Inhibition of Pokeweed Antiviral Protein (PAP) by TurnipMosaic Virus Genome-linked Protein (VPg)*□S

Received for publication, March 29, 2012, and in revised form, July 3, 2012 Published, JBC Papers in Press, July 6, 2012, DOI 10.1074/jbc.M112.367581

Artem V. Domashevskiy‡§, Hiroshi Miyoshi¶, and Dixie J. Goss‡1

From the ‡Department of Chemistry, Hunter College and the Graduate Center of the City University of New York, New York,New York 10065, the §Department of Science, John Jay College of Criminal Justice and the Graduate Center of the City University ofNew York, New York, New York 10019, and the ¶Department of Microbiology, St. Marianna University School of Medicine,Kawasaki 216-8511, Japan

Background: PAP is a ribosome-inactivating protein that depurinates RNA and inhibits protein synthesis.Results: Turnip mosaic VPg inhibits enzymatic activity of PAP in wheat germ extract.Conclusion: VPg may play a role in overcoming viral resistance by suppressing the plant defense mechanism.Significance: Depurination inhibition by VPg suggests a novel viral strategy to evade host cell defense and possible anti-cytotoxic activity against RIPs.

Pokeweed antiviral protein (PAP) from Phytolacca ameri-cana is a ribosome-inactivating protein (RIP) and an RNAN-glycosidase that removes specific purine residues from thesarcin/ricin loop of large rRNA, arresting protein synthesis atthe translocation step. PAP is also a cap-binding protein and is apotent antiviral agent against many plant, animal, and humanviruses. To elucidate the mechanism of RNA depurination, andto understand how PAP recognizes and targets various RNAs,the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as acap analog in cap-independent translation and potentially tar-get PAP to uncapped IRES-containing RNA. In this work, fluo-rescence spectroscopy andHPLC techniqueswere used to quan-titatively describe PAP depurination activity and PAP-VPginteractions. PAP binds to VPg with high affinity (29.5 nM); thereaction is enthalpically driven and entropically favored. Fur-ther, VPg is a potent inhibitor of PAP depurination of RNA inwheat germ lysate and competes with structured RNA derivedfrom tobacco etch virus for PAP binding. VPg may confer anevolutionary advantage by suppressing one of the plant defensemechanisms and also suggests the possible use of this proteinagainst the cytotoxic activity of ribosome-inactivating proteins.

Pokeweed antiviral protein (PAP)2 is a ribosome-inactivatingprotein (RIP) that is isolated from the extracts of pokeweed

plant leaves (Phytolacca americana) (1). It is known to reduceinfectivity of tobaccomosaic virus (2) by inhibiting protein syn-thesis (3). PAP, ricin, abrin, and otherRIPs inactivate ribosomesand inhibit cell-free protein synthesis by means of arresting thefunction of elongation factor EF-2 (4, 5) in the translocationstep (6–8). The N-glycosidase domain of RIPs recognizes aspecific and highly conserved region, the sarcin/ricin (S/R) loop(9), within the large 28S rRNA, and cleaves a distinct A4324residue on the RNA (for rat liver ribosome). This depurinationarrests cellular protein synthesis and leads to the activation ofapoptotic pathways (10). Ribosomal proteins and structural dif-ferences between RIPs themselves account for the diversifiedactivity of RIPs and ribosome substrate specificity (11, 12).The mode of action for the antiviral activity of RIPs is poorly

understood, but there is evidence that this activity does notdepend solely on ribosomal inactivation. PAP isoforms cause aconcentration-dependent depurination of HIV-1 (13), tobaccomosaic virus (14), poliovirus (15), HSV (16), influenza (17), andbrome mosaic virus RNAs (18).PAP inhibits the in vitro translation of brome mosaic virus

and potato virus X RNAs without ribosomal depurination (19)by binding to the cap structure and depurinating the RNA. Thismay be the primary mechanism for PAP antiviral activity (20);however, it does not clarify the inhibitory effects of PAP on thereplication of uncapped viruses, such as influenza (17) andpoliovirus (15). Further, PAP does not depurinate every cappedRNA, and it can inhibit translation of uncapped viral RNAs invitro without causing detectable depurination at multiple sites(21). Thus, recognition of the cap structure alone is not suffi-cient for depurination of RNA (21).In the presence of wheat germ lysate, PAP depurinates

uncapped barley yellow dwarf virus transcripts containing afunctional WT 3�-translation enhancer sequence but does notdepurinate messages containing a non-functional mutant

* This work was supported by National Science Foundation Grants MCB0814051 and MCB 1118320 (to D. J. G.) and MCB 0919626 (to D. E. F.). Thiswork was also supported by PSC-CUNY Faculty Awards (to D. J. G. andD. E. F.), a CUNY Collaborative Award (to D. J. G. and D. E. F.), and Grant-in-aid for Scientific Research 2368042 from the Ministry of Education, Scienceand Culture of Japan (to H. M.).

This paper is dedicated to Dr. Diana E. Friedland, who died in January 2011. Dr.Friedland co-mentored Dr. Domashevskiy and contributed to the designand interpretation of these experiments.

□S This article contains supplemental Fig. 1.1 To whom correspondence should be addressed: Dept. of Chemistry, Hunter

College, City University of New York, New York, NY 10065. Tel.: 212-772-5383; Fax: 212-772-5332; E-mail: dgoss@hunter.cuny.edu.

2 The abbreviations used are: PAP, pokeweed antiviral protein; RIP, ribosome-inactivating protein; VPg, viral protein linked to the genome; S/R, sarcin-

ricin; TEV, tobacco etch virus; TuMV, turnip mosaic virus; m7G, 7-methyl-guanosine; ant-m7GTP, anthranoyl 7-methylguanosine triphosphate; eIF,eukaryotic initiation factor; IRES, internal ribosome entry site; NHS, N-hy-droxysuccinimide; NHS-fluorescein, N-hydroxysuccinimide ester of fluo-rescein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 35, pp. 29729 –29738, August 24, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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3�-translation enhancer sequence (22). This suggests that PAPbinding to eIF4G/iso4G provides a mechanism for PAP toaccess both uncapped and capped viral RNAs for depurination.PAP binds to the eIFiso4G, and the presence of cap analogincreases these protein-protein interactions (23), suggestingthat PAP binds to the 5�-m7G cap of mRNA.

To understand how PAP recognizes and selectively targetsRNAs, interactions between PAP and TuMV genome-linkedprotein (VPg) were investigated. VPg is a 22-kDa potyviral pro-tein, covalently attached via a Tyr residue (24) to the genomesof one-quarter of the plant positive strand RNA viruses, includ-ing the Potyviridae genus. VPg plays a pivotal role in the viralinfection cycle, replication, and cell-to-cell movement and alsohas been implicated in overcoming viral resistance in plants(25). Interactions between VPg of TuMV and the eIFiso4E/iso4F of Arabidopsis thaliana (26, 27) suggest that VPg isimportant in the initiation of protein synthesis (28). Interac-tions between VPg and plant eIFiso4E and effects of eIFiso4Gon these interactions have been characterized (29–31). VPgstimulates the in vitro translation of uncapped IRES-containingRNA by targeting eIF4F to the IRES and inhibits capped RNAtranslation in wheat germ extracts (32).In this study, fluorescence spectroscopy and HPLC tech-

niques were used to quantitatively describe PAP-VPg interac-tions. PAP interacts strongly with VPg in a mixed type compe-tition for m7GTP cap analog. PAP binds to and depurinates anS/R oligonucleotide, capped and uncapped tobacco etch virus(TEV), and luciferase mRNA, supporting previous conclusionsthat the cap structure is not the only determinant within theRNA for depurination by PAP. PAP binds to VPg at a differentsite from the eIFiso4F/eIF4F binding site. The effect of VPg onthe depurination of selected RNA molecules, including struc-tured RNA derived from TEV (33–35), showed that VPgdecreases depurination of RNAs and competes with IRES con-taining TEV RNA for PAP binding. These findings correlatewith the inhibition of PAP enzymatic activity by VPg in wheatgerm lysate. Depurination inhibition by VPg may confer anadvantage for viral replication, and may be a novel mechanismto overcome plant defenses.

EXPERIMENTAL PROCEDURES

Materials—All chemicals used (unless otherwise noted)wereof molecular biology grade. Tris-HCl, HEPES, KCl, dithiothre-itol (DTT), phenylmethylsulfonyl fluoride (PMSF), aprotinin,soybean trypsin inhibitor, diethylpyrocarbonate, EDTA, andthe m7GTP analog were purchased from Sigma. Polyvinylpyr-rolidonewas purchased fromSpectrum. PromegaRiboMAXTM

large scale RNAproduction systemT7 and SP6were used for invitro RNA and S/R oligonucleotide synthesis. Peptone, yeastextract, and NaCl were purchased from Fisher. The SalI andNcoI restriction endonucleaseswere purchased fromNewEng-land Biolabs. HiTrap FF chromatography columns (Mono Qand SP) were from GE Healthcare. Plasmid isolation kits andnickel-nitrilotriacetic acid Superflow column were purchasedfrom Qiagen.Purification of Pokeweed Antiviral Protein—PAP used for

these experiments was isolated from the spring leaves of thepokeweed plant (Phytolacca americana) as described previ-

ously (23). PAP was purified using the AKTApurifier systemfrom GE Healthcare, equipped with pump P-900, monitorUV-900, monitor UPC-900, valve INV-907, and mixer M-925.The protein fractions were analyzed by 12% SDS-PAGE forpurity, and the protein concentration was determined using aPierce Coomassie assay with BSA as the standard.Expression and Purification of Wild Type (WT) VPg-His6,

VPg-71-His6, and VPg-220-His6—Purification of TuMV VPgwas described previously (29). VPg was purified with theAKTApurifier system. Purification of VPg-71-His6 and VPg-220-His6 truncated proteins followed an analogous procedure.The purity of all of the proteins was confirmed by 12% SDS-PAGE, and the protein concentrationwas determined using thePierceCoomassie assaywith BSA as the standard. Prior to spec-troscopic measurements, all samples were dialyzed againstbuffer E (20 mM HEPES-KOH, pH 7.6, 100 mM KCl, 1.0 mM

MgCl2, 1.0mMDTT, 1.0mMEDTA), passed through a 0.22-�mMillipore filter, and concentrated with a Centricon 10 concen-trator (Amicon Co.) as necessary.Expression and Purification of Eukaryotic Translation Initia-

tion Factors (eIFs)—The cap binding and scaffolding initiationfactors (eIFiso4G and eIFiso4E) were expressed in Escherichiacoli containing the constructed pET-3d vector in BL21(DE3)-pLysS cells, as described previously (31). All samples were ana-lyzed by 12%SDS-PAGEand showedhomogeneously pure pro-teins. All protein purification steps were carried out in a coldroom at 4 °C.InVitro Synthesis of TEVRNA, S/ROligonucleotide RNA, and

Luciferase mRNA—TEVDNA constructs were kindly providedby Daniel R. Gallie (University of California, Riverside, CA).The full-length TEV construct was cloned as described previ-ously (33). The TEV1–143 leader sequence was positioned nextto the SP6 promoter of the PTL7SN.3 GUS vector. DNA waslinearizedwithNcoI. The linearizedDNAwas treatedwith pro-teinase K (100�g/ml) and 0.5% SDS in 50mMTris-HCl, pH 7.5,5 mM CaCl2 for 30 min at 37 °C. DNA was further purified byextraction with phenol/chloroform/isoamyl alcohol (25:24:1)at pH 8.0 followed by ethanol precipitation. Purity of theresulted DNA was checked on a 1% agarose gel, and the con-centration was quantified spectrophotometrically and broughtto 0.5 mg/ml. In vitro transcription of the TEV DNA used Pro-mega RiboMAXTM large scale RNAproduction system SP6 fol-lowing the manufacturer’s protocol. Cap analog, m7G(5�)ppp-(5�)G, was incorporated into the TEV transcript during theRiboMAXTM transcription reaction. The ratio of cap analog/GTPwas 5:1 to increase the efficiency of the transcription reac-tion. Capped and uncapped transcripts were analyzed on 20%denaturing polyacrylamide, 8 M urea gels, and the synthesizedproducts were visualized by ethidium bromide staining. Underthe conditions of transcription, more than 75% of RNA tran-scripts were capped, as determined by the fluorescence inten-sity of ethidium bromide. Capped RNA transcripts were slicedfrom the gels, redissolved in a buffer solution, precipitated withethanol, and repurified. The concentration of TEV RNA wasdetermined bymeasuring the optical density at 260 nm, and thepurity of the synthesized RNAwas confirmed bymeasuring theabsorbance ratio A260/A280 in diethylpyrocarbonate-treatedwater. The S/R oligonucleotide dsDNA template (5�-GGATC-

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CTAATACGACTCACTATAGGGTGAACTTAGTACGAG-AGGAACAGTTCA-3�; 53 nucleotides) was purchased fromGene LinkTM with the sequence corresponding to the univer-sally conserved S/R loop of the large rRNA. The linear DNAtemplate was treated with proteinase K and purified by phenol/chloroform extraction as for TEVDNA. In vitro synthesis of theS/R oligonucleotide RNA used Promega RiboMAXTM largescale RNA production system T7 following the manufacturer’sprotocol. Cap analog, m7G(5�)ppp(5�)G, was incorporated intothe RNA transcripts during the RiboMAXTM transcriptionreaction. The plasmid pLUC0 (36) containing the luciferasegene was linearized with DraI and used as template for synthe-sis of the in vitro transcript. pLUC0 contains a linker sequence(GGCCTAAGCTTGTCGACC) between the T7 promoter andthe ATG of luciferase. Following the TAA stop codon of lucif-erase is a poly(A) tail of 50A nucleotides immediately upstreamof the DraI site. Both capped and uncapped luciferase RNAswere synthesized as run-off transcripts of aT7polymerase reac-tion (Promega), as described previously (20).Fluorescence Assay for Adenine Released by PAP—Experi-

ments were performed by incubating RNA (10 nmol/ml) inDepurination Buffer (20 mM Tris-HCl, pH 7.5, 100 mMNH4Cl,7mMmagnesium acetate, and 1mMDTT) for 15min at 37 °C inthe absence and presence of PAP and VPg in 100-�l reactionvolumes. At the end of the incubations, 1 volume of cold etha-nol was added, and after 10 min at �80 °C, the ethanol-solublefractions were recovered by centrifugation for 15 min at 14,000rpm. Free adenine present in the ethanol-soluble fractions wasconverted into its etheno derivative (37–39); 150 �l portions ofthe ethanol-soluble fractions were each diluted to 1 ml withdiethylpyrocarbonate-treated water, and 0.4 ml of a mixture of0.14 M chloroacetaldehyde and 0.1 M sodium acetate buffer, pH5.1, was added to each. The samples were heated in awater bathat 80 °C for 40min, extracted four timeswith 1 volumeofwater-saturated diethyl ether, and passed through 0.45-�m pore sizefilters. Fractions were analyzed with a Waters high-pressureliquid chromatograph equipped with a Waters 2487 dual �absorbance detector (set at 254 nm), a Waters 2475 multi �fluorescence detector (excitation, 315 nm; emission, 415 nm), aWaters 600 controller, and a Waters 717plus autosampler. Thecolumn (4.6 � 150 mm) was a reversed-phase XBridgeTM C18(particle size 5 �m) purchased from Waters Associates. Thecolumnwas eluted isocratically with 50mM ammonium acetatebuffer, pH 5.0, plus methanol (89:11, v/v) at room temperature.Elution profiles were analyzed byWaters EmpowerTM chroma-tography software. Each experiment included a standard ofN6-ethenoadenine in the appropriate buffer and internal stan-dards obtained by adding known amounts ofN6-ethenoadenineto the ethanol-soluble fractions from the control and PAP-treated RNA. The amount of adenine released from PAP-treatedRNA was calculated from the standards after subtraction of thefluorescence reading given by control RNA.Wheat Germ Lysate Translational Assay—Wheat germ

extract was purchased fromPromega. Translation of TEVRNAwas determined in luciferase assay buffer (25 mM Tricine, pH8.0, 5 mM MgCl2, 0.1 mM ETDA supplemented with 33 mM

DTT, 0.25 mM coenzyme A, and 0.5 mM ATP) for luciferaseactivity. A 1.0-�g sample of TEV1–143-luc-A50 RNA was trans-

lated in a 200-�l reactionmixture containing 50 �l of completewheat germ lysate extract, 50 units of RNase inhibitor, and 10�Mcomplete amino acidmixture (Promega). Luciferase activityfor brome mosaic virus RNA provided by Promega was used asa control. Equimolar concentrations of PAP, WT VPg, or VPg-220 were added to the translational mixture as described under“Results.” Light emissionwasmeasured after the addition of 0.5mM luciferin as a function of time using the PerkinElmerGelience 600 imaging system.Synthesis of the Fluorescent Anthranoyl-m7GTP—The fluo-

rescent anthranoyl-m7GTP cap analog was synthesized asdescribed previously (40, 41) with the following modifications.Them7GTP cap (10mg) was dissolved in 1ml of distilled waterat 37 °C. The pH of the resulting solution was adjusted to 9.6with 2 NNaOH. To this solution with continuous stirring, crys-talline isatoic anhydride (5 mg) was added. The pH of the mix-ture wasmaintained at 9.6 by titrating 2 NNaOHduring the 2-hreaction. After the reaction was complete, the pH of the reac-tion mixture was adjusted to 7.0 with 1 N HCl solution. Thereaction mixture containing the products and the unreactedmaterials was loaded onto a Sephadex LH-20 (2.4 � 56-cm)column equilibrated with autoclaved distilled water. The col-umn was eluted with the same solvent at a flow rate of about 6ml/h. Fractions of 1 ml were collected and assayed by TLC onsilica gel. The plateswere developed in systemA (n-propyl alco-hol/ammonia/water (6:3:1, v/v/v), containing 0.5 g/literEDTA). The ant-m7GTP analog had brilliant blue fluorescence(monitored by a UV lamp), whereas anthranilic acid (a byprod-uct of this reaction) showed dark violet fluorescence. Peak frac-tions of the fluorescent analog were pooled, combined, andlyophilized in vacuo at liquid nitrogen temperature to preventdegradation. The resulting residue was then dissolved in amin-imum amount of water (0.5 ml), and an excess of cold ethanolwas added to induce the precipitation of the compound. Thefluorescent cap analog was then dried in vacuo over phosphoricanhydride at 4 °C, giving an amorphous powder.Labeling of PAP with NHS-fluorescein—Pokeweed antiviral

protein was labeled with fluorescent N-hydroxysuccinimide(NHS)-fluorescein reagent using the Pierce NHS-fluoresceinantibody labeling kit (Thermo Scientific) according to theman-ufacturer’s protocol. NHS-ester labeling reagents react effi-ciently with primary amines in the side chains of lysine residuesof PAP. PAP storage buffer S was replaced with 50 mM sodiumborate, pH 8.5, buffer, containing 1 mM DTT using Microcon�centrifugal filters (Millipore). The fluorophore/protein ratiowas estimated spectrophotometrically in phosphate-bufferedsaline (PBS) by measuring absorbance at 280 and 495 nm (i.e.Amax of NHS-fluorescein), A280/Amax � 0.3. The averageamount of labeling was determined to be 65%. Protein concen-tration was calculated as follows,

Moles of fluorophore/moles of protein

�Amax of the labeled protein

�fluor � �protein�� DF (Eq. 1)

where Amax is the maximum absorbance of the labeled protein,measured at 495 nm, �fluor � 70,000 (NHS-fluorescein molar

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extinction coefficient), [Protein] is the molar protein concen-tration, and DF is the dilution factor. The degree of PAP label-ing was calculated as follows,

�Protein��M� �A280 � � Amax � CF�

�protein� DF (Eq. 2)

where A280 is the absorbance of the non-labeled protein, mea-sured at 280 nm, Amax is the maximum absorbance of thelabeled protein, measured at 495 nm, �protein is the molarextinction coefficient of the non-labeled protein, correctionfactor (CF) � A280/Amax � 0.3, and DF is the dilution factor.Fluorescence Data Acquisition and Analysis—Steady state

fluorescence was used to monitor protein-protein and protein-nucleic acid interactions (42). Acquisition of steady state fluo-rescence in the ultraviolet region allows the use of intrinsicprotein fluorophores to determine equilibrium constants. AHoriba Jobin Yvon FluoroMax�-3 fluorometer with a 150-wattxenon lamp with photodiode array detectors was used for allfluorescence measurements. Fluorescence changes (quenchingor enhancement, depending on the titrations) were monitoredusing an excitation wavelength of 280 nm and an emissionwavelength of 332 nm (intrinsic protein fluorescence) or usingexcitation of 332 nm for the anthranoyl group and 493 nm forNHS-fluorescein and an emissionwavelength of 420 nm for theanthranoyl group and 516 nm for NHS-fluorescein (for extrin-sic fluorophores). All samples were thermoregulated, and thetemperatures weremonitored by a thermocouple in the samplechamber. All titrations were performed in a titration buffer (20mM HEPES-KOH, pH 7.5, 100 mM KCl, 1 mM DTT). For eachdata point, three samples were prepared. The fluorescenceintensity of a protein (e.g. PAP) was measured in the first sam-ple. A second sample containing specific amount of titrant pro-tein (e.g. VPg) was also measured, and the corrected intensitiesof the two samples were summed together (Fs). A third samplecontaining the same amount of PAP and VPg proteins wasmixed together, and the corrected fluorescence intensity forthis complex was obtained (Fc). The difference in fluorescenceintensity related to the complex was defined as F � Fc � Fs.Similarmeasurements were also performed for other titrations.The inner filter corrections for the RNA experiments wereapplied as described previously (34) using the following equa-tion (42),

Fc � Fo � antilog�Aex Aem

2 � (Eq. 3)

where Fc and Fo are the corrected and observed fluorescenceintensities, respectively. Aex and Aem are the absorbance of theexcitation and emission wavelengths, respectively. Correctionsfor the dilutions of the titrated samples were taken into consid-eration as well. The absorbance of the sample was measuredusing an Ultrospec 1100 Pro UV-visible absorption spectro-photometer. The normalized fluorescence difference (F/Fmax) between the protein-protein and protein-RNA com-plexes and the sum of the individual fluorescence spectra wereused to determine the equilibriumdissociation constant (Kd). Adouble reciprocal plot was used for determination of Fmax.The details of the data fitting are described elsewhere (32, 43).

PRISM�, version 5, was used to analyze and plot the data. Non-linear least-squares fitting of plotted normalized data wereused; one-site and two-site binding models were tested.Evaluation of Thermodynamic Parameters—Thermody-

namic parameters, H (van’t Hoff enthalpy), S (entropy), andG (free energy), were determined using the followingequations,

�ln Keq �H0

RT�

S0

R(Eq. 4)

G0 � �RTlnKeq � H0 � TS0 (Eq. 5)

where R and T are the universal gas constant and absolute tem-perature, respectively. Keq is the association equilibrium con-stant,H andSwere determined from the slope and the inter-cept of a plot of lnKa against 1/T, andGwas determined fromEquation 5.Determination of the Number of Binding Sites—The quench-

ing of the native fluorescence emission maximum upon theaddition of a ligand was monitored for the fluorescence changerelative to untitrated PAP or eIFiso4F. VPg has very low intrin-sic protein fluorescence. The fractional quench, Q, was deter-mined at each PAP/VPg or eIFiso4F/VPgmolar ratio (R). For anobserved fluorescence intensity, F, the fractional quench, Q,was obtained from Equation 6 (44).

Q �F0 � F

m(Eq. 6)

Herem is the maximal quench.Fractional quench is linearly related to ligand binding,

�Ligand � Protein�

�Protein�T� Q (Eq. 7)

where [Protein]T represents the total PAP or eIFiso4F proteinconcentration. The average number of binding sites (n) wasdetermined from the x intercept of the Scatchard plotQ versusQ/(R � Q)[Protein]T (44).

RESULTS

PAPDepurinates bothm7GpppG-capped andUncapped S/R,TEV, and LuciferasemRNA—Because PAP can bind to cap ana-logs, we determined the extent to which the presence of a capon the RNA affected depurination. To examine the extent towhich PAP discriminates between capped and uncapped RNAtranscripts, a synthetic S/R oligonucleotide RNA, TEV RNA,and luciferase mRNA were used as substrates for PAP enzy-matic activity. PAP recognized a specific and highly conservedregion, the S/R loop within the large rRNA, and cleaves a dis-tinct adenine residue (A4324) on the RNA (for the rat liverribosome) (9). Previous reports showed that PAP was able torecognize the cap structure on RNA transcripts (19). It waspostulated that PAP binding to the cap structure promotes thedepurination of capped mRNAs (20). Other findings indicatedthat PAP is able to inhibit translation of uncapped RNAs with-out detectable levels in depurination (21). Both findings are notmutually exclusive but in fact imply that the cap structure itselfis not enough to promote the depurination of RNA or inhibi-

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tion of RNA translation. To determine whether the cap struc-ture affects depurination of S/R and TEV RNAs and luciferasemessenger RNA, the above RNAs were capped during run-offtranscription reactions with an m7GpppG cap analog. Separa-tion using HPLC techniques and identification by means offluorescence allowed for construction of a linear relationshipbetween the amount of 1-N6-ethenoadenosine derived fromthe depurination of RNAs and the integrated peak area over awide range of 1-N6-ethenoadenosine concentrations (from 10to 200 pmol) (supplemental Fig. 1A). The amount of adeninereleased from WT S/R and WT TEV RNA versus m7GpppG-capped S/R and TEV RNAs with an addition of increasingamounts of PAP was determined. Under the conditions ofHPLC, loaded fractions gave a single fluorescent peak with aretention time of 4.5 min (supplemental Fig. 1, B and C). Theamount of adenine released upon depurination of capped ver-sus uncapped RNAs was of the same order of magnitude. PAPdepurination did not discriminate between either capped oruncapped S/R oligonucleotide and capped or uncapped TEVRNAs (Fig. 1). The amount of adenine released from cappedand uncapped S/R RNA was 14.9 � 0.8 and 13.7 � 0.7 nM,respectively. The amount of adenine released from capped anduncapped TEV RNA was 6.0 � 0.4 and 4.1 � 0.1 nM, respec-tively. Depurination of uncapped cellular luciferase mRNAyielded 27.5 � 0.6 nM adenine released compared with 35.6 �1.8 nM for m7GpppG-capped RNA. These results indicate thatthe cap itself had little effect on depurination for naturallyuncapped RNA and only a modest effect on luciferase mRNA.PAPHas Different Kinetics from Ricin A Chain—To examine

the rates at which PAP depurinates WT TEV RNA, standardquantification of adenine in the discontinuous assay formatwasperformed. Analysis of the fractions on the HPLC indicatedthat RNA depurination by PAP was virtually 80% completeafter 3 min (Fig. 2A). To establish catalytic constants of TEVRNA depurination by PAP, the RNA concentrations were var-ied. The progress of the reaction was monitored by the appear-ance of a UV-absorbing product at the saturating conditions.Calculated depurination rates were plotted against the RNA

concentrations, resulting in a Michaelis-Menten type behavior(Fig. 2B). The catalytic constant, kcat, was calculated to be 2.5min�1 (0.042 s�1). Fluorescence titrations of NHS-labeled PAPwith RNA produced a Km of 13.6 nM. The specificity constant,kcat/Km, was calculated to be 3.1 � 106 M�1 s�1. This is com-pared with the specificity constant of ricin A chain for 80S rab-bit ribosomes of 1.4 � 108 M�1 s�1 (45).

FIGURE 1. VPg inhibits PAP depurination of S/R oligonucleotide and TEV RNA. Shown are comparisons of WT (uncapped) S/R oligonucleotide RNA (A)depurination by PAP (black bars) with that of m7GpppG-capped S/R oligonucleotide RNA (gray bars), WT (uncapped) TEV RNA (B) (black bars) with that ofcapped TEV RNA (gray bars), and uncapped and capped luciferase mRNA (C) in the presence and absence of VPg. Error bars, S.E.

FIGURE 2. Kinetics of WT TEV RNA depurination by PAP. A, time course ofadenine released during depurination of RNA by PAP as measured by thefluorescence of 1-N6-ethenoadenine. Aliquots of PAP-RNA depurination mix-tures were withdrawn at different times and loaded directly onto the HPLCcolumn. Excitation and emission wavelengths were 315 and 415 nm, respec-tively. B, 1-N6-ethenoadenine assay kinetic curve for depurination catalysis ofRNA by PAP. 100 nM PAP was treated with increasing concentrations of rRNA,and the amount of released adenine was monitored as described under“Experimental Procedures.”

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PAP Binds to VPg with Higher Affinity than to eIFiso4F andm7GTP—The equilibrium binding constants for PAP and VPginteraction over a range of different temperatures were deter-mined from fluorescence titration studies (Fig. 3). The equilib-rium constant for PAP-VPg interaction was determined to be29.5 � 1.8 nM at 25 °C (Table 1). This compares with VPg-eIFiso4FKd of 81.3 nM (31) and PAP-m7GTP binding of 43.3 nM(23).PAP Binding to VPg Is Enthalpically Driven and Entropically

Favored—To establish the forces that drive PAP-VPg interac-tions, the thermodynamics of PAP-VPg binding were deter-mined. Table 1 shows that the affinity of PAP for VPg decreaseswith the increase in temperature (Kd � 29.5 � 1.8 nM at 25 °Cversus 12.5 � 0.6 nM at 5 °C). The values of H0 and S0 wereobtained from the intercept and the slope, respectively, of avan’t Hoff plot (Fig. 3, inset) (correlation coefficient of �0.98).The van’t Hoff analyses showed that the VPg binding to PAP isenthalpy-driven (H0 � �29.2 � 0.9 kJ/mol) and entropy-fa-vored (S0 � 46.0 � 3.0 J/mol), leading to a negative G0

(�43.0 � 1.8 kJ/mol). The TS van’t Hoff component contrib-utes 32% overall to the value of G0 at 25 °C.PAP and VPg Bind in a 1:1 Ratio—To determine the stoichi-

ometry of PAP-VPg binding, direct fluorescence titration stud-ies of PAP with VPg were performed (Fig. 4). The slope andintercept of the Scatchard plotQ/[VPg]� 10�6 versus Q (Fig. 4,inset) gave the binding constant (Kd � 29.5� 1.8 nM) and bind-ing capacity (n� 0.99� 0.01) of PAP forVPg (44).We concludethat the PAP and VPg interact in a 1:1 stoichiometric ratio.PAP and eIFiso4F Bind VPg at Different Sites—VPg-71 is a

truncated variant of wild type VPgwhere theN-terminal aminoacids 1–70 are removed so that it lacks the eIF4F and eIFiso4F

binding sites (32). PAP exhibits 2.8 times stronger bindingaffinity for VPg (29.5 � 1.8 nM) than eIFiso4F (81.3 nM) (31).The equilibrium constant for PAP�VPg-71 was found to be37.4 � 3.0 nM at 25 °C. Because VPg-71 has the eIF4F/iso4Fbinding sites removed yet still binds PAP with high affinity, weconclude that the PAP binding site on VPg differs from theeIF4F binding site on VPg.VPg and Cap Analog Bind PAP in a Mixed Type Competi-

tion—Competitive binding of VPg and cap to PAP was deter-mined by employing a fluorescent cap analog, ant-m7GTP (40,41). The competitive substitution reactions were performed atconstant ant-m7GTP concentration (100 nM), monitoring thefluorescence change of the analog and increasing amounts ofPAP in the absence and presence of VPg. ant-m7GTP was asuitable candidate to study these competition interactionsbecause excitation (332 nm) and emission (420 nm) of thisextrinsic fluorophore are far removed from the protein fluores-cence, and Kd for the PAP-ant-m7GTP interactions was essen-tially the same as reported previously for m7GTP interactionwith PAP (Kd � 43.3 nM) (23). Lineweaver-Burk plots (Fig. 5A)meet at the left of the y axis intercept, indicative of mixed typecompetitive ligand binding between ant-m7GTP and VPg, sug-

FIGURE 3. Binding isotherms for the interactions of PAP with VPg. Thenormalized fluorescence values (�ex � 280 nm and �em � 332 nm) for thefraction of the bound ligand are plotted versus VPg concentration at 5 °C (E),15 °C (�), and 25 °C (‚) (at 25 °C (�) for PAP�VPg-220, control). PAP concen-tration was 100 nM in titration buffer. The excitation and emission wave-lengths were 280 and 332 nm, respectively. The curves were fit to obtaindissociation constants (Kd) as described under “Experimental Procedures.”Inset, van’t Hoff plot for the interactions of PAP with VPg. ƒ, 10 °C; �, 20 °C.

TABLE 1Equilibrium dissociation constants for the interactions of PAP withVPg/VPg-71

ComplexEquilibrium dissociation constant (Kd)

5 °C 10 °C 15 °C 20 °C 25 °C

nMPAP�VPg 12.5 � 0.6 17.0 � 0.7 20.9 � 1.2 26.7 � 1.3 29.5 � 1.8PAP�VPg-71 NDa ND ND ND 37.4 � 3.0

a ND, not determined.

FIGURE 4. VPg binds more tightly to PAP than to eIFiso4F (complex ofeIFiso4E�eIFiso4G). Intrinsic PAP (E) or eIFiso4F (�) fluorescence was moni-tored upon binding to VPg. VPg has negligible intrinsic fluorescence. Solidlines, fitted theoretical curves. Inset, Scatchard plots showing one binding sitefor eIFiso4F and PAP with VPg. The slope and intercept of the straight lineobtained on the plot Q/[VPg] � 10�6 versus Q provided the binding constant(Ka) and binding capacity (n) of the above proteins with VPg. Q is the fractionalquench of fluorescence in titration. n for the PAP�VPg was determined as0.99 � 0.01 and, for eIFiso4F�VPg, was 1.05 � 0.01 (T � 25 °C, �ex � 280 nm,�em � 332 nm) (31).

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gesting that VPg binds to PAP at a site distinct from the capbinding site.VPg-71 and eIFiso4F Bind PAP Competitively—To deter-

mine if binding of VPg and eIFiso4F to PAP was competitive ornoncompetitive, VPg-71 (a truncated variant of VPg that lacksthe eIFiso4F binding site and does not interact with eIFiso4F),and N-hydroxysuccinamide (NHS)-fluorescein-labeled PAPwere utilized. The Kd for the NHS-fluorescein-labeled PAP-m7GTP interactions (63.8 � 7.9 nM at 25 °C; data not shown) isin agreement with a previously published WT PAP-m7GTPvalue (23), showing that labeling did not affect the protein. Theapparent affinity of labeled PAP for VPg-71 was found todecrease in the presence of 150 nM eIFiso4F (at 25 °C). Fluores-cence data were also represented as a double-reciprocal plot(Fig. 5B) where the lines meet on the y axis, indicating a com-petitive type ligand binding between VPg and eIFiso4F.VPg Competes with TEV RNA for PAP Binding—Zeenko and

Gallie (33) have previously demonstrated that the uncapped 5�TEV (tobacco etch virus) UTR contains pseudoknot 1 (PK1)within its structure that is sufficient to confer cap-independenttranslation. VPg had previously been shown to enhance TEVtranslation (32) by a mechanism where VPg enhances eIF4Fbinding to the TEVmRNA. In order to determine if VPg interac-

tion with PAP affected TEV binding, competition experimentswere performed, where PAP binding to TEV in the presence ofincreasing amounts of VPg was determined. A double-reciprocalplot (Fig. 5C) shows that the lines meet on the y axis, indicating acompetitive type ligand binding between VPg and TEV for PAP.Unlike eIF4F, VPg competes with PAP for TEV binding.VPg Decreases PAP-mediated Depurination of S/R, TEV, and

Luciferase mRNA—Having determined that PAP binds to VPgwith a high affinity, and VPg stimulates the in vitro translationof uncapped IRES-containing RNAby increasing eIF4F binding(32), we investigated the extent to which the binding of VPg toPAP could affect PAP activity, possibly by targeting PAP to theIRES of uncapped viral RNA. VPg in the depurination reactionsdecreased depurination of both WT (uncapped) and m7GpppG-capped RNA constructs. The presence of equimolar concentra-tionsofVPg toPAP in thedepurinationreactionscompletely abol-ished the enzymatic activity of PAP, regardless of whether theRNA transcripts were capped or not (supplemental Fig. 1, B andC). Fig. 1 summarizes these findings.The results fromthese exper-iments, consistent with the competitive binding experiments,show that VPg does not target PAP to IRES-containing RNA norS/R RNA but rather acts as a potent inhibitor of PAP activity.VPg Inhibits Activity of PAP in theWheatGermLysate Trans-

lation System—To examine the PAP depurination activity ofuncapped TEV RNA in wheat germ lysate and investigate howVPg affects this depurination, uncapped TEV RNA constructcontaining the TEV-untranslated region (143 nucleotides,including an IRES) with the luciferase reporter gene was used(46). Fig. 6 shows that the addition of PAP to the wheat germlysate system after 1 min caused depurination of the RNA andinhibited translation of the luciferase reporter. The presence ofVPg in the system prior to the addition of PAP caused neutral-

FIGURE 5. A, ant-m7GTP cap analog and VPg show mixed competition bindingfor PAP. Shown is a Lineweaver-Burk plot for competition of ant-m7GTP andVPg with PAP. The fluorescence change of ant-m7GTP cap analog was mea-sured with increasing concentrations of PAP. VPg concentrations were 0 nM

(E), 15 nM (‚), and 30 nM (�). The excitation wavelength was 332 nm, andemission was 420 nm. The spectrum was measured in buffer containing 100nM ant-m7GTP and VPg as indicated. Data points were fitted using leastsquare analysis. B, eIFiso4F and VPg-71 bind competitively to NHS-fluoresce-in-labeled PAP. Lineweaver-Burk plots of competitive binding are presented.The fluorescence change of NHS-fluorescein-labeled PAP (200 nM) was mon-itored (�ex � 493 nm, �em � 516 nm) with increasing concentrations ofVPg-71 in the presence and absence of eIFiso4F (0 (E), 50 (�), 100 (‚), and 150nM (�)). Data points were fitted using least square analysis. C, VPg and TEVRNA bind competitively to NHS-fluorescein-labeled PAP. Lineweaver-Burkplots show competitive binding. The fluorescence change of NHS-fluoresce-in-labeled PAP was monitored (�ex � 493 nm, �em � 516 nm) with increasingconcentrations of TEV RNA in the presence and absence of VPg (0 (E), 30 (ƒ),50 (�), and 100 nM (‚)). Data points were fitted using least square analysis.

FIGURE 6. Translation of luciferase reporter TEV RNA constructs in wheatgerm extracts. Luciferase relative light units (RLU) were measured for TEV(1–143)-luc-A50 RNA (E), TEV(1–143)-luc-A50 RNA WT VPg (�), TEV(1–143)-luc-A50 RNA PAP after 1 min (ƒ), TEV(1–143)-luc-A50 RNA WT VPg PAP after1 min (Œ), and TEV(1–143)-luc-A50 RNA VPg-220 PAP after 1 min (�) inwheat germ translational extract as a function of time. The proteins wereadded in the stoichiometric concentrations (10 nM) in the presence of 1.0 �gof TEV(1–143)-luc-A50 RNA, and light emission was measured after the addi-tion of 0.5 mM luciferase substrate.

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ization of the enzymatic activity of PAP and rapid translation ofthe luciferase reporter by thewheat germ translationalmachin-ery. This indicates that VPg serves as an inhibitor of PAP inwheat germ lysate. VPg-220, a truncated mutant of VPg thatlacks the C-terminal portion of the protein, was used as a neg-ative control for the PAP-VPg interactions. The VPg-220 vari-ant did interact with PAP with 10-fold lower affinity (Fig. 3);however, it did not affect PAPenzymatic activity, as determinedby the fluorescence assay. The presence of VPg-220 in thetranslational system did not show any increase in translation ofthe luciferase reporter compared with WT VPg.

DISCUSSION

PAP is a highly toxic protein produced by the pokeweed cellsand exported outside the cells once synthesized (47, 48). Stor-age of PAP within extracellular spaces ensures close proximityof PAP to ribosomes. When a pathogen infects the cell, PAPalso gains entrance and disrupts cellular protein synthesis, thuskilling the pathogen-infected cell and thereby preventingpathogen replication (49).Khan et al. (32) have characterized interactions between

VPg, plant eIFiso4E/iso4F, eIF4F, and TEVRNA and concludedthat VPg increases the binding affinity of eIF4F for TEV RNA.The requirement for eIF4G in cap-independent translation (34)has been demonstrated, and a mechanism was proposed whereVPg substitutes for the cap analog and enhances formation ofan eIF4F complex with viral IRES (31, 32). We thereforehypothesized that VPgmay interact with PAP and possibly tar-get it to uncapped RNA.The rationale of our investigation was that PAP, being a cap-

binding protein, will bind to VPg that functions as a cap analog,and these interactions would affect depurination of uncappedviral RNA or capped cellular RNA. VPg stimulates the in vitrotranslation of uncapped IRES-containing RNA and inhibitscapped RNA translation. Our research indicated that PAP has ahigh affinity for VPg and that this affinity is almost twice that ofthem7GTP analog (23). Greater affinity of PAP forVPg than forthe cap structurewould produce an advantage for the cell if VPgwere to localize PAP to viral RNA for depurination. However,VPg inhibits PAP activity, providing ameans to avoid one of thepotential host cell defense mechanisms.The thermodynamic parameters of PAP-VPg binding are

similar in magnitude to those of eIFiso4E- or eIFiso4F-VPgbinding. Both interactions are enthalpically driven and entropi-cally favored. The TS van’t Hoff component contributesnearly one-third to the overall value of G0 (at 25 °C), suggest-ing lesser dependence on electrostatic contributions and agreater conformational contribution in the PAP-VPg bindingwith hydrophobic residues less solvent-exposed in the com-bined structure. The fact that PAP-VPg interactions are enthal-pically driven and entropically favored at biological tempera-tures supports previous observations by Baldwin et al. (23) that,because PAP is a plant defense protein, it should be able toperform under unpredictable temperature conditions, given itsaccepted function as a ribosome depurinating agent (23).Different equilibrium dissociation constant (Kd) values for

PAP-VPg compared with eIFiso4E- or eIFiso4F-VPg bindingsuggest differences between the active site of PAP and the cap-

binding sites of eIFs. Leonard et al. (26, 30) have establishedpreviously the interactions between VPg and various isoformsof eIF4E, and Khan et al. (31) have quantified eIF4E/iso4E-VPginteractions as competitive with the m7GTP cap analog (26).Moreover, the binding domain on VPgwasmapped to a stretchof 35 amino acids, and substitution of aspartic acid residuefound within this region completely abolished interactions ofVPg with eIF4E/iso4E (26). Plants infected with a TuMVinfectious cDNA (p35Tunos) showed viral symptoms withp35Tunos, whereas plants infected with p35TuD77N, amutantthat contained the aspartic acid substitution in the VPg domainthat abolished the interaction with eIF4E/iso4E, remainedsymptomless, suggesting that VPg-eIF4E/iso4E interaction is acritical element for virus production (26). VPg-71 lacks theeIF4F/iso4F binding sites (32); however, PAP was able to bindVPg-71, indicating that the PAP binding site remains present inVPg-71 and does not include, at least partially, the amino ter-minus of the protein.Having determined the binary interactions between PAP,

VPg, and eIFiso4F, we have examined the ternary interactions.A summary of directly measured binary and ternary complexesis schematically presented in Fig. 7. The equilibrium associa-tion constants K1, K2, and K5 were directly measured by fluo-rescence titration experiments; K3 was fromKhan et al. (31). InFig. 7, K4 and K6 were chosen as the thermodynamicallydependent equilibrium constants and were calculated from therelationships shown in Equations 8 and 9.

K4 �K2K5

K1(Eq. 8)

K6 �K2K5

K3(Eq. 9)

A comparison of the cross-terms in Fig. 7 shows that thebinding of eIFiso4F to PAP diminishes the binding of VPg(comparingK1 andK5); similarly, the binding of eIFiso4F toVPgdiminishes the binding of PAP (comparing K3 and K5).

FIGURE 7. Schematic representation of the interactions between PAP,VPg, and eIFiso4F. K1, K2, K3, and K5 were determined experimentally; K4 andK6 were calculated as described under “Discussion.” Binding and couplingfree energies (kJ/mol) for PAP, VPg, and eIFiso4F were calculated according tothe Kd values. All equilibrium constants cited are 106

M�1.

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In order to quantitate these interactions, coupling energieswere calculated according to the method of Weber (50) and aspreviously described in detail (51, 52). The coupling energiesreflect the overestimation and underestimation of the freeenergy of binding for the formation of ternary eIFiso4F�VPg�PAP complex, G0

(eIFiso4F�VPg�PAP), calculated from theaddition of the component binary energies for the interac-tion of eIFiso4F with PAP, G0

(eIFiso4F�PAP), or with VPg,G0

(eIFiso4F�VPg), and PAP with VPg, G0(PAP�VPg). These cou-

pling energies therefore represent different binding perspec-tives and are defined by Equations 10–12.

G�eIFiso4F, VPg�PAP)0 � G(eIFiso4F�VPg�PAP)

0

� G�eIFiso4F�VPg)0 � G�eIFiso4F�PAP)

0(Eq. 10)

G�PAP, eIFiso4F�VPg)0 � G�eIFiso4F, VPg�PAP)

0

� G�eIFiso4F�VPg)0 � G�PAP�VPg)

0(Eq. 11)

G�VPg, eIFiso4F�PAP)0 � G�eIFiso4F, VPg�PAP)

0

� G�eIFiso4F�PAP)0 � G�PAP�VPg)

0(Eq. 12)

The values for G0(eIFiso4F�PAP), G0

(VPg�PAP), andG0

(eIFiso4F�VPg) were determined from K1, K2, and K3 in Fig. 7,respectively. G0

(eIFiso4F�VPg�PAP) values were determined fromthe addition of theG0 values calculated fromK2 andK5. Theseinteraction energies indicate how the binding of one compo-nent to its site affects the binding of a second component to itssite; thus, each component (PAP, VPg, and eIFiso4F) is createdas if it possesses two binding sites. For instance, Go

(PAP,eIFiso4F�VPg) shows how the binding of eIFiso4F to one site onPAP affects the affinity of the VPg for its binding site on PAP.The coupling energies may be positive, negative, or zero,depending on whether the interactions are anticooperative,cooperative, or noncooperative due to the binding of the sec-ond component, respectively (52). The coupling energies calcu-lated in this manner are presented in Fig. 7.The binding of either PAP or VPg to eIFiso4F enhances

the subsequent binding of the second factor to eIFiso4F,G0(eIFiso4F, VPg�PAP) is�1.5 kJ/mol, which is indicative of coop-erative heterotropic interaction between these proteins. On theother hand, the binding of either VPg or eIFiso4F to PAP isanticooperative (G0

(PAP, eIFiso4F�VPg) � 1.1 kJ/mol) and sup-ports competitive type binding between VPg and eIFiso4F, aspreviously determined. This suggests that the eIFiso4F-VPginteraction may prevent VPg from interacting with structuralfeatures of the PAP. The binding of PAP or eIFiso4F to VPg(G0(VPg, eIFiso4F�PAP) � 0.1 kJ/mol) is relatively indifferent tothe subsequent binding of the second component to VPg. Fromthese data, a mechanism can be proposed for the sequence ofevents leading to the formation of PAP�VPg�eIFiso4F complexwith subsequent inhibition of depurination of TEV-derivedRNA. Two models are possible, where PAP first forms a binarycomplex with eIFiso4F initiation factor, with the subsequentbinding to VPg, or PAP binds to a preformed eIFiso4F�VPgbinary complex. Both models lead to a ternary PAP�VPg�eIFiso4Fcomplex formation, which brings PAP to close proximity with

viralRNA.However, theabovecooperative interactionshinder theenzymatic site of PAP from the depurination of RNA, thus pro-moting inhibition of the plant’s defense mechanism.Because cap-binding proteins bind to VPg similarly to cap

analogs, andVPg stimulates the in vitro translation of uncappedIRES-containing RNA and inhibits capped RNA translation inwheat germ extracts (32), we have analyzed the extent to whichVPg can selectively target PAP to uncapped IRES-containingviral RNA, in contrast to its ability to target eIF4F to TEV RNA(32). Instead, VPg inhibits depurination of both capped anduncapped S/R oligonucleotides and IRES-containingTEV tran-scripts. Inhibition of enzymatic activity of PAP is supported inthe wheat germ lysate translational system (Fig. 6), indicatingthat VPg can inhibit PAP even in the presence of other cellularcomponents. This inhibition of the depurinating activity ofPAP is concentration-dependent; equimolar concentrations ofVPg completely abolish the enzymatic activity of PAP. VPg hasbeen implicated in overcoming viral resistance in plants (25).Extreme toxicity of PAP to plant cells does not allow expressionof the protein in vivo and subsequent studies of PAP interactionwith VPg. However, in support of the in vivo relevance of ourfindings is the fact that pokeweed mosaic virus, a VPg-linkedviral species, is the only virus to our knowledge reported toinfect P. americana (53).Our findings further support thenotionthatVPgmayplayarole

in overcoming viral resistance by suppressing the defensemecha-nism of the plant. Furthermore, depurination inhibition by VPgalso suggests the possible use of this protein against cytotoxicactivity of RIPs and inhibition of their biological potency.

REFERENCES1. Obrig, T. G., Irvin, J. D., and Hardesty, B. (1973) The effect of an antiviral

peptide on the ribosomal reactions of the peptide elongation enzymes,EF-I and EF-II. Arch. Biochem. Biophys. 155, 278–289

2. Duggar, B. M., and Armstrong, J. K. (1925) The effect of treating virus oftobacco mosaic with juice of various plants. Ann. Mol. Bot. Gard. 12,359–365

3. Dallal, J. A., and Irvin, J. D. (1978) Enzymatic inactivation of eukaryoticribosomes by the pokeweed antiviral protein. FEBS Lett. 89, 257–259

4. Osborn, R. W., and Hartley, M. R. (1990) Dual effects of the ricin A chainon protein synthesis in rabbit reticulocyte lysate. Inhibition of initiationand translocation. Eur. J. Biochem. 193, 401–407

5. Brigotti, M., Rambelli, F., Zamboni, M., Montanaro, L., and Sperti, S.(1989) Effect of �-sarcin and ribosome-inactivating proteins on the inter-action of elongation factors with ribosomes. Biochem. J. 257, 723–727

6. Mansouri, S., Nourollahzadeh, E., and Hudak, K. A. (2006) Pokeweed an-tiviral protein depurinates the sarcin/ricin loop of the rRNAprior to bind-ing of aminoacyl-tRNA to the ribosomal A-site. RNA 12, 1683–1692

7. Tumer, N. E., Parikh, B. A., Li, P., and Dinman, J. D. (1998) The pokeweedantiviral protein specifically inhibits Ty1-directed 1 ribosomal frame-shifting and retrotransposition in Saccharomyces cerevisiae. J. Virol. 72,1036–1042

8. Hudak, K. A., Hammell, A. B., Yasenchak, J., Tumer, N. E., and Dinman,J. D. (2001) A C-terminal deletion mutant of pokeweed antiviral proteininhibits programmed 1 ribosomal frameshifting and Ty1 retrotranspo-sition without depurinating the sarcin/ricin loop of rRNA. Virology 279,292–301

9. Barbieri, L., Battelli, M. G., and Stirpe, F. (1993) Ribosome-inactivatingproteins from plants. Biochim. Biophys. Acta 1154, 237–282

10. Nielsen, K., and Boston, R. S. (2001) Ribosome-inactivating Proteins: APlant Perspective. Annu Rev. Plant Physiol. Plant Mol. Biol. 52, 785–816

11. Hudak, K. A., Dinman, J. D., and Tumer, N. E. (1999) Pokeweed antiviralprotein accesses ribosomes by binding to L3. J. Biol. Chem. 274, 3859–3864

Inhibition of PAP by VPg

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12. Di, R., and Tumer, N. E. (2005) Expression of a truncated form of ribo-somal protein L3 confers resistance to pokeweed antiviral protein and theFusarium mycotoxin deoxynivalenol. Mol. Plant Microbe Interact. 18,762–770

13. Rajamohan, F., Venkatachalam, T. K., Irvin, J. D., and Uckun, F. M. (1999)Pokeweed antiviral protein isoforms PAP-I, PAP-II, and PAP-III depuri-nate RNA of human immunodeficiency virus (HIV)-1. Biochem. Biophys.Res. Commun. 260, 453–458

14. Chen, Z., Antoniw, J. F., andWhite, R. F. (1993) A possible mechanism forthe antiviral activity of pokeweed antiviral protein. Physiol. Mol. PlantPathol. 42, 249–258

15. Ussery, M. A., Irvin, J. D., and Hardesty, B. (1977) Inhibition of poliovirusreplication by a plant antiviral peptide.Ann. N.Y. Acad. Sci. 284, 431–440

16. Aron, G. M., and Irvin, J. D. (1980) Inhibition of herpes simplex virusmultiplication by the pokeweed antiviral protein.Antimicrob. Agents Che-mother. 17, 1032–1033

17. Tomlinson, J. A., Walker, V. M., Flewett, T. H., and Barclay, G. R. (1974)The inhibition of infection by cucumber mosaic virus and influenza virusby extracts from Phytolacca americana. J. Gen. Virol. 22, 225–232

18. Picard, D., Kao, C. C., and Hudak, K. A. (2005) Pokeweed antiviral proteininhibits brome mosaic virus replication in plant cells. J. Biol. Chem. 280,20069–20075

19. Hudak, K. A., Wang, P., and Tumer, N. E. (2000) A novel mechanism forinhibition of translation by pokeweed antiviral protein. Depurination ofthe capped RNA template. RNA 6, 369–380

20. Hudak, K. A., Bauman, J. D., and Tumer, N. E. (2002) Pokeweed antiviralprotein binds to the cap structure of eukaryotic mRNA and depurinatesthe mRNA downstream of the cap. RNA 8, 1148–1159

21. Vivanco, J. M., and Tumer, N. E. (2003) Translation inhibition of cappedand uncapped viral RNAs mediated by ribosome-inactivating proteins.Phytopathology 93, 588–595

22. Wang,M., andHudak, K. A. (2006)Anovel interaction of pokeweed antiviralprotein with translation initiation factors 4G and iso4G. A potential indirectmechanism to access viral RNAs.Nucleic Acids Res. 34, 1174–1181

23. Baldwin, A. E., Khan, M. A., Tumer, N. E., Goss, D. J., and Friedland, D. E.(2009) Characterization of pokeweed antiviral protein binding to mRNAcap analogs. Competition with nucleotides and enhancement by transla-tion initiation factor iso4G. Biochim. Biophys. Acta 1789, 109–116

24. Murphy, J. F., Rychlik, W., Rhoads, R. E., Hunt, A. G., and Shaw, J. G.(1991) A tyrosine residue in the small nuclear inclusion protein of tobaccovein mottling virus links the VPg to the viral RNA. J. Virol. 65, 511–513

25. Keller, K. E., Johansen, I. E., Martin, R. R., and Hampton, R. O. (1998)Potyvirus genome-linked protein (VPg) determines pea seed-borne mo-saic virus pathotype-specific virulence in Pisum sativum. Mol. Plant Mi-crobe Interact. 11, 124–130

26. Leonard, S., Plante, D., Wittmann, S., Daigneault, N., Fortin, M. G., andLaliberte, J. F. (2000) Complex formation between potyvirus VPg andtranslation eukaryotic initiation factor 4E correlates with virus infectivity.J. Virol. 74, 7730–7737

27. Wittmann, S., Chatel, H., Fortin, M. G., and Laliberte, J. F. (1997) Interac-tion of the viral protein genome linked of turnipmosaic potyvirus with thetranslational eukaryotic initiation factor (iso) 4E of Arabidopsis thalianausing the yeast two-hybrid system. Virology 234, 84–92

28. Herbert, T. P., Brierley, I., and Brown, T. D. (1997) Identification of aprotein linked to the genomic and subgenomic mRNAs of feline calicivi-rus and its role in translation. J. Gen. Virol. 78, 1033–1040

29. Miyoshi, H., Suehiro, N., Tomoo, K., Muto, S., Takahashi, T., Tsukamoto,T., Ohmori, T., and Natsuaki, T. (2006) Binding analyses for the interac-tion between plant virus genome-linked protein (VPg) and plant transla-tional initiation factors. Biochimie 88, 329–340

30. Leonard, S., Viel, C., Beauchemin, C., Daigneault, N., Fortin, M. G., andLaliberte, J. F. (2004) Interaction of VPg-Pro of turnip mosaic virus withthe translation initiation factor 4E and the poly(A)-binding protein inplanta. J. Gen. Virol. 85, 1055–1063

31. Khan, M. A., Miyoshi, H., Ray, S., Natsuaki, T., Suehiro, N., and Goss, D. J.(2006) Interaction of genome-linked protein (VPg) of turnip mosaic viruswith wheat germ translation initiation factors eIFiso4E and eIFiso4F.

J. Biol. Chem. 281, 28002–2801032. Khan, M. A., Miyoshi, H., Gallie, D. R., and Goss, D. J. (2008) Potyvirus

genome-linked protein, VPg, directly affects wheat germ in vitro transla-tion. Interactions with translation initiation factors eIF4F and eIFiso4F.J. Biol. Chem. 283, 1340–1349

33. Zeenko, V., and Gallie, D. R. (2005) Cap-independent translation of to-bacco etch virus is conferred by an RNA pseudoknot in the 5�-leader.J. Biol. Chem. 280, 26813–26824

34. Ray, S., Yumak, H., Domashevskiy, A., Khan,M. A., Gallie, D. R., andGoss,D. J. (2006) Tobacco etch virus mRNA preferentially binds wheat germeukaryotic initiation factor (eIF) 4G rather than eIFiso4G. J. Biol. Chem.281, 35826–35834

35. Kneller, E. L., Rakotondrafara, A. M., and Miller, W. A. (2006) Cap-inde-pendent translation of plant viral RNAs. Virus Res. 119, 63–75

36. Gallie, D. R., Feder, J. N., Schimke, R. T., and Walbot, V. (1991) Post-transcriptional regulation in higher eukaryotes. The role of the reportergene in controlling expression.Mol. Gen. Genet. 228, 258–264

37. Zamboni, M., Brigotti, M., Rambelli, F., Montanaro, L., and Sperti, S.(1989) High-pressure liquid chromatographic and fluorimetric methodsfor the determination of adenine released from ribosomes by ricin andgelonin. Biochem. J. 259, 639–643

38. Zhang, Y., Geiger, J. D., and Lautt, W. W. (1991) Improved high-pressureliquid chromatographic-fluorometric assay for measurement of adeno-sine in plasma. Am. J. Physiol. 260, G658–G664

39. Miura, K., Okumura, M., Yukimura, T., and Yamamoto, K. (1991) Fluoro-metric determination of plasma adenosine concentrations using high-performance liquid chromatography. Anal. Biochem. 196, 84–88

40. Ren, J., andGoss, D. J. (1996) Synthesis of a fluorescent 7-methylguanosineanalog and a fluorescence spectroscopic study of its reaction with wheatgerm cap-binding proteins. Nucleic Acids Res. 24, 3629–3634

41. Hiratsuka, T. (1983) New ribose-modified fluorescent analogs of adenineand guanine nucleotides available as substrates for various enzymes.Biochim. Biophys. Acta 742, 496–508

42. Lakowicz, J. R. (2006) Principles of Fluorescence Spectroscopy, 3rd Ed., pp.52–54, Springer Science

43. Firpo, M. A., Connelly, M. B., Goss, D. J., and Dahlberg, A. E. (1996)Mutations at two invariant nucleotides in the 3�-minor domain of Esche-richia coli 16 S rRNA affecting translational initiation and initiation factor3 function. J. Biol. Chem. 271, 4693–4698

44. Levine, R. (1977) Fluorescence-quenching studies of the binding of biliru-bin to albumin. Clin. Chem. 23, 2292–2301

45. Sturm, M. B., and Schramm, V. L. (2009) Detecting ricin. Sensitive lumi-nescent assay for ricin A-chain ribosome depurination kinetics. Anal.Chem. 81, 2847–2853

46. Niepel, M., and Gallie, D. R. (1999) Identification and characterization ofthe functional elements within the tobacco etch virus 5� leader requiredfor cap-independent translation. J. Virol. 73, 9080–9088

47. Bonness, M. S., Ready,M. P., Irvin, J. D., andMabry, T. J. (1994) Pokeweedantiviral protein inactivates pokeweed ribosomes. Implications for theantiviral mechanism. Plant J. 5, 173–183

48. Tumer, N. E., Hudak, K., Di, R., Coetzer, C., Wang, P., and Zoubenko, O.(1999) Pokeweed antiviral protein and its applications. Curr. Top. Micro-biol. Immunol. 240, 139–158

49. Ready, M. P., Brown, D. T., and Robertus, J. D. (1986) Extracellular local-ization of pokeweed antiviral protein. Proc. Natl. Acad. Sci. U.S.A. 83,5053–5056

50. Weber, G. (1975) Energetics of ligand binding to proteins. Adv. ProteinChem. 29, 1–83

51. Goss, D. J., Parkhurst, L. J., Mehta, A. M., and Wahba, A. J. (1984) Coop-erative interactions in the system ribosomes-ribosomal protein S1-poly-nucleotide triplets. Biochemistry 23, 6522–6529

52. Carberry, S. E., and Goss, D. J. (1991) Interaction of wheat germ proteinsynthesis initiation factors eIF-3, eIF-(iso)4F, and eIF-4F with mRNA an-alogues. Biochemistry 30, 6977–6982

53. Kim, K. S., and Fulton, J.P. (1969) Electron microscopy of pokeweed leafcells infected with pokeweed mosaic virus. Virology 37, 297–308

Inhibition of PAP by VPg

29738 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 35 • AUGUST 24, 2012

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Artem V. Domashevskiy, Hiroshi Miyoshi and Dixie J. GossGenome-linked Protein (VPg)

Inhibition of Pokeweed Antiviral Protein (PAP) by Turnip Mosaic Virus

doi: 10.1074/jbc.M112.367581 originally published online July 6, 20122012, 287:29729-29738.J. Biol. Chem. 

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