUBA1B, TUBA1C, TUBB3 2.96 × 10−3

a) Database for Annotation, Visualization, and Integrated Discovery (DAVID) was applied to functionally annotate enriched proteins. Theknowledge base used was the KEGG pathway database. Processes with at least five protein members and p values less than 0.01 wereconsidered significant.


Proteomics 2016, 16, 2351–2362 2357

Table 2. Enrichment analysis of biological processes for EV71 5′UTR-associated proteins in differentiated SH-SY5Y cells

Biological processa) Identified proteins p value

RNA splicing/processing KHDRBS1, STRAP, PTBP1, RBM4, ELAVL1, HSPA1A,ELAVL4, HNRNPA1, HNRNPR, YBX1, PRPF19,HNRNPA3, HNRNPL, HNRNPM, PCBP3, PCBP2,KHSRP, HNRNPC

6.82 × 10−10

Glycolysis ALDOA, GPI, LDHB, ALDOC, MDH2, PDHB, MDH1,PGM2

8.70 × 10−7

Nuclear mRNA splicing, viaspliceosome

HNRNPL, HNRNPA3, HNRNPM, PTBP1, PCBP2,HNRNPC, HNRNPA1, HNRNPR, YBX1

1.18 × 10−5

Protein folding CCT7, CCT4, FKBP4, DNAJC7, CCT2, DNAJB1,STUB1, AIP

2.45 × 10−4

Cellular protein complex assembly SRP54, FKBP4, TUBB2C, H2AFY, TUBA1B, TUBA1C,TUBB3

5.64 × 10−4

Negative regulation of proteinmetabolic process

GPS1, GNAI3, GNAI2, PPP2CA, PSMC1, PPP2R4,SFN, PSMD7, UBA52

2.39 × 10−3

a) Database for Annotation, Visualization, and Integrated Discovery (DAVID) (version 6.7) was applied to functionally annotate enrichedproteins using the annotation category GOTERM_BP_FAT. Processes with at least seven protein members and p values less than 0.01 wereconsidered significant.CCT: chaperonin-containing TCP1 complex.

3.4 FBP3 interacts with the EV71 5′UTR

In addition to the hnRNP family, FBP family members werealso identified, including FBP1, FBP2, and FBP3. Amongthese, FBP1 and FBP2 have been demonstrated to influencethe activity of the EV71 5′UTR IRES [17, 18]. To confirm theinteraction between FBP3 and the EV71 5′UTR, an RNA-protein pull-down assay was performed using differentiatedSH-SY5Y cell extracts. As shown in Fig. 2B, the presence ofFBP3 proteins in the pull-down product was clearly observed.In addition to FBP3, FBP1 and FBP2 were also detected.Furthermore, FBP3 was found to be mainly located in thenucleus of differentiated SH-SY5Y cells, which was revealedby immunofluorescence staining (Fig. 2C).

3.5 EV71 infection redistributes FBP3 in host cells

Although FBP3 is primarily located in the cell nucleus ofdifferentiated SH-SY5Y cells, it could also be detected in thecytoplasm. As shown in Fig. 4, in the early stage of infection,viral protein 3D was only detected in the cytoplasm, and FBP3was mainly located in the cell nucleus. However, the amountof FBP3 in the nucleus was decreased after 8 h of infection.Interestingly, FBP3 appeared as foci in the perinuclear area ofEV71-infected cells (Fig. 4A). Similar results were observedin other tested cells, including SF268, HeLa, and RD cells(Fig. 4B), suggesting that the relocalization of FBP3 uponEV71 infection is a general event in infected cells.

3.6 FBP3 affects EV71 replication

FBP1 and FBP2 have been demonstrated to affect EV71 repli-cation by interacting with the EV71 IRES region. However,

the role of FBP3 in the replication of this virus is unknown.FBP3 siRNA was transfected into differentiated SH-SY5Ycells to inhibit FBP3 expression, and the levels of FBP3 pro-tein were confirmed by Western blotting after 48 h. The re-sults showed that FBP3 was significantly downregulated inthe transfected cells (Fig. 5A). The FBP3 knockdown cellswere then infected with EV71 at an MOI of 2; total cell lysateswere collected at 24 hr and 48 hr p.i. As measured by a plaqueassay, the virus titer in the FBP3 knockdown cells was lessthan in the control cells (Fig. 5B). To examine whether overex-pression of FBP3 exerts beneficial effect in EV71 replication,plasmids encoded FBP3 was transfected into the differenti-ated SH-SY5Y cells. These transfected cells were then infectedwith EV71 at 2 MOI. The protein samples were harvestedand analyzed by Western blotting. Our results showed theexpression levels of viral protein were not affected by overex-pression of FBP3 (Fig. 5C). However, about 28% increase ofproduced viral particles was observed in FBP3-overexpressedcells (Fig. 5D). Therefore, FBP3 might serve as a positiveregulator of EV71 replication.

4 Discussion

A positive-stranded RNA virus releases an uncoated RNAgenome interacting with many host RNA binding proteinsto assist in viral replication. Certain host proteins have beendemonstrated to affect picornavirus replication through as-sociations with the 5′UTR, which contains a cloverleaf-likestructure and an IRES region. For example, polypyrimi-dine tract binding protein (PTB) is essential for translationdriven by the IRES regions of FMDV and Theiler′s murineencephalomyelitis virus [31, 32]. Previous studies haveshown that EV71 replication could be regulated by various



Figure 3. Protein–protein interaction network analysis of proteins identified in EV71 5′UTR pulled down mixtures. The protein–protein interaction network of the proteins listed in Supporting Information Table 3 was constructed using the STRING v10 database(http://string-db.org/), depicting 154 interaction links between individual nodes/proteins (solid lines). Three modules, involving more than10 PPIs, are labeled in red.

RNA-binding proteins, such as PCBP2, hnRNP A1, hnRNPK, FBP1, and FBP2 [17–20, 33]. However, knowledge regard-ing protein interaction with the EV71 5′UTR in neural lineagecells is limited. Differentiated SH-SY5Y cells were chosen inthis study because their properties are similar to neuronalcells [27]. Using a biotinylated RNA-protein pull-down assaycoupled with LC–MS/MS analysis, we identified 109 proteinsin this study. The EV71 5′UTR-associated proteins previouslyhave been profiled in RD and SF268 cells using MALDI-TOFMS. Among the identified proteins, seven proteins, includ-ing PTB1, PTB2, PCBP1, PCBP2, glycyl-tRNA synthase, hn-RNP A1, and hnRNP K, were found in both cell lines. PTB1,PCBP2, and hnRNP A1 were also found in this study, sug-gesting that parts of EV71 5′UTR-interacting proteins may be

commonly expressed in various cell types. Compared to pre-vious reports, we identified more proteins, and were thus ableto perform STRING analysis to illustrate interactions amongputative EV71 5′UTR-interacting proteins.

We divided the identified proteins into three modulesaccording to function: the RNA splicing/mRNA process-ing, the protein folding/metabolic process, and the glycoly-sis/gluconeogenesis groups. The RNA splicing and mRNAprocessing group contains proteins able to bind to nu-cleotides. The proteins in this group include hnRNP A1, hn-RNP A3, hnRNP AB, hnRNP C, hnRNP L, hnRNP M, hnRNPR, PCBP2 (hnRNP E2), and PTBP1 (hnRNP I). Some of thoseproteins have been shown to be capable of modulating viralreplication through different mechanisms [34, 35]. Previous


http://string-db.org/

Proteomics 2016, 16, 2351–2362 2359

Figure 4. Distribution of FBP3 in differentiated SH-SY5Y cells is affected in response to EV71 infection. (A) Differentiated cells were infectedwith EV71 at an MOI of 40. After 8 h, the cells were harvested and double immunofluorescence staining was performed to detect thedistribution of FBP3 in EV71-infected cells. Anti-EV71 3D was used to detect the expression of viral antigen in infected cells; anti-FBP3 Abwas applied to examine the presence of this host protein. Mock-infected cells served as negative controls. A higher magnification of thewhite boxes was shown. The white arrow indicates a cell with a positive reaction for the anti-EV71 3D and anti-FBP3 antibodies (200×; thepicture in third lane, 200× objective with additional 4× computer-generated magnification). (B) HeLa, SF268, and RD cells were infectedwith EV71 at the MOI of 40. Cells were harvested and subjected for immunofluorescence staining to detect the cellular distribution of FBP3in EV71 infected cells (magnification = 200×).



Figure 5. Effect of FBP3 downregulationand overexpression on EV71 replication.(A) Differentiated SH-SY5Y cells (control)were transfected with FBP3 siRNA (siFBP3) or scramble siRNA (scramble), andthe efficiency of knockdown was con-firmed by Western blot analysis. (B) Af-ter transfection, the cells were infectedwith EV71 at an MOI of 2, and the totallysates were collected at 24 and 48 hr p.i.A plaque assay was performed to deter-mine the virus titers in the cell lysates. (C)Control vector and vector encoding FBP3(3×FLAG-FBP3) was transfected into differ-entiated SH-S5Y cells. The expression lev-els of FBP3 were confirmed using Westernblot analysis. (D) These cells were then in-fected with EV71 at MOI of 2. Total lysateswere harvested for plaque analysis.

studies showed PCBP and hnRNP A1 in this module havebeen demonstrated to function as ITAFs in modulating thereplication of EV71 [18–20,33,36]. However, hnRNP M facil-itates enterovirus infection without being involved in IRES-dependent translation or RNA stability [14]. In addition tothe family Picornaviridae, hnRNPs are also implicated in thereplication of other RNA viruses. For example, hnRNP C1/C2functions as a positive regulator in modulating dengue viruspropagation [37]. These results show that hnRNPs may playessential roles in modulating the life cycles of many positive-strand RNA viruses [38].

Proteins associated with protein folding and metabolic pro-cess are grouped into the second module, including heatshock proteins and constituents of the proteasome or ribo-some. Several proteins of the chaperonin-containing TCP1complex are also observed in this category. These proteinsare known to assist the folding of cytoskeleton proteins, suchas actin and tubulin [39]. Intriguingly, cytoskeleton proteinswere previously demonstrated to be associated with RNA-binding proteins and are related to their transport, whichmay be important for RNA localization [40]. Further evi-dence also shows that ribonucleoprotein particles can asso-ciate with cytoskeletal filaments; such cytoskeletal-associated

ribonucleoprotein granules are present in neurons and con-tain RNA, ribosomal subunits, and elongation factors [41].In addition, it has been shown that cytoskeleton-associatedpolysomes are able to efficiently translate protein, which isof great importance for the translation of viral RNA [42]. Thethird module contains proteins that relate to glycolysis. Manyproteins in this category are moonlighting proteins that areable to perform multiple biochemical functions. However, itis currently unknown whether other glycolysis proteins areinvolved in viral replication.

As a member of the FUSE-binding protein family, FBP3,similar to FBP1 and FBP2, is able to bind to single-strandedDNA and regulates the expression of c-myc [43]. All FBPsshare sequence similarity [44]. The present study demon-strates that FBP3 is able to bind to the EV71 5′UTR. Toexamine whether FBP3 influences EV71 replication, a viralgrowth assay was performed using FBP3 knockdown cells.Our data revealed decreased viral titers in FBP3 knockdowncells, indicating FBP3 may be involved in EV71 replication.Previous studies have shown FBP1 and FBP2 can modulatethe EV71 life cycle via different mechanisms [17, 18]. FBP2can serve as an ITAF and negatively modulate viral transla-tion, whereas FBP1 positively regulates EV71 IRES activity


Proteomics 2016, 16, 2351–2362 2361

and thus functions as a positive regulator of viral translation[17,18]. However, the mechanisms associated with the abilityof FBP3 in modulating EV71 replication are not clear.

Our results revealed that FBP3 is redistributed in responseto EV71 infection. In infected cells, the FBP3 protein was ab-sent in the cell nucleus and appeared as foci in the cytoplasm.Many host RNA binding proteins are nuclear shuttling pro-teins, and their distribution is altered upon infection. Theobserved changes in subcellular distribution may due to thedisruption of nuclear cytoplasmic traffic. The shuttling ofFBP3 in infected cells occurs in the early stages of infec-tion. It has been demonstrated that several RNA viruses candisrupt nuclear pore trafficking, resulting in the appearanceof many nuclear factors in the cytoplasm. The 2A proteaseof enteroviruses is capable of cleaving nuclear pore proteinsand thus may be associated with dysregulation of the porecomplex [45]. However, the mechanisms associated with theFBP3 relocalization in EV71 infected cells remain unclear.

In this study, we employed a proteomic technique to iden-tify cellular proteins that may be associated with the EV715′UTR. Furthermore, we showed that FBP3 is able to interactwith the EV71 5′UTR and affects viral replication. Therefore,our results provide useful information for elucidating cellularfactors that may interact with the EV71 5′UTR and supportthe notion that FBP3 regulates EV71 replication.

We thank the Proteomics Core Laboratory, Chang Gung Uni-versity, Taiwan for the technical assistance. This work was sup-ported by grants to Chih-Ching Wu from the Ministry of Scienceand Technology (MOST), Taiwan (102-2320-B-182-029-MY3,103-2325-B-182-007, and 103-2632-B-182-001) and ChangGung Memorial Hospital (CGMH), Taiwan (CLRPD190016,CMRPD2B0053, and BMRPC77), and grants to Hsing-I Huangfrom the MOST (104-2320-B-182-024-MY3) and CGMH, Tai-wan (CMRPD1C0601-3, CMRPD1E0431-4, and BMRPB33).

The authors have declared no conflict of interest.

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