RoleofProteinPhosphatase1inDephosphorylationofEbola ... · 2014-08-08 ·...

Role of Protein Phosphatase 1 in Dephosphorylation of EbolaVirus VP30 Protein and Its Targeting for the Inhibition of ViralTranscription*

Received for publication, April 18, 2014, and in revised form, June 7, 2014 Published, JBC Papers in Press, June 16, 2014, DOI 10.1074/jbc.M114.575050

Philipp A. Ilinykh‡§1, Bersabeh Tigabu‡§1, Andrey Ivanov¶1, Tatiana Ammosova¶�, Yuri Obukhov¶, Tania Garron‡§**,Namita Kumari¶, Dmytro Kovalskyy‡‡§§, Maxim O. Platonov‡‡§§, Vasiliy S. Naumchik‡‡§§, Alexander N. Freiberg‡§,Sergei Nekhai¶�¶¶2, and Alexander Bukreyev‡§**3

From the Departments of ‡Pathology and **Microbiology and Immunology, University of Texas Medical Branch at Galveston,Galveston, Texas 77555, §Galveston National Laboratory, Galveston, Texas 77555, the ¶Center for Sickle Cell Disease andDepartments of �Medicine and ¶¶Microbiology, Howard University, Washington, D. C. 20059, ‡‡Kiev National Taras ShevchenkoUniversity, Kiev 01601, Ukraine, and §§Enamine Ltd., Kiev 01103, Ukraine

Background: The Ebola VP30 is required for viral transcription and can exist in phosphorylated (inactive) and dephosphor-ylated (active) forms.Results: VP30 is dephosphorylated by PP1; the small PP1-targeting molecule 1E7-03 inhibits VP30 dephosphorylation and viraltranscription, thereby blocking viral replication.Conclusion: PP1 plays an important role in Ebola virus transcription.Significance: Targeting PP1 is a feasible approach for inhibition of Ebola virus.

The filovirus Ebola (EBOV) causes the most severe hemor-rhagic fever known. The EBOV RNA-dependent polymerasecomplex includes a filovirus-specific VP30, which is critical forthe transcriptional but not replication activity of EBOV poly-merase; to support transcription, VP30 must be in a dephosphor-ylated form. Here we show that EBOV VP30 is phosphorylatednot only at the N-terminal serine clusters identified previouslybut also at the threonine residues at positions 143 and 146. Wealso show that host cell protein phosphatase 1 (PP1) controlsVP30 dephosphorylation because expression of a PP1-bindingpeptide cdNIPP1 increased VP30 phosphorylation. Moreover,targeting PP1 mRNA by shRNA resulted in the overexpressionof SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, andincreased EBOV transcription, suggesting that cytoplasmicaccumulation of PP1 induces EBOV transcription. Further-more, we developed a small molecule compound, 1E7-03, thattargeted a non-catalytic site of PP1 and increased VP30 dephos-phorylation. The compound inhibited the transcription butincreased replication of the viral genome and completely sup-pressed replication of EBOV in cultured cells. Finally, mutationsof Thr143 and Thr146 of VP30 significantly inhibited EBOV tran-scription and strongly induced VP30 phosphorylation in theN-terminal Ser residues 29 – 46, suggesting a novel mechanismof regulation of VP30 phosphorylation. Our findings suggestthat targeting PP1 with small molecules is a feasible approach to

achieve dysregulation of the EBOV polymerase activity. Thisnovel approach may be used for the development of antiviralsagainst EBOV and other filovirus species.

EBOV,4 along with the closely related Sudan, Bundibugyo,Taï Forest, Reston, Marburg, and Ravn viruses, is a member ofthe family Filoviridae and a biosafety level 4 pathogen, whichcauses the most severe hemorrhagic fevers known in humansand non-human primates, with a mortality rate in humans of upto 90% (1). The latest outbreak of EBOV started several monthsago in Guinea and spread to Liberia and Sierra Leone (2). Cur-rently, there are no approved treatments against filoviruses.Thus, there is an urgent need for universal treatments againstall diverse species and strains of filoviruses.

The ribonucleoprotein complex of EBOV consists of nucleo-protein (NP), phosphoprotein (VP35), and the large subunit ofpolymerase (L). In addition, it also includes the VP30 protein(reviewed in Ref. 3). The polymerase complex can mediate boththe transcription of individual genes and replication of thewhole genome. In the transcription mode, the polymerasesequentially transcribes each gene, starting at the 3�-proximalgene, initiating the transcription at the gene-start signal andterminating at the gene-end signal. In the replication mode, thepolymerase uses the genomic RNA to produce a complemen-tary strand of anti-genome and subsequently generates a newgenomic RNA strand using antigenome as a template. Al-though the exact mechanism of the polymerase switch betweenthe transcription and replication modes is unknown, several

* This work was supported, in whole or in part, by National Institutes of HealthGrants 1 U19 AI109664-01 (to A. B.), 1SC1GM082325, 1P50HL118006-01,8G12MD007597, and 1 U19 AI109664-01(to S. N.). This work was also sup-ported by a departmental start-up grant from the University of Texas Med-ical Branch (to A. B.) and by District of Columbia Developmental Center forAIDS Research Grant P30AI087714 (to S. N.).

1 These authors contributed equally to this work.2 Co-senior author. To whom correspondence may be addressed. Tel.: 202-

865-4545; Fax: 202-865-7861; E-mail: [email protected] Co-senior author. To whom correspondence may be addressed. Tel.: 409-

772-2829; Fax: 409-747-2429; E-mail: [email protected].

4 The abbreviations used are: EBOV, Ebola virus; NP, nucleoprotein; PP, pro-tein phosphatase; eGFP, enhanced GFP; RSV, respiratory syncytial virus;KD, knockdown; MOI, multiplicity of infection; S29 – 46A, mutation of ser-ines 29, 30, 31, 42, 44, and 46 to alanine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 33, pp. 22723–22738, August 15, 2014Published in the U.S.A.

AUGUST 15, 2014 • VOLUME 289 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 22723

by guest on May 30, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

studies pointed to VP30 as a transcription activation factorunique for filoviruses (4 – 6).

EBOV VP30 protein was shown to be phosphorylated at twoserine clusters at positions 29 –31 and 42– 46 and at a threoninein position 52, located close to the RNA-binding domain (7).Phosphorylation of VP30 blocks the ability of the viral poly-merase to function during transcription but not genome repli-cation (5–7). VP30 that was expressed and phosphorylated incultured cells and then immunoprecipitated was dephosphor-ylated in vitro by added catalytic subunits of protein phospha-tase 1, 2A, or 2C (PP1, -2A, or -2C) (7). However, the identity ofthe phosphatase that might dephosphorylate VP30 in culturedcells and control EBOV transcription remained unknown.

The PP1 holoenzyme consists of a constant catalytic subunit(PP1�, PP1�/�, or PP1�) and a variable regulatory subunit thatdetermines the localization, activity, and substrate specificity ofthe phosphatase (8). Major regulatory subunits of PP1, such asNIPP1 (nuclear inhibitor of PP1) or PNUTS (phosphatasenuclear targeting subunit), bind the PP1 catalytic subunit withnanomolar affinity (8). The binding occurs through one or acombination of short binding motifs, such as the well estab-lished RVXF motif and the recently identified SILK, MyPhoNE,SpiDoC, and iDoHA motifs (9). Engagements of different com-binations of these motifs define both the composition of PP1holoenzymes and their unique specificity for different sub-strates in various cell compartments.

We previously showed that PP1 activates human immuno-deficiency virus 1 (HIV-1) transcription and that expression ofNIPP1 or a short central domain of NIPP1 (cdNIPP1) inhibitsHIV-1 transcription and replication (10 –12). We recentlydeveloped a small molecule, 1H4, that targets the RVXF-bind-ing pocket of PP1 and efficiently inhibits HIV-1 (13).

In the present study, we determined whether PP1 regulatesVP30 phosphorylation and whether PP1 can be therapeuticallytargeted for EBOV inhibition. We analyzed EBOV VP30 phos-phorylation in cultured cells and in EBOV virions using high-resolution mass spectrometry that resulted in identification ofthe novel Thr143 and Thr146 phosphorylation sites in additionto the previously identified phosphorylation at the N-terminalSer clusters in positions 29 –31 and 42– 46. We next deter-mined whether PP1 controls VP30 phosphorylation by express-ing its inhibitor cdNIPP1 or using PP1-targeting shRNA.

These experiments showed that expression of PP1-inhibitorycdNIPP increases VP30 phosphorylation and that overexpres-sion of SIPP1, a splicing factor that interacts with PQBP1 (poly-glutamine tract-binding protein 1) and PP1 and shuttles PP1 tocytoplasm (14), decreases it. Next, we developed about 300small molecule compounds based on the 1H4 structure (13)that were targeted to PP1. We showed that the non-toxic com-pound 1E7-03 increases EBOV VP30 phosphorylation andeffectively suppresses replication of EBOV particles in cell cul-tures. Furthermore, we analyzed the effect of 1E7-03 on EBOVtranscription and replication using the mini-genome systemand showed that the molecule reduces the transcription ofEBOV genes but not replication of EBOV genome. Finally, weanalyzed the role of the identified VP30 Thr143 and Thr146

phosphorylation sites. We found that disabling of these sitesincreases VP30 phosphorylation within the serine clusters in

residues 29 –31 and 42– 46 and inhibits EBOV transcription.Our study demonstrates a novel role of PP1 in regulation ofVP30 phosphorylation and suggests a new approach to inhibitEBOV by targeting PP1 by the identified small molecules thatcan be further developed as an EBOV-inhibitory drug.

EXPERIMENTAL PROCEDURES

Design of the 1H4 Derivative Library—The Enamine stockcollection, which included about 2 million small molecule com-pounds at the time the study was initiated, was used for thevirtual screening. After excluding the compounds with reactivegroups, the remaining 600,000 compounds were virtuallydocked to PP1 as we described recently (13). About 300 uniquecompounds that were identified in the virtual screening werefurther evaluated biologically for inhibition of HIV-1 and thelack of toxicity. As a result, a single compound, 1H4, inhibitingHIV-1 transcription was identified. The compound competedwith the RVXF-containing peptide for the binding to PP1,which effectively blocked the interaction between HIV-1 Tatand PP1 (13). To improve binding potency and reduce cytotox-icity, we designed multiple 1H4 derivatives, as described under“Results.”

Analysis of the PP1-targeted Compounds’ Cytotoxicity—Vero-E6 cells and 293T cells were grown to confluence in96-well tissue culture plates. Each compound (1E7, 1E7-03, and1E7-04) was serially diluted in cell medium and added to the cellmonolayer in triplicates. The plates were incubated for 1 or 3days at 37 °C and 5% CO2. After incubation, the effect of eachcompound or DMSO control on the cell viability was deter-mined using Cell Titer-Glo reagent (Promega) according to themanufacturer’s protocol. Non-treated control wells wereassumed to represent 100% cell viability and were used to cal-culate the percentage viability of cells treated with thecompounds.

In Vitro Binding of Recombinant PP1 to the ImmobilizedCompound 1E7—100 nmol of 1E7 analog with an additionalNH2 group were coupled with N-hydroxysuccinimide-acti-vated Sepharose (GE Healthcare) according to the manufactu-rer’s protocol. N-hydroxysuccinimide-activated Sepharosebeads that were blocked with Tris-HCl buffer served as a con-trol. 10 units of recombinant protein PP1 (New England Bio-labs) were incubated for 2 h at 4 °C with the beads containingimmobilized 1E7 in 50 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM

DTT, 0.01% Nonidet P-40; washed three times; and thentrypsinized overnight at 37 °C. Solution above the beads wascollected, dried, and dissolved in 0.1% trifluoroacetic acid(TFA), and the peptides were purified using C18 Ziptips (Mil-lipore) according to the manufacturer’s protocol.

Plasmids—The expression vectors for cdNIPP1, amino acids143–224, fused to eGFP, or its inactive mutant V201A/F203Awere described previously (15). The plasmid pCEZ-VP30expressing EBOV VP30 (16) was kindly provided by Dr. Yoshi-hiro Kawaoka (University of Wisconsin). The EBOV VP30 cod-ing sequence (amino acids 1–246) was amplified by PCR andsubcloned in EcoRI and BamHI sites of PrecisionShuttle mam-malian vector pCMV6-AC-IRES-GFP-Puro (Origene) in framewith Myc and DDK (FLAG) sequences.

Targeting Dephosphorylation of Ebola Virus VP30 by PP1

22724 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 33 • AUGUST 15, 2014


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Analysis of VP30 Phosphorylation—Phosphorylation ofEBOV VP30 in cells was analyzed as described previously (17).Briefly, FLAG-tagged VP30 was expressed in 293T cells orVero-E6 cells. The cells were pulsed with [32P]orthophosphatefor 2 h with or without the addition of okadaic acid or 1E7-03.VP30 was immunoprecipitated with anti-FLAG antibodies(Sigma) and resolved on SDS-PAGE. VP30 expression was ana-lyzed by immunoblotting, and VP30 phosphorylation was ana-lyzed using a Packard Cyclone PhosphorImager (PerkinElmerLife Sciences).

Mass Spectrometry Analysis—FLAG-tagged VP30 was ex-pressed in 293T cells and immunoprecipitated with anti-FLAGantibodies. Recombinant EBOV expressing enhanced greenfluorescent protein (EBOV-eGFP) (18) was propagated inVero-E6 cells, purified on a sucrose gradient, lysed with 10%SDS, and heat-inactivated at 95 °C for 10 min. RecombinantFLAG-tagged VP30 or proteins of the purified virus wereresolved on 10% SDS-PAGE. The gel was stained with Simple-Blue safe stain (Invitrogen), washed with water, and cut intopieces. Gel pieces were crushed into smaller pieces, dehydratedwith acetonitrile (MP Biochemicals), and rehydrated with 50mM ammonium bicarbonate for 5 min. Dehydration and rehy-dration were repeated three times. Reduction and alkylation ofthe cysteine residues was conducted by treating the sampleswith 10 mM DTT for 1 h at 60 °C followed by 50 mM iodoacet-amide in the dark at room temperature for 30 min. TrypsinGold (Promega) reconstituted in 50 mM ammonium bicarbon-ate was added to the gel pieces at a final concentration 10 ng/�l,and the mixture was incubated at 37 °C overnight. Elutedpeptides were collected and dried on a Savant SpeedVac con-centrator (ThermoFisher). Samples were reconstituted in 0.1%TFA and purified using C18 Ziptips (Millipore) according tothe manufacturer’s protocol. Peptides were eluted with 80%acetonitrile containing 0.1% TFA and dried on a SpeedVac. Forthe liquid chromatography-mass spectrometry (LC-MS) pro-cedure, samples were reconstituted in 40 �l of 0.1% formic acid.Next, samples were separated by reversed-phase liquid chro-matography (HPLC), using microcapillary column C18, cou-pled in line with nanospray and tandem mass spectrometerThermoFisher LTQ Orbitrap XL. The LC gradient was run for60 min from 2 to 80% of acetonitrile containing 0.1% formicacid at a flow rate of 400 nl/min. One Fourier transform MSscan and three data-dependent Fourier transform MS/MSscans on major multicharged MS peaks with resolution 30,000were performed in a single measurement block. The normal-ized collision-induced dissociation energy was 35%. TheMS/MS spectra were analyzed against the NCBI human proteindatabase using Proteome Discover version 1.2 software (Ther-moFisher) with the SEQUEST (Scripps Research Institute)search engine. Because the FLAG-tagged VP30 was not presentin the database due to the modifications (the addition of FLAGand Myc sequences), its sequence was manually added to thedatabase. Only peptides having X-correlation (Xcorr) cut-offs of1.0 for [M � 2H]2�, 1.5 for [M � 3H]3�, and higher charge statewere considered. These SEQUEST criteria thresholds resultedin less than 1% false discovery rate.

PP1� Knockdown Cell Lines—Vero-E6 cells were infectedwith pan-PP1 shRNA (human) lentiviral particles and a control

shRNA lentiviral particle (Santa Cruz Biotechnology, Inc.).About 2 � 104 cells were inoculated at 1.1 pfu/1,000 cells. Stableclones were selected in the presence of 5 �g/ml puromycin.Two clones, which demonstrated a significant decrease in PP1�mRNA expression, were selected and used for further studies.

Analysis of PP1� and PP1 Regulatory Subunit mRNAExpression—Total RNA was extracted using TRIzol reagent(Invitrogen). 100 ng of total RNA was reverse-transcribed tocDNA with hexamers and oligo(dT) primers using the Super-scriptTM RT-PCR kit (Invitrogen). Real-time PCR analysis wasconducted on a Roche LightCycler 480 detection system(Roche Applied Science) with SYBR Green. 45 cycles of dena-turation were performed with primers for GAPDH, PP1�,SIPP1, Sds22, or PNUTS with 10 s of denaturation at 95 °C, 10 sof annealing at 60 °C, and 10 s of extension at 72 °C; sequencesof the primers can be provided upon request. Mean Cp valuesand expression levels were determined using ��Cp analysiswith GAPDH as a reference. Unpaired t test was used to evalu-ate statistical significance.

Experiments with EBOV—Experiments with live EBOV-eGFP were performed in BSL-4 facilities of the GalvestonNational Laboratory and Robert E. Shope Laboratory (Univer-sity of Texas Medical Branch). To measure titers of EBOV-eGFP in supernatants of infected Vero-E6 cells, aliquots weretaken every 24 h, frozen, and titrated in Vero-E6 cell monolay-ers under 0.9% methylcellulose/minimum Eagle’s mediumoverlay. After 3– 4 days at 37 °C, fluorescent viral plaques werecounted under a UV microscope.

Experiments with Respiratory Syncytial Virus (RSV)—Tomeasure titers of RSV in HEp-2 cell supernatants, aliquots weretaken on days 4 and 6 postinfection, frozen, and titrated inHEp-2 cell monolayers under a 0.9% methylcellulose/minimumEagle’s medium overlay. After 4 days at 37 °C, viral plaques weredetected using monoclonal antibodies 1243 and 1129 againstRSV F protein (kindly provided by Dr. Peter Collins, NationalInstitutes of Health), followed by incubation with a secondaryHRP-labeled anti-mouse IgG (KPL) and stained with the chro-mogenic 4CN two-component peroxidase substrate system(KPL).

Construction of the Bicistronic Minigenome—To generate thebicistronic minigenome, we used the monocistronic mini-genome construct (19) previously modified by replacement ofthe chloramphenicol gene with the firefly luciferase gene (20),kindly provided by Dr. Elke Mühlberger (19). The NsiI restric-tion endonuclease site was generated downstream of the fireflyluciferase gene by insertion of a G nucleotide using theQuikChange site-directed mutagenesis kit (Stratagene). TheRenilla luciferase gene was PCR-amplified with the EBOV Ltranscriptional termination signal (gene-end) added down-stream the open reading frame and flanking NsiI restrictionendonuclease sites upstream of the open reading frame anddownstream of the gene-end signal and inserted into the NsiIsite of the minigenome plasmid. Thereafter, a C nucleotide wasintroduced to create a BglII restriction endonuclease siteupstream of the Renilla luciferase open reading frame as above,for subsequent insertion of the authentic stem-loop structurerequired for transcription (4). To insert the stem-loop struc-ture, the NP-VP35 gene junction PCR product (nucleotides




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

2,690 –3,128 in the EBOV genome), incorporating the non-coding parts of the genes and the intergenic sequence that formthe stem-loop structure, with NotI and BglII sites introduced atthe termini during amplification, was inserted into the mini-genome using the respective restriction enzyme sites.

Construction of the Mutated VP30 Plasmids—The EBOVVP30 was reported to contain two clusters of serine residuesthat can be phosphorylated: serines 29, 30, and 31 and serines42, 44, and 46 (7). To generate constructs encoding perma-nently “phosphorylated” (VP30phos) or “dephosphorylated”(VP30dephos) forms of VP30 protein, Ser codons in pCEZ-VP30were replaced with either Asp (GAT in amino acid positions 29,30, 31, 42, 44, and 46) or Ala (GCA, GCA, GCC, GCA, GCC, andGCA in positions 29, 30, 31, 42, 44, and 46), respectively, usingthe QuikChange site-directed mutagenesis kit (Stratagene);primer sequences can be provided upon request.

To generate plasmids pCEZ-VP30143D, pCEZ-VP30146D,pCEZ-VP30143D146D, and pCEZ-VP30143A146A with the newlyidentified sites of phosphorylation in positions 143 and 146mutagenized, the two Thr codons were replaced with the Aspcodon GAC (amino acid position 143) and Asp codon GAT(position 146) or Ala codon GCC (position 143) and Ala codonGCA (position 146) using standard molecular biological meth-ods; primer sequences can be provided upon request. To gen-erate constructs expressing mutated forms of VP30 for analysisof their phosphorylation, the VP30 ORFs from the pCEZ-VP30dephos, pCEZ-VP30143D146D, and pCEZ-VP30143A146Aplasmids were cloned in pCMV6-AC-IRES-GFP-Puro vector(OriGene). An additional construct, VP30 S29 – 46AT143A,T146A, was obtained from the VP30 S29 – 46A con-struct by PCR mutagenesis using the same pair of primers as forgeneration of the pCEZ-VP30143A146A plasmid.

Minigenome Experiments—The plasmids pCEZ-NP, pCEZ-VP35, pCEZ-VP30, pCEZ-L, and pC-T7 (16) were kindly pro-vided by Dr. Yoshihiro Kawaoka. For minigenome experiments,the following amounts of plasmids were used: pCEZ-NP, 0.25�g; pCEZ-VP35, 0.25 �g; pCEZ-VP30 or its mutants, 0.15 �g;pCEZ-L, 2.0 �g; pC-T7, 0.5 �g; mono- or bicistronic mini-genome, 0.5 �g. The plasmids were transfected in 293T cellswith Mirus transfection reagent (Mirus Bio). In 48 h, transcrip-tion was measured by a dual luciferase reporter assay (Pro-mega), and RNA was isolated for the analysis of replication ofthe minigenome by Northern blotting. RNA samples wereseparated in 1.5% agarose (Fisher) with 0.66 M formaldehyde(Fisher) and transferred to Protran BA 85 nitrocellulosemembranes (GE Healthcare), UV-immobilized, and hybrid-ized overnight with a luciferase-specific RNA probe of neg-ative polarity, labeled with digoxigenin (Roche Applied Sci-ence). RNA bands were visualized by incubation of themembranes with anti-digoxigenin antibodies labeled withalkaline phosphatase (Roche Applied Science), followed byincubation with ECF substrate (GE Healthcare), and quanti-fied with a Typhoon Tryo PhosphorImager.

RESULTS

Analysis of VP30 Phosphorylation by Mass Spectrometry—We used mass spectrometry to analyze phosphorylation of vec-tor-expressed VP30 and also VP30 in EBOV viral particles. To

analyze the vector-expressed protein, FLAG-tagged VP30 wasexpressed in 293T cells, immunoprecipitated, and resolved bySDS-PAGE (Fig. 1A, lane 2). The high resolution LC-MS/MSanalysis of tryptic peptides extracted from the gel identified thecontinuous VP30 protein sequence with the exception of theC-terminal part, although the C-terminal Myc and FLAG tagswere detected (65% coverage; Fig. 1B, thin lines). Analysis ofpost-translational modifications indicated phosphorylation ofSer29 and Ser30 (Fig. 1, B and C) and also Thr143 and Thr146 (Fig.1, B and D). We next analyzed whether VP30 packaged in EBOVparticles is also phosphorylated. Proteins from purified EBOVparticles were resolved on 10% SDS-PAGE (Fig. 1E) and in-geltrypsinized, and the extracted peptides were analyzed by highresolution LS-MS/MS. The continuous VP30 sequence wasidentified with the exception of the C-terminal part (49% cov-erage; Fig. 1B, thick lines). Analysis of post-translational modi-fications indicated the phosphorylation of Ser42 (Fig. 1F) andalso Thr63, Thr96, Thr143, and Thr146 (not shown; outlined inFig. 1B). These data confirmed the previously reported phos-phorylation of VP30 in the serine clusters of Ser29–Ser31 andSer42–Ser46 (7) and also indicated that Thr63, Thr96, Thr143,and Thr146 might also be phosphorylated. The phosphorylatedThr143 and Thr146 were identified both in the VP30 expressedin cultured cells and that packaged in EBOV (Fig. 1B).

EBOV VP30 Is Dephosphorylated by PP1—To confirm thatVP30 is phosphorylated in cultured cells, FLAG-tagged VP30was expressed in 293T cells, and the cells were treated with[32P]orthophosphate and also with 100 nM okadaic acid. Treat-ment with okadaic acid induced VP30 phosphorylation (Fig.2A, compare lanes 2 and 3). However, okadaic acid is knownto preferentially inhibit PP2A over PP1 (21). Thus, to deter-mine whether VP30 is dephosphorylated by PP1 in culturedcells, PP1-binding peptide, cdNIPP (10), was co-expressedwith FLAG-tagged VP30 in 293T cells. Expression of cdNIPPsignificantly increased VP30 phosphorylation as comparedwith cdNIPP inactive mutant that does not bind PP1 (10)(Fig. 2B), suggesting that PP1 dephosphorylates VP30 in cul-tured cells.

To further analyze the involvement of PP1 in VP30 dephos-phorylation and therefore in EBOV transcription, we generateda PP1� knockdown (PP1 KD) Vero-E6 stable cell line. Cellsexpressing PP1�-targeting shRNA expressed about 2.5 timesless PP1� mRNA compared with the parental cells or a controlcell line that expressed a non-targeting shRNA (Fig. 2C). Unex-pectedly, analysis of VP30 phosphorylation showed a signifi-cantly reduced VP30 phosphorylation in PP1 KD cells com-pared with parental or control shRNA-expressing cells (Fig.2D). Because PP1 functions as a heterodimer, we analyzed PP1distribution in the cells by quantifying expression of its cyto-plasmic and nuclear regulatory subunits. Specifically, we ana-lyzed the expression of SIPP1, a nucleocytoplasmic shuttlingsubunit of PP1 that accumulates in the cytoplasm in UV orX-irradiated cells (14); PNUTS, a major nuclear subunit of PP1(22); and Sds22, a subunit of PP1 that shuttles PP1 to thenucleus (23). SIPP1 mRNA expression was significantlyincreased in PP1 KD cells, whereas the expressions of Sds22 andPNUTS mRNAs were not altered (Fig. 2E). Thus, knockdown of




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

FIGURE 1. EBOV VP30 has two clusters of phosphorylation. A, purification of FLAG-tagged VP30 for MS/MS analysis. FLAG-tagged VP30-expressing 293T cellswere treated with 0.1 �M okadaic acid for 3 h. VP30 was immunoprecipitated, resolved on 10% SDS-PAGE, and stained with colloidal Coomassie Blue. Lane 1,molecular weight markers; lane 2, VP30; lane 3, mock-transfected control. The position of VP30 is shown by an arrow. B, MS/MS analysis of virion-associated andrecombinant VP30, the SEQUEST search results. VP30 was in-gel-digested with trypsin, and the eluted peptides were subjected to MS analysis on a Thermo LTQOrbitrap XL mass spectrometer. Peptides identified with high, median, and low probability are underscored with green, red, and blue lines, respectively; the thinand thick lines depict data for recombinant and virion-associated VP30, respectively. Peptides that were found to be phosphorylated in virion-associated andrecombinant VP30 are overscored with brown and black lines, respectively. The positions of phosphorylated amino acids in recombinant and virion-associatedVP30 are indicated with black and brown arrows, respectively. Note that threonines 143 and 146 were found to be phosphorylated in both virion-associated andrecombinant VP30. C, MS/MS spectrum of the VP30 peptide 29 –36. The colored peaks indicate matched MS/MS fragments. Green indicates precursors, asoutlined in the figure; blue and red indicate y and a ions, respectively. The spectrum gives positive identification of SSSRENYR peptide with the indicatedphosphorylation sites. D, MS/MS spectrum of the VP30 peptide 142–149. The colored peaks indicate matched MS/MS fragments (as described in C). Thespectrum gives positive identification of the ITLLTLIK peptide with the indicated phosphorylation sites. E, electrophoretic separation of the purified EBOV forMS/MS analysis. EBOV was resolved in 10% SDS-PAGE and stained with colloidal Coomassie Blue. Lane 1, molecular weight markers; lane 2, proteins of purifiedinactivated EBOV. F, MS/MS analysis of the peptide 41– 49 from the EBOV-incorporated VP30. The colored peaks indicate matched MS/MS fragments (asdescribed in C). The spectrum gives positive identification of QSESASQVR with the indicated phosphorylation site.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

PP1� leads to a significant increase in PP1 holoenzyme in cyto-plasm, most likely as a compensation for the knockout of thecatalytic subunit. Therefore, the observed reduction in VP30phosphorylation is probably caused by the increased concen-tration of PP1 holoenzyme in the cytoplasm.

Because disabling the transcriptional activity of EBOVpolymerase will prevent synthesis of the viral proteins and virusreplication, it cannot be easily analyzed in virus-infected cells.We therefore analyzed the effects of PP1 KD on EBOV tran-scription by using the previously developed EBOV minigenomesystem (Fig. 2F) (19, 24). The system includes the monocis-tronic minigenome, under control of the T7 promoter contain-ing the firefly luciferase reporter gene under control of EBOV-specific transcription initiation (gene-start) and termination(gene-end) signals, and plasmids expressing the viral proteinsinvolved in transcription and replication (L, NP, VP35, andVP30) under the control of the chicken �-actin promoter.Importantly, without the VP30 transcription activation factor,it is still fully capable of replication but not transcription (19).The T7 polymerase, required to drive the minigenome system,was provided by a co-expressed plasmid. The transcriptionalactivity was analyzed by the luciferase assay. Consistently withthe reduced phosphorylation of VP30 in PP1 KD cells (Fig. 2D),the EBOV transcription was significantly increased (Fig. 2G).Taken together, these results clearly indicate that PP1 isinvolved in regulation of EBOV by controlling the VP30phosphorylation.

Development of PP1-targeted Small Molecules—We previ-ously developed PP1-targeted small molecule 1H4 (13). Toimprove the binding potency and reduce cytotoxicity of thecompound, we designed multiple 1H4 derivatives. To enhancethe interaction with Asp242, Lys260, and Gln292 residues of PP1,the 4th position of the 1,2,3,4-tetrahydroacridine heterocyclewas varied by including aromatic hydrocarbons with differentfunctional groups. To provide more rich hydrogen bondingwith Tyr255, Arg261, and Cys291 and van der Waals interactionswith Phe257, Met290, and Phe293 of PP1 and to facilitate dockinginto the RVXF-accommodating groove, the 9-carboxylic tailwas modified by extending it with aromatic rings while retain-

ing the flexibility. In total, 42 compounds were generated.Based on the ability of the compounds to inhibit HIV-1, whichis sensitive to PP1 inhibition (25), three compounds wereselected for the current study: 1E7, 1E7-03, and 1E7-04 (Fig.3A). To confirm that the 1E7 family of compounds interactswith PP1, an analog of 1E7 was synthesized with an unsubsti-tuted terminal NH2 group that was used to couple the com-pound to N-hydroxysuccinimide-Sepharose beads. Mass spec-trometer analysis of the purified enzyme incubated with the1E7 analog immobilized on the beads or the control beadsshowed a specific association of PP1 to the beads with theimmobilized 1E7 analog (Fig. 3B).

Comparison of Antiviral Activities of Individual Compoundsof the 1E7 Family—The antiviral effects of the compoundsagainst EBOV-eGFP, which is an infectious virus with replica-tion characteristics identical to those of WT EBOV in culturedcells (18), were tested in Vero-E6 cells. Treatments with 1E7,1E7-03, or 1E7-04 for 30 min prior to the infection with a mul-tiplicity of infection (MOI) of 0.01 pfu/cell resulted in a dose-dependent inhibition of the virus replication (Fig. 3C). Forexample, the addition of 1E7-03 at 10 �M resulted in the reduc-tion of the viral titers on days 2 and 3 by 200- and 147-fold,respectively.

Cytotoxicity of the PP1-targeted Compounds—To test thecytotoxicity of 1E7, 1E7-03, and 1E7-04, Vero-E6 and 293T cellswere treated with the compounds at different concentrations,and cell viability was analyzed on days 1 and 3 (Fig. 4). 1E7-04significantly reduced the viability of both the Vero-E6 and 293Tcells at an IC50 of 10.6 and 32.5 �M, respectively, at day 3. Incontrast, treatment with 1E7 had no effect on cell viability atday 1 and slightly decreased viability of Vero-E6 but not 293Tcells at the greatest concentration tested, 73 �M, at day 3. Treat-ment with 1E7-03 resulted in cell viability similar to that in cellstreated with 1E7 except that the reduction of viability ofVero-E6 on day 3 was even less (Fig. 4).

1E7-03 Increases Phosphorylation of VP30 —We next ana-lyzed the effect of 1E7-03 on EBOV VP30 phosphorylationusing FLAG-tagged VP30 expressed in 293T cells. We observedthat treatment of cells with 1E7-03 at 10 �M concentration

FIGURE 2. EBOV VP30 is dephosphorylated by PP1. A, phosphorylation of EBOV VP30 is increased in the presence of okadaic acid. FLAG-tagged VP30 wasexpressed in 293T cells and metabolically labeled with [32P]orthophosphate with or without 0.1 �M okadaic acid (OA). VP30 was immunoprecipitated withanti-FLAG antibodies, resolved on 10% SDS-PAGE, and either exposed to a PhosphorImager screen (top) or probed for VP30 with anti-FLAG antibodies(bottom). The positions of VP30 are shown by arrows. B, PP1 inhibition induces VP30 phosphorylation: phosphorylation of EBOV VP30 in the presence of WT orthe mutated non-active form of cdNIPP1. Top, FLAG-tagged VP30 and cdNIPP1-eGFP (WT or mutant) were expressed in 293T cells and metabolically labeledwith [32P]orthophosphate. VP30 was immunoprecipitated, resolved on 10% SDS-PAGE, and either exposed to a PhosphorImager screen (top) or probed forVP30 with anti-FLAG antibodies (bottom). Bottom, averages of five independent experiments � S.D. (error bars). The p value was calculated by Student’s pairedt test. C, expression of PP1� mRNA is reduced in PP1 KD cells. Concentrations of PP1 RNA in Vero-E6 cells stably transduced with vectors expressing controlnon-targeting shRNA or PP1�-targeting shRNA were determined by real-time RT-PCR with 18 S ribosomal RNA as an internal control. Shown are means oftriplicate values � S.D. The p value was calculated by Student’s paired t test. D, VP30 phosphorylation is reduced in PP1 KD cells. FLAG-tagged VP30 wasexpressed in Vero-E6 cells untreated (WT) or stably transduced with non-targeting shRNA (Sh) or PP1�-targeting shRNA (KD). The cells were metabolicallylabeled with [32P]orthophosphate, and VP30 was immunoprecipitated with anti-FLAG antibodies, resolved on 10% SDS-PAGE, and either exposed to aPhosphorImager screen (top) or probed for VP30 with anti-FLAG antibodies (bottom). E, expression of SIPP1 is increased in PP1 KD Vero-E6 cells. Concentrationsof the regulatory subunits of PP1 SIPP1, Sds22, and PNUTS were determined by real-time RT-PCR with 18 S ribosomal RNA as an internal control. Shown aremeans of triplicate values � S.D. F, EBOV minigenome. Top, the EBOV genome; overlapping of some genes is shown; the EBOV-specific transcriptionalgene-start and gene-end signals are highlighted in green and red, respectively. Left, the EBOV monocistronic minigenome used in the present experiment andthe bicistronic minigenome used in the experiments shown in Fig. 7. The EBOV leader sequence, which includes the promoter, non-coding sequence, andgene-start, and the EBOV trailer sequence, including the gene-end and non-coding sequence, flank the firefly luciferase ORF in the monocistronic minigenomeand flank the firefly and the Renilla luciferase ORFs in the bicistronic minigenome. Right, the support plasmids encoding the EBOV polymerase complex and T7polymerase. Transcription was analyzed by luminometry; in addition, in the experiments shown in Fig. 7C, the minigenome was used for analysis of replicationby Northern blotting. G, EBOV transcription is increased in PP1 KD cells. Transcription of the monocystronic minigenome in Vero-E6 cells stably transduced withvector expressing PP1�-targeting shRNA: the mean luciferase signals based on triplicate values � S.E. The p value was calculated by Student’s paired t test. WB,Western blot.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

increased phosphorylation of VP30 to a level comparable withthat in cells treated by okadaic acid (Fig. 3D). VP30 expressed inVero-E6 cells was also phosphorylated, and the phosphoryla-tion was increased in the cells treated with okadaic acid (Fig.3E). We also compared VP30 phosphorylation in Vero-E6 and293T cells. VP30 phosphorylation in the presence of okadaicacid or 10 �M 1E7-03 in Vero-E6 cells was comparable with thatin the presence of 10 �M 1E7-03 in 293T cells (Fig. 3F).

The Leading Compound 1E7-03 Effectively Inhibits Replica-tion of EBOV—Focusing on the non-cytotoxic compound 1E7-03, cells pretreated with 1E7-03 at 3 �M and infected with MOI

0.001 pfu/cell did not display any visible eGFP fluorescence upto 5 days postinfection (Fig. 5A, middle). In time-of-additionexperiments, 10 �M 1E7-03 was added to the cells at 24 h priorto the infection, during the infection, and 24 h or 48 h postin-fection with EBOV-eGFP at MOI 0.01 pfu/cell. Adding thecompound 24 h prior to the infection resulted in a lack of anydetectable virus in the medium up to 3 days postinfection andvery low viral titers on the subsequent days. For example, ondays 4 and 6 postinfection, the relative reduction of the viraltiters was 2,750-fold and 5,537-fold, respectively (Fig. 5B). Add-ing the compound at 0, 24, or 48 h postinfection resulted in a

FIGURE 3. Effect of the PP1-targeting compounds on EBOV replication and VP30 phosphorylation. A, chemical structures of the compounds 1E7, 1E7-03,and 1E7-04. B, binding of PP1 to 1E7 analog in vitro. An analog of 1E7 containing an additional NH2 group was coupled with N-hydroxysuccinimide-Sepharoseand incubated with PP1. The beads were treated with trypsin, and the eluted peptides were analyzed by nano-LC connected to an LTQ XL Orbitrap massspectrometer. The PP1 peptide EIFLSQPILLELEAPLK, z � 2, m/z � 977.07 Da, was detected by SEQUEST with high confidence. The bar represents the LC signalamplitude for this mass with tolerance 0.03 Da. C, multistep growth kinetics of EBOV-eGFP in Vero-E6 cells, which were treated with the indicated compounds30 min prior to the infection with EBOV-eGFP at MOI 0.01 pfu/cell. Following 1-h-long adsorption, media were removed, and fresh media containing theoriginal concentrations of the molecules were added. For the samples in which no virus was detected, the values 2-fold below the limit of detection wereassigned. Shown are mean EBOV-eGFP titers � S.E. in the medium, based on triplicate monolayers. D, induction of VP30 phosphorylation by 1E7-03. 293T cellsexpressing FLAG-tagged VP30 were treated with 0.1 �M okadaic acid (OA) or 10 �M 1E7-03 and labeled with [32P]orthophosphate. VP30 was immunoprecipi-tated, resolved on 10% SDS-PAGE, and either exposed to a PhosphorImager screen (top) or probed for VP30 with anti-FLAG antibodies (bottom). TheImageQuant software was used to quantify the PhosphorImager data, which were normalized by dividing phosphorylation values by protein values. The barsshow averages of three independent experiments � S.D. (error bars). The p values were calculated by Student’s paired t test. E, induction of VP30 phosphor-ylation in Vero-E6 cells. Vero-E6 cells expressing FLAG-tagged VP30 were treated with 0.1 �M okadaic acid (OA) and labeled with [32P]orthophosphate. VP30 wasimmunoprecipitated, resolved on 10% SDS-PAGE, and either exposed to a PhosphorImager screen (top) or probed for VP30 with anti-FLAG antibodies (bottom).The bars show unadjusted PhosphorImager mean values � S.D. The p value was calculated by Student’s paired t test. F, comparison of 1E7-03-inducedphosphorylation of VP30 in Vero-E6 and 293T cells; the experiment was performed similarly to that shown in E. WB, Western blot.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

reduction of the viral titers at day 4 by 77-, 33-, and 79-fold,respectively, and at day 6 by 384-, 221-, and 31-fold, respectively(Fig. 5B). Analysis of 10 �M 1E7-03 compound stability in aque-ous solution showed a decrease by 50% after 3 h of incubation at37 °C (Fig. 5C). Consequently, we tested the effect of 1E7-03addition at a 3 �M dose every 24 h, with the first dose adminis-tered 24 h prior to or during infection with MOI 0.01 pfu/cell.We found that each treatment regimen resulted in a dramaticreduction in virus replication. Adding the compound starting at24 h prior to infection resulted in the most effective inhibition.For example, no virus was detected up to 3 days postinfection,equating to a �68,000-fold reduction of the viral titers; on days4, 5, and 6, the relative reductions of the viral titers were25,000-, 136,000-, and 147,000-fold, respectively. When treat-ment started at 0 h (i.e. concurrent with infection), the relativereductions of the viral titers on days 4, 5, and 6 were 794-,

1,080-, and 858-fold, respectively (Figs. 5D and 6). To checkwhether the ability of 1E7-03 to inhibit EBOV replication wasspecific to this virus and not due to an intrinsic toxicity thatmight not be easily detected by viability tests, we analyzed thereplication of a non-related RSV in the presence of 10 �M 1E7-03. The compound did not affect replication of RSV (Fig. 5E),suggesting that it specifically targets replication of EBOV.

1E7-03 Reduces the Transcription but Not Replication ofEBOV Genome—The increase in VP30 phosphorylation wasexpected to disable the transcriptional but not the replicationactivity of EBOV polymerase. We therefore analyzed the effectsof 1E7-03 on transcription and replication by using the EBOVminigenome system described above (Fig. 2F). Because VP30 isrequired for reinitiation of transcription of downstream genes(6), we also generated a bicistronic minigenome (Fig. 2F). Thesecond transcriptional unit, a Renilla luciferase gene, was

FIGURE 4. Viability of Vero-E6 and 293T cells treated with the indicated compounds at the indicated concentrations on days 1 and 3. The valuesrepresent mean percentages of cells with viability of untreated cells considered to be 100% based on triplicate monolayers � S.D. (error bars). Note that theerror bars for Vero-E6 cells are not seen due to their small size.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

flanked by the EBOV-specific gene-start and gene-end tran-scriptional signals and placed immediately downstream of thefirefly luciferase transcriptional unit. Previous studies demon-strated that filovirus non-translated regions upstream of eachopen reading frame form stem-loop structures, which includetranscriptional gene-start signals (26 –28). These stem-loopstructures were shown to be important for VP30-mediatedtranscription (4). We therefore also inserted the authenticEBOV Zaire NP-VP35 gene junction (nucleotides 2,690 –3,128), which includes the non-coding parts of the genes andthe intergenic sequence, and which forms the stem-loop struc-ture (nucleotides 3,029 –3,045), required for transcription (4)(data not shown). As a control, a phosphorylation mimickingVP30 (VP30phos), which abrogates the transcription but notreplication of the mini-genome (7), was created by replacingserines at positions 29, 30, 31, 42, 44, and 46 (7) with aspartic

acid residues. A dephosphorylated form, VP30dephos, whichpromotes transcription, was created by replacing the serineclusters with alanines (7). The construct, along with the plas-mids expressing NP, VP35, L, and VP30 as well as the T7 poly-merase-expressing plasmid, was transfected to 293T cells,which resulted in an effective transcription of both the fireflyluciferase gene (transcriptional unit 1) and the Renilla lucifer-ase gene (transcriptional unit 2). We analyzed the effects of1E7-03, VP30dephos, and VP30phos on transcription of themonocistronic (Fig. 7A) or the bicistronic (Fig. 7B) mini-genomes and replication of the monocistronic minigenome(Fig. 7C). This was achieved by transfecting 293T cells with themono- or bicistronic minigenome; plasmids NP, VP35, L, andone of the following plasmids: VP30, VP30dephos, and VP30phos;and the T7 plasmid. In addition, the cells were also treated with3 �M 1E7-03 where indicated. On day 2 posttransfection, the

FIGURE 5. Effect of the small molecules on replication of EBOV-eGFP in Vero-E6 cells. A, cells on day 5 postinfection with EBOV-eGFP at MOI 0.001 pfu/cell:untreated (left) or treated with 1E7-03 (3 �M) 30 min prior to infection under UV (left, middle) or bright field (BF) microscopy (right). B, effect of the time of 1E7-03addition on multistep growth kinetics of EBOV at MOI 0.01 pfu/ml. 1E7-03 was added at 10 �M 24 h prior to, during, or 24 or 48 h after infection. After 1-h-longadsorption, the medium was removed, cells were washed with phosphate-buffered saline, and fresh medium was added; in the case of treatment, 24 h priorto the infection or during the infection with 1E7-03, fresh medium contained 10 �M 1E7-03. C, concentration of 1E7-03 in water solution. Freshly dissolved1E7-03 at a 1 �M concentration was aliquoted in 50-�l samples. Each sample was incubated at 37 °C for the indicated time and frozen until analyzed. Thesamples were analyzed by electrospray ionization-MS, and the amplitudes of peaks m/z � 504.2123 Da, z � 1 were recorded. D, effect of adding the 1E7-03compound at 3 �M every 24 h starting at the indicated time. B and D, mean EBOV-eGFP titers � S.E. (error bars) in the medium, based on triplicate monolayers.The dotted lines indicate the limit of detection. E, the 1E7-03 compound has no effect on replication of RSV. HEp-2 cells were infected with RSV at MOI 0.1 pfu/mland treated with 1E7-03 at 3 �M daily, as described for EBOV (D). Medium aliquots were collected daily and flash-frozen; RSV was titrated in HEp-2 monolayers,and on day 2, plaques were immunostained using mouse monoclonal antibodies specific for the F protein of the virus and counted.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

cells were harvested, and transcriptional activities of the mono-cistronic and bicistronic minigenomes were analyzed by a sin-gle or dual luciferase assay, respectively. To quantify replicationof the minigenome, total RNA was isolated from collected cells,and the positive-sense anti-genome was analyzed by Northernblotting by hybridizing with the digoxigenin-labeled RNAprobes of negative polarity. Bands were detected by incubationwith alkaline phosphatase-labeled anti-digoxigenin antibodies,followed by incubation with the substrate and phosphorimag-ing (Fig. 7C). The following effects were observed. Consistentwith the previous data (7), expression of VP30 was required fortranscription because the absence of VP30 abrogated the lucif-erase signal (Fig. 7, A and B, column 8 versus column 2). Expres-sion of VP30phos abrogated the transcription (Fig. 7, A and B,column 4 versus column 2), whereas expression of VP30dephoshad no inhibitory effect on transcription (Fig. 7, A and B, col-umn 5 versus column 2), as reported previously (7). VP30phosincreased genome replication (Fig. 7C, lane 7 versus lane 5); theinhibition of EBOV transcription and increase in genome rep-lication by the phosphorylated VP30 was recently demon-strated by Biedenkopf et al. (5). The VP30-dependent regula-tion of EBOV transcription/replication balance appears to besimilar to the regulation of the transcription/replication bal-ance of RSV, for which the transcription anti-termination fac-tor, M2-1, promotes transcription, and the negative regulatorof transcription, M2-2, shifts the balance toward replication(29, 30). However, to our knowledge, no phosphorylation-de-pendent mechanism of regulation of transcription/replicationhas been identified for RSV so far. Treatment of cells with1E7-03 reduced transcription (Fig. 7, A and B, column 3 versuscolumn 2) but increased replication (Fig. 7C, lane 4 versus lane5; the quantitative data are provided in the figure legend). Theseresults are consistent with the observed increase in phosphor-ylation of VP30 in the presence of 1E7-03 (Fig. 3D) and theobserved abrogation of transcription in the presence ofVP30phos. The effects of VP30 and their mutated forms as well

as 1E7-03 were similar for both the upstream and the down-stream genes (Fig. 7B, top and bottom, respectively). Thus,1E7-03 prevents VP30 dephosphorylation by PP1 and preventsthe effective transcription of the viral genes, which in turn pre-vents the effective replication of the viral particles.

VP30 Phosphorylation at Thr143 and Thr146 Inhibits EBOVTranscription—We next characterized the effects of Thr143 andThr146, which we found to be phosphorylated in VP30expressed in cultured cells or packaged in EBOV virions (Fig. 1).We generated a set of VP30 mutants, in which the threonineresidues were mutated to asparagine residues to mimic phos-phorylation (T143D,T146D) or to alanine to mimic theirdephosphorylation (T143A,T146A). To exclude the effect ofphosphorylation at the N-terminal serine residues (7), we alsogenerated mutants in which the N-terminal serine clusters(Ser29, Ser30, Ser31, Ser42, Ser44, and Ser46) were replaced withalanines to make them permanently dephosphorylated, with orwithout the mutations T143A,T146A. To analyze the effect ofthe Thr143 and Thr146 mutations on VP30 phosphorylation, theFLAG-tagged mutated proteins were expressed in 293T cells.Unexpectedly, not only T143D,T146D but also T143A,T146Amutations led to a significant increase in VP30 phosphorylation(Fig. 8A, lanes 3 and 4). This effect was comparable with theincrease of VP30 phosphorylation induced by 1E7-03 treat-ment (Fig. 8A, lane 5). The increase in VP30 phosphorylationappeared to be due to the phosphorylation of the N-terminalserine clusters because substitution of serines 29, 30, 31, 42, 44,and 46 to alanines (the S29 – 46A mutant) prevented thisincreased phosphorylation (Fig. 8A, lanes 6 and 7). Similarly,treatment with 1E7-03 had no effect on phosphorylation ofVP30 S29 – 46A and VP30 S29 – 46A T143A,T146A mutants(Fig. 8A, lanes 8 and 9). Thus, phosphorylation of Thr143 andThr146 affected the phosphorylation of the N-terminal serinecluster.

We next validated the functional importance of Thr143 andThr146. We generated three VP30 mutants in which threonine

FIGURE 6. UV microscopy of Vero-E6 cells, which were infected with EBOV-eGFP at 0.01 pfu/cell and treated daily with 1E7-03 at 3 �M starting 24 hprior to the infection or during infection. Days 5 and 8 postinfection are shown. No eGFP-positive cells were observed in 1E7-03-treated monolayers on days1– 4, which are therefore not shown.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

residues were replaced with aspartic acid to mimic phosphory-lation, VP30143D, VP30146D, and VP30143D146D, and analyzedthe effects of the mutations on transcription using the EBOVminigenome system described above. Each of the two individ-ual mutations or their combination significantly reduced thelevel of EBOV transcription. Specifically, T143D reduced thetranscription by 61%, T146D by 81%, and their combination by81% (Fig. 8B). Thus, phosphorylation of VP30 Thr143 andThr146 inhibits EBOV transcription, suggesting that theseamino acid residues play a regulatory role in the transcription/replication balance.

DISCUSSION

The present study demonstrated multiple novel findings.First, using high resolution mass spectrometry, we identifiednew phosphorylation sites in the VP30 component of the EBOVpolymerase complex and demonstrated their role in regulation

of EBOV transcription/replication balance. Second, we pro-vided evidence that dephosphorylation of VP30 is controlled byPP1. Third, we designed a small molecule compound, 1E7-03,which effectively binds PP1, induces VP30 phosphorylation,and effectively inhibits replication of EBOV particles. We alsodemonstrated that 1E7-03 shifts the transcription/replicationbalance of the EBOV polymerase toward replication, suggestingthat its antiviral effect results from the imbalance of EBOVpolymerase it causes.

Previously, plasmid-expressed VP30 was shown to be phos-phorylated in cultured cells, and its phosphorylation was signif-icantly decreased with alanine mutations of serines 29 –31 andserines 42, 44, and 46, suggesting phosphorylation of these res-idues by cellular kinases (7). In our study, phosphorylation onSer29 and Ser30 in VP30 expressed in cultured cells and Ser42 inVP30 incorporated in EBOV virions were demonstrateddirectly by mass spectrometry analysis. This is the first study in

FIGURE 7. Effect of 1E7-03 on the transcription and replication of EBOV genome. A and B, analysis of transcription of the monocistronic (A) and bicistronic(B) minigenome by the luciferase assay: mean values based on triplicate samples � S.E. (error bars). Shown is statistical significance of the 1E7-03-mediatedreduction of the transcription (Student’s unpaired t test), lane 3 versus lane 2 (A and B). A, p 0.01; B, gene 1, p � 0.03; gene 2, p � 0.05. C, analysis of replicationof the monocistronic minigenome by Northern blotting; the EBOV miniantigenome bands were quantified by a PhosphorImager. Please note that irrelevantlanes between lanes 6 and 7 were removed. The following values were obtained after background subtractions (lanes 1–7): 228, 1,439, 6,788, 33,881, 25,696,23,621, and 52,504. The experiment was repeated with results essentially similar to those shown in the figure.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

which phosphorylation of virion-incorporated VP30 was con-ducted. Although we observed different residues being phos-phorylated in the N-terminal serine clusters in the recombinantVP30 and the VP30 incorporated in EBOV virions, we cannotexclude the possibility that additional residues in these clusterswere also phosphorylated but not detected in our MS analysis.Furthermore, the levels of phosphorylation of various sites maybe different, as demonstrated for several viruses, including RSV(31), and detection of sites with a lesser level of phosphorylationmay be complicated. Further detailed phosphopeptide map-

ping analysis is needed to analyze VP30 phosphorylation. Also,the fact that EBOV was assembled in Vero-E6 cells and VP30was expressed in 293T cells may have led to the differences inthe N-terminal serine cluster phosphorylation. In addition,VP30 Thr143 and Thr146 residues were found to be phosphory-lated both in cultured cells and virions, and Thr63 and Thr96

were phosphorylated in VP30 expressed in cultured cells. Ourdata complement the previous findings by extending the num-ber of VP30 residues that may be phosphorylated. We furtheranalyzed the phosphorylation and functional importance ofThr143 and Thr146; the role of the other potential sites of phos-phorylation remains to be analyzed.

Previously published studies demonstrated that VP30expressed in cultured cells is effectively dephosphorylated invitro by added purified catalytic subunits of PP1 or PP2A, and,somewhat less effectively, by PP2C, suggesting that in vivo,phosphorylation of VP30 may be controlled by PP1 or PP2A (7).The present study specifically investigated the role of PP1 inphosphorylation of VP30. Treatment of cultured cells with highconcentrations of okadaic acid (IC50 100 nM) over a 24 –36-h-long period was previously shown to inhibit EBOV transcrip-tion (7). Okadaic acid inhibits PP2A at low nanomolar IC50 andPP1 at high nanomolar IC50 (11, 21). However, at high concen-trations (100 nM and above), okadaic acid blocks cellular tran-scription by promoting phosphorylation of cellular RNApolymerase II (32). Thus, a long term incubation of cells withhigh concentration of okadaic acid is likely to produce artifactsdue to its strong cellular effects. We showed that short, 2-h-long, treatment with 100 nM okadaic acid induced VP30 phos-phorylation, which has not been noted before. Moreover, weused a specific PP1 inhibitor, cdNIPP1, which we previouslydemonstrated to be non-toxic when stably expressed in 293Tcells (10), to analyze the role of PP1 in VP30 phosphorylation.Expression of cdNIPP1 increased phosphorylation of VP30,suggesting that it is controlled by PP1. Thus, our study specifi-cally demonstrates for the first time that PP1 is involved indephosphorylation of VP30 under physiological conditions.The PP1� knockdown in cultured cells led to up-regulation ofthe PP1 cytoplasmic shuttling regulatory subunit, SIPP1, andreduction of VP30 phosphorylation. Because SIPP1 has highaffinity to PP1 (33), the increased expression of SIPP1 willtranslate to the higher levels of SIPP1/PP1 holoenzyme in thecytoplasm. This effect also coincided with the increase in EBOVtranscription, suggesting that SIPP1 is likely to target PP1 toVP30. Because PP1 exists as a heterodimer or even a hetero-trimer (PP1/Sds22/inhibitor-3) (23), and some of its catalyticsubunits are interchangeable, complete PP1 knockout is diffi-cult to achieve. The use of PP1-targeting peptides, such ascdNIPP1, is clearly a more straightforward approach. Further-more, in an early study in vivo, NIPP1 was shown to co-purifywith PP1�/� from rat liver extracts (34). The recent crystalstructure of the NIPP1-PP1 complex was determined for PP1�(35). Thus, cdNIPP1 is likely to bind equally well PP1� andPP1�/�. Whether both of these catalytic subunits are involvedin VP30 dephosphorylation remains to be determined. Never-theless, our PP1 KD experiments clearly indicate that PP1 playsa role in EBOV transcription. Further studies are needed toconfirm the role of PP1 holoenzyme in VP30 dephosphory-

FIGURE 8. Role of Thr143 and Thr146 in EBOV transcription. A, mutations ofThr143 and Thr146 increase VP30 phosphorylation. FLAG-tagged VP30 (WT)and its mutated forms T143D,T146D, VP30 T143A,T146A, VP30 S29 – 46A, andVP30 S29 – 46A T143A,T146A were expressed in 293T cells followed by treat-ment of cells with 10 �M 1E7-03. The cells were metabolically labeled with[32P]orthophosphate. VP30 was immunoprecipitated with anti-FLAG anti-bodies, resolved on 10% SDS-PAGE, and either exposed to a PhosphorImagerscreen (top) or probed for VP30 with anti-FLAG antibodies (middle). The quan-titative phosphorylation data were normalized to VP30 expression by divid-ing phosphorylation values by protein values; mean values � S.D. (error bars)are indicated (bottom). The p values were calculated by Student’s paired t test.B, mutations in VP30 Thr143 and Thr146 residues reduce EBOV transcription:transcription of the monocistronic minigenome in the presence of VP30 inwhich threonines in position 143 and/or 146 were substituted with asparticacids to mimic permanent phosphorylation. Mean luciferase signals arebased on six samples � S.E. The p values were calculated by Student’s pairedt test; comparison with non-mutated VP30: *, p � 0.028, **, p � 0.006.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

lation and to identify the regulatory cytoplasmic PP1 subunitthat may target PP1 to VP30. Also, we cannot fully exclude thepossibility that PP2A may also participate in dephosphorylationof certain residues of VP30.

Either alanine or aspartic acid mutation in Thr143 and Thr146

led to a strong induction of VP30 phosphorylation, whichapparently took place in the N-terminal serine clusters becausetheir mutations prevented the induction of phosphorylation,similar to the phenomenon known as “cascade phosphoryla-tion” described for vesicular stomatitis virus (36) and RSV (37).Consistent with that, the phosphorylation mimic mutationsstrongly inhibited transcription of the EBOV genome. Itappears that targeted phosphorylation of Thr143 and/or Thr146

has a strong regulatory effect on VP30 phosphorylation. Iden-tifying a kinase that targets these two residues would be impor-tant for the development of antivirals against filoviruses andmay represent a novel regulatory pathway that may be targetedfor the development of antivirals.

When our study was in progress, Biedenkopf et al. (5)reported that phosphorylation of VP30 shifts the transcription/replication balance of the EBOV polymerase toward replica-tion. Whereas the study of Biedenkopf et al. used strand-spe-cific quantitative PCR, our study used Northern blotting, whichis a more direct method and which complements the study of

Biedenkopf et al. (5). VP30 proteins of filoviruses do not haveany analogs among other non-segmented negative strandviruses, which typically have three proteins: nucleoprotein,phosphoprotein (its filovirus analog is called VP35), and L. Toour best knowledge, RSV and pneumonia virus of mice are theonly other non-segmented negative strand viruses, which haveproteins (M2-1 and M2-2) with functions somewhat similar tofilovirus VP30 (30, 38, 39). However, the M2-1-regulated tran-scription/replication balance is controlled by phosphorylationof the P protein, which interferes with its interaction withM2-1, resulting in the reduction of transcription elongation(40). The other important difference includes the effects ofthese factors on transcription. The RSV M2-1 protein serves asa transcription elongation factor whose absence results in apremature termination of transcription (39), and M2-2 medi-ates a regulatory switch from transcription to replication (30).In contrast, EBOV VP30 is involved in initiation of transcrip-tion, and its absence results in a lack of transcription but not thepremature termination of transcription (4, 19).

Importantly, the small molecule compound generated in thestudy affected the EBOV transcription/replication balance thesame way as permanent phosphorylation of VP30 (i.e. reducingthe transcription and increasing replication of the viralgenome). Martinez et al. (41) demonstrated indirectly that the

FIGURE 9. A model of the effects of 1E7-03 on EBOV. A, during the EBOV life cycle, VP30 exists in two modes: active (dephosphorylated), in which it promotestranscription of the viral genome by the viral polymerase complex, and inactive (phosphorylated), when the polymerase switches to the replication mode. The1E7-03 compound prevents dephosphorylation of VP30 by PP1, thereby skewing the transcription/replication balance toward replication and reducingtranscription, which restricts viral growth. The viral genome of the negative polarity is shown in black with the seven genes depicted as thick bars; the sevenmRNAs are depicted in red, and the anti-genomes of the positive polarity are depicted in blue. B, comparison of the phosphorylation sites of VP30 of variousspecies of filoviruses. The phosphorylated serine (S) residues of EBOV (7) and Marburg virus (48) and the putative phosphorylated residues of additional filovirusspecies are highlighted in red. Shown in the order of PubMed accession numbers are the VP30 N-terminal 60 amino acids of EBOV, Q05323; Sudan virus (SUDV),AY729654.1; Bundibugyo virus (BDBV), YP_003815438.1; Taï Forest virus (TAFV), FJ217162.1; Marburg virus (MARV), P35258; and Ravn virus (RAVV), DQ447649.1.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

dephosphorylated, but not the phosphorylated, form reinitiatesEBOV transcription based on the ability of the permanentlyphosphorylated and dephosphorylated forms of VP30 to sup-port recovery of the virus. In contrast to the study by Martinezet al. (41), this study used a bicistronic minigenome to providedirect evidence that dephosphorylated form of VP30 is requirednot only for initiation but also for reinitiation of transcription.

Our study demonstrated that 1E7-03 strongly inhibits repli-cation of EBOV and provides evidence that the moleculeincreases phosphorylation of the viral VP30. Because the tran-scription of EBOV, and probably other filoviruses, is criticallydependent on VP30 dephosphorylation, the molecule preventsthe effective transcription but increases replication of theEBOV genome. As a result of the transcription/replicationimbalance, viral replication is effectively prevented (Fig. 9A).

The small molecule preventing the effective dephosphory-lation of VP30 presented here, which provides the completeinhibition of EBOV in vitro, represents a novel approach for thedevelopment of antivirals against EBOV, which are urgentlyneeded to combat the devastating disease caused by the virus.The present study suggests that 1E7-03 can be used as a startingpoint for the development of a highly effective drug againstEBOV. A remarkable similarity in the phosphorylation sites atthe N termini of the VP30 proteins of multiple filovirus species(Fig. 9B) suggests that the approach is feasible for developmentof a pan-filoviral drug.

The study demonstrates that with repeated treatments, 1E7effectively suppresses concentration of EBOV at 3 �M concen-tration (Fig. 5D). Our recent study showed that mice injectedwith 25 mg/kg 1E7-03 accumulated 90 �M plasma concentra-tion of the compound up to 8 h (25). Thus, concentrations of 10and 3 �M, which effectively suppress replication of EBOV invitro (Fig. 4, B and D), will be achieved by administering thecompound at 3 and 1 mg/kg, respectively. These doses arecomparable with the active doses of the anti-influenza drugoseltamivir, when administered to children (2 mg/kg) (42), andthe anti-influenza drug zanamivir tested in cynomolgusmacaques (10 –20 mg/kg) (43). Importantly, our leading com-pound 1E7-03 is non-toxic, presumably because it targets onlyone PP1 non-catalytic site and probably prevents the interac-tion of PP1 holoenzyme with EBOV VP30, whereas the host cellPP1 regulatory subunits typically bind PP1 through severalinteracting sites with a much higher affinity (44). Several linesof evidence suggest that sequestering of PP1 per se does notresult in a general toxic effect, unlike the toxic effects of PP1inhibitors, such as microcystin and okadaic acid, that bind tothe active site of PP1 with global PP1 inhibition and cannot beused as therapeutics (45). First, our recent study showed thatPP1-targeted compound 1H4 did not prevent the interaction ofPP1 with major regulatory subunits, such as NIPP1 or PNUTS(13). Second, we recently demonstrated that high doses of1E7-03 administered to mice by injections do not result in atoxic effect (25). Third, a stable expression of PP1-sequesteringcdNIPP1, although inhibitory for HIV-1, did not compromisecell growth (10). Fourth, viable cell lines stably expressingcdNIPP1 (10) and transgenic mice in which genetic PP1 inhibi-tion through induced expression of inhibitor-1 (I-1) resulted in70% reduction of the PP1 activity (46) were developed. There-

fore, it is unlikely that the compound interferes with interac-tions of PP1 with its cellular regulatory subunits and its cellularsubstrates. Although direct targeting of a RNA virus frequentlyresults in selection for drug-resistant mutants (47), targeting ofan interaction of a viral protein with a cellular componentdescribed here is unlikely to have such effect. Thus, targeting ofPP1 for inhibition of its interaction with filovirus VP30 mayrepresent a viable approach for development of an antiviralmolecule.

Acknowledgments—We thank Dr. Elke Mühlberger (Boston Univer-sity) for providing the EBOV minigenome and for the useful advice;Dr. Yoshihiro Kawaoka (University of Wisconsin) for providing theplasmids expressing EBOV NP, VP35, L, VP30, and the T7 polymer-ase; and Dr. Mathieu Bollen and Dr. Monique Beullens (University ofLeuven, Belgium) for cdNIPP1-expressing vectors. We thank Dr.Chandrasekhar Yallampalli (University of Texas Medical Branch)for kindly providing access to the Typhoon Tryo PhosphorImager. Wethank Dr. Krishna Narayanan and Dr. Cheng Huang (University ofTexas Medical Branch) for valuable suggestions in Northern blotanalysis. We thank Anand Soorneedi for technical assistance. We alsothank Dr. Michelle Meyer for reading the manuscript.

REFERENCES1. World Health Organization (1978) Ebola haemmorhagic fever in Zaire,

1976. Bull. World Health Organ. 56, 271–2932. Centers for Disease Control and Prevention (2014) Outbreak of Ebola in

Guinea, Liberia, and Sierra Leone, Centers for Disease Control and Pre-vention, Atlanta

3. Kuhn, J. H. (2008) Filoviruses, 1st Ed., pp. 190 –251, Springer, New York4. Weik, M., Modrof, J., Klenk, H. D., Becker, S., and Muhlberger, E. (2002)

Ebola virus VP30-mediated transcription is regulated by RNA secondarystructure formation. J. Virol. 76, 8532– 8539

5. Biedenkopf, N., Hartlieb, B., Hoenen, T., and Becker, S. (2013) Phosphor-ylation of Ebola virus VP30 influences the composition of the viral nucleo-capsid complex: impact on viral transcription and replication. J. Biol.Chem. 288, 11165–11174

6. Martınez, M. J., Biedenkopf, N., Volchkova, V., Hartlieb, B., Alazard-Dany,N., Reynard, O., Becker, S., and Volchkov, V. (2008) Role of Ebola virusVP30 in transcription reinitiation. J. Virol. 82, 12569 –12573

7. Modrof, J., Muhlberger, E., Klenk, H. D., and Becker, S. (2002) Phosphor-ylation of VP30 impairs Ebola virus transcription. J. Biol. Chem. 277,33099 –33104

8. Bollen, M., Peti, W., Ragusa, M. J., and Beullens, M. (2010) The extendedPP1 toolkit: designed to create specificity. Trends Biochem. Sci. 35,450 – 458

9. Heroes, E., Lesage, B., Gornemann, J., Beullens, M., Van Meervelt, L., andBollen, M. (2013) The PP1 binding code: a molecular-Lego strategy thatgoverns specificity. FEBS J. 280, 584 –595

10. Ammosova, T., Yedavalli, V. R., Niu, X., Jerebtsova, M., Van Eynde, A.,Beullens, M., Bollen, M., Jeang, K. T., and Nekhai, S. (2011) Expression ofa protein phosphatase 1 inhibitor, cdNIPP1, increases CDK9 threonine186 phosphorylation and inhibits HIV-1 transcription. J. Biol. Chem. 286,3798 –3804

11. Ammosova, T., Jerebtsova, M., Beullens, M., Lesage, B., Jackson, A.,Kashanchi, F., Southerland, W., Gordeuk, V. R., Bollen, M., and Nekhai, S.(2005) Nuclear targeting of protein phosphatase-1 by HIV-1 Tat protein.J. Biol. Chem. 280, 36364 –36371

12. Ammosova, T., Jerebtsova, M., Beullens, M., Voloshin, Y., Ray, P. E., Ku-mar, A., Bollen, M., and Nekhai, S. (2003) Nuclear protein phosphatase-1regulates HIV-1 transcription. J. Biol. Chem. 278, 32189 –32194

13. Ammosova, T., Platonov, M., Yedavalli, V. R., Obukhov, Y., Gordeuk,V. R., Jeang, K. T., Kovalskyy, D., and Nekhai, S. (2012) Small moleculestargeted to a non-catalytic “RVxF” binding site of protein phosphatase-1




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

inhibit HIV-1. PLoS One 7, e3948114. Llorian, M., Beullens, M., Lesage, B., Nicolaescu, E., Beke, L., Landuyt, W.,

Ortiz, J. M., and Bollen, M. (2005) Nucleocytoplasmic shuttling of thesplicing factor SIPP1. J. Biol. Chem. 280, 38862–38869

15. Beullens, M., Vulsteke, V., Van Eynde, A., Jagiello, I., Stalmans, W., andBollen, M. (2000) The C-terminus of NIPP1 (nuclear inhibitor of proteinphosphatase-1) contains a novel binding site for protein phosphatase-1that is controlled by tyrosine phosphorylation and RNA binding. Biochem.J. 352, 651– 658

16. Neumann, G., Feldmann, H., Watanabe, S., Lukashevich, I., and Kawaoka,Y. (2002) Reverse genetics demonstrates that proteolytic processing of theEbola virus glycoprotein is not essential for replication in cell culture.J. Virol. 76, 406 – 410

17. Ammosova, T., Obukhov, Y., Kotelkin, A., Breuer, D., Beullens, M., Gor-deuk, V. R., Bollen, M., and Nekhai, S. (2011) Protein phosphatase-1 acti-vates CDK9 by dephosphorylating Ser175. PLoS One 6, e18985

18. Towner, J. S., Paragas, J., Dover, J. E., Gupta, M., Goldsmith, C. S., Huggins,J. W., and Nichol, S. T. (2005) Generation of eGFP expressing recombi-nant Zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening. Virology 332, 20 –27

19. Muhlberger, E., Weik, M., Volchkov, V. E., Klenk, H. D., and Becker, S.(1999) Comparison of the transcription and replication strategies of mar-burg virus and Ebola virus by using artificial replication systems. J. Virol.73, 2333–2342

20. Watanabe, S., Noda, T., Halfmann, P., Jasenosky, L., and Kawaoka, Y.(2007) Ebola virus (EBOV) VP24 inhibits transcription and replication ofthe EBOV genome. J. Infect. Dis. 196, S284 –S290

21. Ammosova, T., Washington, K., Debebe, Z., Brady, J., and Nekhai, S.(2005) Dephosphorylation of CDK9 by protein phosphatase 2A and pro-tein phosphatase-1 in Tat-activated HIV-1 transcription. Retrovirology 2,47

22. Allen, P. B., Kwon, Y. G., Nairn, A. C., and Greengard, P. (1998) Isolationand characterization of PNUTS, a putative protein phosphatase 1 nucleartargeting subunit. J. Biol. Chem. 273, 4089 – 4095

23. Lesage, B., Beullens, M., Pedelini, L., Garcia-Gimeno, M. A., Waelkens, E.,Sanz, P., and Bollen, M. (2007) A complex of catalytically inactive proteinphosphatase-1 sandwiched between Sds22 and inhibitor-3. Biochemistry46, 8909 – 8919

24. Muhlberger, E., Lotfering, B., Klenk, H. D., and Becker, S. (1998) Three ofthe four nucleocapsid proteins of Marburg virus, NP, VP35, and L, aresufficient to mediate replication and transcription of Marburg virus-spe-cific monocistronic minigenomes. J. Virol. 72, 8756 – 8764

25. Ammosova, T., Platonov, M., Ivanov, A., Kont, Y. S., Kumari, N., Kehn-Hall, K., Jerebtsova, M., Kulkarni, A. K., Uren, A., Kovalskyy, D., andNekhai, S. (2014) 1E7– 03, a small molecule targeting host protein phos-phatase-1, inhibits HIV-1 transcription. Br. J. Pharmacol. 10.1111/bph.12863

26. Sanchez, A., Kiley, M. P., Holloway, B. P., and Auperin, D. D. (1993) Se-quence analysis of the Ebola virus genome: organization, genetic elements,and comparison with the genome of Marburg virus. Virus Res. 29,215–240

27. Bukreyev, A. A., Belanov, E. F., Blinov, V. M., and Netesov, S. V. (1995)Complete nucleotide sequences of Marburg virus genes 5 and 6 encodingVP30 and VP24 proteins. Biochem. Mol. Biol. Int. 35, 605– 613

28. Muhlberger, E., Trommer, S., Funke, C., Volchkov, V., Klenk, H. D., andBecker, S. (1996) Termini of all mRNA species of Marburg virus: sequenceand secondary structure. Virology 223, 376 –380

29. Collins, P. L., Hill, M. G., Camargo, E., Grosfeld, H., Chanock, R. M., andMurphy, B. R. (1995) Production of infectious human respiratory syncytialvirus from cloned cDNA confirms an essential role for the transcriptionelongation factor from the 5� proximal open reading frame of the M2mRNA in gene expression and provides a capability for vaccine develop-ment. Proc. Natl. Acad. Sci. U.S.A. 92, 11563–11567

30. Bermingham, A., and Collins, P. L. (1999) The M2-2 protein of humanrespiratory syncytial virus is a regulatory factor involved in the balancebetween RNA replication and transcription. Proc. Natl. Acad. Sci. U.S.A.96, 11259 –11264

31. Asenjo, A., Rodrıguez, L., and Villanueva, N. (2005) Determination ofphosphorylated residues from human respiratory syncytial virus P proteinthat are dynamically dephosphorylated by cellular phosphatases: a possi-ble role for serine 54. J. Gen. Virol. 86, 1109 –1120

32. Washington, K., Ammosova, T., Beullens, M., Jerebtsova, M., Kumar, A.,Bollen, M., and Nekhai, S. (2002) Protein phosphatase-1 dephosphorylatesthe C-terminal domain of RNA polymerase-II. J. Biol. Chem. 277,40442– 40448

33. Llorian, M., Beullens, M., Andres, I., Ortiz, J. M., and Bollen, M. (2004)SIPP1, a novel pre-mRNA splicing factor and interactor of protein phos-phatase-1. Biochem. J. 378, 229 –238

34. Jagiello, I., Beullens, M., Stalmans, W., and Bollen, M. (1995) Subunitstructure and regulation of protein phosphatase-1 in rat liver nuclei.J. Biol. Chem. 270, 17257–17263

35. O’Connell, N., Nichols, S. R., Heroes, E., Beullens, M., Bollen, M., Peti, W.,and Page, R. (2012) The molecular basis for substrate specificity of thenuclear NIPP1:PP1 holoenzyme. Structure 20, 1746 –1756

36. Barik, S., and Banerjee, A. K. (1992) Sequential phosphorylation of thephosphoprotein of vesicular stomatitis virus by cellular and viral proteinkinases is essential for transcription activation. J. Virol. 66, 1109 –1118

37. Barik, S., McLean, T., and Dupuy, L. C. (1995) Phosphorylation of Ser232

directly regulates the transcriptional activity of the P protein of humanrespiratory syncytial virus: phosphorylation of Ser237 may play an acces-sory role. Virology 213, 405– 412

38. Dibben, O., Thorpe, L. C., and Easton, A. J. (2008) Roles of the PVM M2–1,M2–2 and P gene ORF 2 (P-2) proteins in viral replication. Virus Res. 131,47–53

39. Collins, P. L., Hill, M. G., Cristina, J., and Grosfeld, H. (1996) Transcriptionelongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus. Proc. Natl. Acad. Sci. U.S.A. 93, 81– 85

40. Asenjo, A., Calvo, E., and Villanueva, N. (2006) Phosphorylation of humanrespiratory syncytial virus P protein at threonine 108 controls its interac-tion with the M2–1 protein in the viral RNA polymerase complex. J. Gen.Virol. 87, 3637–3642

41. Martinez, M. J., Volchkova, V. A., Raoul, H., Alazard-Dany, N., Reynard,O., and Volchkov, V. E. (2011) Role of VP30 phosphorylation in the Ebolavirus replication cycle. J. Infect. Dis. 204, S934 –S940

42. Sugaya, N., and Ohashi, Y. (2010) Long-acting neuraminidase inhibitorlaninamivir octanoate (CS-8958) versus oseltamivir as treatment for chil-dren with influenza virus infection. Antimicrob. Agents Chemother. 54,2575–2582

43. Stittelaar, K. J., Tisdale, M., van Amerongen, G., van Lavieren, R. F., Pis-toor, F., Simon, J., and Osterhaus, A. D. (2008) Evaluation of intravenouszanamivir against experimental influenza A (H5N1) virus infection in cyn-omolgus macaques. Antiviral Res. 80, 225–228

44. Bollen, M., and Beullens, M. (2002) Signaling by protein phosphatases inthe nucleus. Trends Cell Biol. 12, 138 –145

45. Fujiki, H., and Suganuma, M. (2009) Carcinogenic aspects of protein phos-phatase 1 and 2A inhibitors. Prog. Mol. Subcell. Biol. 46, 221–254

46. Genoux, D., Haditsch, U., Knobloch, M., Michalon, A., Storm, D., andMansuy, I. M. (2002) Protein phosphatase 1 is a molecular constraint onlearning and memory. Nature 418, 970 –975

47. McKimm-Breschkin, J. L. (2013) Influenza neuraminidase inhibitors: an-tiviral action and mechanisms of resistance. Influenza Other Respir. Vi-ruses 7, 25–36

48. Modrof, J., Moritz, C., Kolesnikova, L., Konakova, T., Hartlieb, B., Randolf,A., Muhlberger, E., and Becker, S. (2001) Phosphorylation of Marburgvirus VP30 at serines 40 and 42 is critical for its interaction with NPinclusions. Virology 287, 171–182




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Vasiliy S. Naumchik, Alexander N. Freiberg, Sergei Nekhai and Alexander BukreyevObukhov, Tania Garron, Namita Kumari, Dmytro Kovalskyy, Maxim O. Platonov,

Philipp A. Ilinykh, Bersabeh Tigabu, Andrey Ivanov, Tatiana Ammosova, Yuriand Its Targeting for the Inhibition of Viral Transcription

Role of Protein Phosphatase 1 in Dephosphorylation of Ebola Virus VP30 Protein

doi: 10.1074/jbc.M114.575050 originally published online June 16, 20142014, 289:22723-22738.J. Biol. Chem.

10.1074/jbc.M114.575050Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/289/33/22723.full.html#ref-list-1

This article cites 46 references, 20 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M114.575050

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;289/33/22723&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/289/33/22723

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=289/33/22723&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/289/33/22723

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/289/33/22723.full.html#ref-list-1

http://www.jbc.org/

RoleofProteinPhosphatase1inDephosphorylationofEbola ... · 2014-08-08 ·...

Documents

Transcript of RoleofProteinPhosphatase1inDephosphorylationofEbola ... · 2014-08-08 ·...