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Article Cite This: Biochemistry XXXX, XXX, XXX-XXX

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Plant Translation Initiation Complex eIFiso4F Directs Pokeweed Antiviral Protein to Selectively Depurinate Uncapped Tobacco Etch Virus RNA Artem V. Domashevskiy,* Shawn Williams, Christopher Kluge, and Shu-Yuan Cheng Department of Sciences, John Jay College of Criminal Justice, the City University of New York, New York, New York 10019, United States S Supporting Information *

ABSTRACT: Pokeweed antiviral protein (PAP) is a ribosome inactivating protein (RIP) that depurinates the sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. PAP depurinates viral RNA, and in doing so, lowers the infectivity of many plant viruses. The mechanism by which PAP accesses uncapped viral RNA is not known, impeding scientists from developing effective antiviral agents for the prevention of the diseases caused by uncapped RNA viruses. Kinetic rates of PAP interacting with tobacco etch virus (TEV) RNA, in the presence and absence of eIFiso4F, were examined, addressing how the eIF affects selective PAP targeting and depurination of the uncapped viral RNA. PAP-eIFs copurification assay and fluorescence resonance energy transfer demonstrate that PAP forms a ternary complex with the eIFiso4G and eIFiso4E, directing the depurination of uncapped viral RNA. eIFiso4F selectively targets PAP to depurinate TEV RNA by increasing PAP’s specificity constant for uncapped viral RNA 12-fold, when compared to the depurination of an oligonucleotide RNA that mimics the SRL of large rRNA, and cellular capped luciferase mRNA. This explains how PAP is able to lower infectivity of pokeweed viruses, while preserving its own ribosomes and cellular RNA from depurination: PAP utilizes cellular eIFiso4F in a novel strategy to target uncapped viral RNA. It may be possible to modulate and utilize these PAP-eIFs interactions for their public health benefit; by repurposing them to selectively target PAP to depurinate uncapped viral RNA, many plant and animal diseases caused by these viruses could be alleviated.

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mutants (e.g., S1-3 and S2-3), suggesting that PAP recognizes specific structural RNA motifs.24 Additionally, PAP binds to eIF4G and its isoform eIFiso4G; a region of the protein between amino acids 511 and 624 is required for PAP binding activity.25 PAP binds to m7GTP-sepharose,25 and this interaction does not diminish PAP binding to eIFiso4G, indicating that a complex can form between the cap structure, PAP, and eIFiso4G. In the presence of wheat germ lysate, PAP depurinates uncapped transcripts containing a functional wild type 3′-translational enhancer sequence (3′-TE), but does not depurinate messages containing a nonfunctional 3′-translational enhancer sequence mutant,25 which supports an earlier hypothesis that the binding of PAP to eIF4G and eIFiso4G may provide an alternative mechanism for how PAP accesses both capped and uncapped viral RNA for depurination. However, the mechanism by which eIFs direct PAP to depurinate uncapped viral RNA remains unknown. PAP not only binds to the initiation factor eIFiso4G, but the binding of cap analogue to PAP is increased by this protein− protein interaction, suggesting a model where PAP interacts

ibosome inactivating proteins (RIPs) are widely distributed plant protein toxins.1−6 RIPs are RNA Nglycosidases7,8 that selectively remove a specific adenine residue from the sarcin/ricin loop (SRL) of large rRNA,3,9 inhibiting protein synthesis 10−14 and resulting in apoptosis.15−17 Ribosome inactivating proteins are important in plant senescence and in the defense mechanisms against foreign pathogens.2 The common pokeweed plant (Phytolacca americana L.) produces several isoforms of the pokeweed antiviral protein (PAP),18−20 a ribosome inactivating protein with prominent antiviral and antifungal properties, in addition to its ribosome depurinating activity. PAP binds the 5′-cap of mRNA and depurinates portions of gene transcripts adjacent to the cap, causing inhibition of the in vitro translation of several viruses, without depurination of the host ribosomes.21,22 PAP’s activity is selective, and the cap structure alone is not sufficient for the depurination of RNA at multiple sites;23 since PAP also lowers infectivity of uncapped viruses, this suggests that a different mechanism may exist allowing PAP to recognize and depurinate uncapped viral RNA. Presently, it is unknown how PAP targets uncapped viral RNA for depurination. PAP depurinates the full length 5′-leader sequence of tobacco etch virus (FLTEV) RNA, but not its nonfunctional © XXXX American Chemical Society

Received: June 23, 2017 Revised: September 6, 2017 Published: October 24, 2017 A

DOI: 10.1021/acs.biochem.7b00598 Biochemistry XXXX, XXX, XXX−XXX

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Mass Spectrometry. The sample of synthesized antm7GTP was subjected to an accurate Mass/HRMS ESI (electrospray ionization in negative mode) analysis, confirming the synthesis of a monosubstituted 2′/3′-ester derivative of antm7GTP. The calculated m/z for [C18H23N6O15P3−H−] is 655.0356 a.m.u., and the experimental m/z was found to be 655.0359 a.m.u. Absence of the diester-substituted 2′,3′-antm7GTP derivative was confirmed in the similar manner (Figure S5 of the Supporting Information). Waters XEVO G2-XS QToF mass spectrometer, equipped with a UPC2 SFC inlet, on-board fluidics, an electrospray ionization (ESI) probe, an atmospheric pressure chemical ionization (APCI) probe, and an atmospheric pressure solids analysis probe (APAP), was employed for the analysis of ant-m7GTP sample (Department of Chemistry, Columbia University, NY). Adenine Fluorescence Quantification Assay. Depurination of FLTEV RNA, S1-3 TEV RNA, SRL oligonucleotide RNA, and luciferase mRNA was assayed as previously described by Zamboni et al.,39 with several modifications outlined in the Supporting Information. Steady-State Fluorescence. Steady-state fluorescence was used to monitor protein−protein interactions.40 Acquisition of steady state fluorescence allows the use of intrinsic and extrinsic protein fluorophores to determine equilibrium constants. A Horiba Jobin Yvon FluoroMax-3 fluorometer, equipped with a 150-W xenon lamp and photodiode array detectors, was used for all fluorescence measurements. Fluorescence changes (quenching or enhancement, depending on the titration) were monitored using λem = 332 nm (intrinsic protein fluorescence), 516 nm (for fluorescein), or 420 nm (antm7GTP). The analysis details are outlined in the Supporting Information. Stopped-Flow Measurements. Stopped flow fluorescence measurements were performed on a Horiba Jobin fluorometer, equipped with a SFA-20 rapid kinetics stopped-flow accessory (Hi-Tech Scientific). The excitation wavelength for fluoresceinlabeled PAP was 495 nm. The details of the experiments are outlined in the Supporting Information. Protein Co-Purification Assay. Protein interactions were analyzed employing m7GTP-agarose resin (Figure S3 of the Supporting Information)41 as previously described,42,43 with several modifications outlined in the Supporting Information. Fluorescence Resonance Energy Transfer (FRET) Assay. For FRET assay, recombinant eIFiso4E and eIFiso4G proteins were labeled with Alexa Fluor 488 and Alexa Fluor 555 fluorescent dyes, respectively; PAP was labeled with Alexa Fluor 647 dye. Labeled proteins were mixed together in 1:1 stoichiometric ratios to produce various combinations; the FRET signal was measured by steady-state fluorescence. All steady-state fluorescence measurements were carried out at 25 °C in buffer E (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.5 mM EDTA, 0.2 μM BSA, and 10% glycerol). Excitation wavelength was 488 nm (blue laser, unless otherwise stated; slit width was 4 nm), and the emission wavelength was 519 nm for the 488-labeled eIFiso4E, 565 nm for the 555-labeled eIFiso4G, and 655 nm for the 647-labeled PAP (slit width was 8 nm). All fluorescence measurements were corrected for lamp fluctuations, eliminating the contributions of a lamp noise and intensity drift. To minimize nonspecific binding of labeled proteins to glass surfaces, and to minimize nonspecific binding of free probe to proteins, BSA was included at 0.2 μM. Details are outlined in the Supporting Information.

with eIFiso4G/eIF4G (as part of the eIFiso4F/eIF4F complex) and binds to the cap region of mRNA.26 Furthermore, the addition of eIFiso4E/eIF4E (as part of the eIFiso4F/eIF4F complex) lowers the binding affinity of PAP for the cap competitively because both are specific cap-binding proteins. These PAP−eIFs interactions possibly unfold the active site of PAP, thus recognizing purine residues for depurination.26 This model was modified to account for the 5′- and 3′-untranslated regions (UTRs) and TEs commonly present within mRNAs, which are important for PAP’s antiviral activity.27 Here we hypothesize that plant eIFiso4F complex is a key element that directs PAP to depurinate uncapped viral RNA, and examine the effects of eIFs (eIFiso4E and eIFiso4G) on PAP’s depurination rates of the uncapped 5′-leader sequence of TEV (FLTEV) RNA (Figure S1 of the Supporting Information); the oligonucleotide that mimics the SRL of large rRNA and cellular m 7 G-capped luciferase RNA were examined.19,24,28,29 Using a combination of biophysical and biochemical techniques, we show that eIFiso4F (complex of eIFiso4E and eIFiso4G) preferentially directs PAP to depurinate TEV RNA by increasing PAP’s specificity constant for uncapped viral RNA nearly 12-fold, when compared to the depurination of nonviral control RNAs.

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MATERIALS AND METHODS Materials and methods for protein expression, purification and labeling, synthesis of a fluorescent cap analogue, in vitro RNA transcription, steady state and stopped-flow fluorescence, and protein copurification assay are described in the Supporting Information. Pokeweed Antiviral Protein (PAP). Spring leaves of the pokeweed plant were used as a raw material for the purification of the PAP-I isoform, employed in these experiments.24,26,30 Purification of the pokeweed antiviral protein from the pokeweed plant is described in the Supporting Information. Translation Initiation Factors: eIFiso4E and eIFiso4G. Wheat eIFiso4E and eIFiso4G recombinant proteins were expressed in BL21(DE3)pLys Escherichia coli by means of a pET3d plasmid, and purified as described previously.31−34 For the purification of recombinant eIFiso4G protein, a HiTrap SP column was employed. Purification of the eIFs is detailed in the Supporting Information. Protein-Fluorophore Labeling. For the titration experiments, PAP and eIFiso4G were labeled with fluorescein, using a Pierce N-hydroxysuccinamide (NHS)-fluorescein antibody labeling kit, according to the manufacture’s protocol, as described previously.30 The APEX antibody labeling kits provided a convenient method for covalently linking Alexa Fluor-488, -555, and -647 fluorescent dyes to the eIFiso4E, eIFiso4G, and PAP proteins, respectively. Protein-fluorophore labeling is described in the Supporting Information. Anthraniloyl-m7GTP Cap Analogue. Synthesis of anthraniloyl-m7GTP cap analogue was accomplished by reacting m7GTP with crystalline isatoic anhydrate at 37 °C, while maintaining steady pH of 9.6, as per the previously published protocol,35,36 and described in the Supporting Information. Typically, the anthraniloyl moiety may be covalently attached to one or both hydroxyl groups of the ribose ring,37,38 producing either mono- or disubstituted regioisomers that may affect properties of the cap-binding proteins. MS ESI of the sample showed that only the monosubstituted derivative was afforded during the reaction (see Figure S5 of the Supporting Information). B

DOI: 10.1021/acs.biochem.7b00598 Biochemistry XXXX, XXX, XXX−XXX

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Figure 1. Kinetic studies of PAP depurinating TEV, SRL, and luciferase RNA, and the effects of eIFiso4F on the rates of the depurination. (A) Time course curves of adenine released during the depurination of TEV 5′-UTR (in the absence (−light blue boxes−) and presence (−green boxes−) of eIFiso4F), SRL RNA (in the absence (−light purple circles−) and presence (−purple circles−) of eIFiso4F), and m7G-capped luciferase RNA (in the absence (−light blue diamonds−) and presence (−blue diamonds−) of eIFiso4F) by PAP, as measured by the fluorescence of N6-ethenoadenine. The controls of the studies were depurination of S1-3 TEV RNA (−orange triangles−) and poly(C) RNA (−red triangles−) in the presence of eIFiso4F. Aliquots of PAP-RNA mixtures (± eIFiso4F) were withdrawn at different times, and loaded onto the HPLC column (excitation and emission wavelengths were 315 and 415 nm, respectively). (B) N6-Ethenoadenine assay kinetic curve for the depurination catalysis of TEV 5′-UTR (in the absence (−□−) and presence (−■−) of eIFiso4F), SRL RNA (in the absence (−○−) and presence (−●−) of eIFiso4F), and m7G-capped luciferase RNA (in the absence (−◊−) and presence (−⧫−) of eIFiso4F) by PAP. The controls of the studies were depurination of S1-3 TEV RNA (−Δ−) and poly(C) RNA (−∇−) in the presence of eIFiso4F. Each reaction included a sample of PAP (100 nM), treated with the increasing concentrations of RNA, and amounts of the released adenines were monitored, as described under Materials and Methods.

Table 1. Effects of eIFiso4F on Kinetic Rates of PAP Depurinating TEV RNA, SRL Oligonucleotide RNA, and Capped Luciferase RNA kcat (min−1)

■

kcat / Km (M−1·s−1)

Km (nM)

RNA

−

+eIFiso4F

−

+eIFiso4F

−

+eIFiso4F

TEV SRL luciferase

4.2 ± 0.7 4.9 ± 0.6 1.8 ± 0.4

3.9 ± 0.3 4.7 ± 0.3 1.1 ± 0.2

91.6 ± 8.9 324 ± 31 89.5 ± 8.2

7.4 ± 1.3 162 ± 18 81.5 ± 6.4

7.6 × 105 2.5 × 105 3.4 × 105

88 × 105 4.8 × 105 2.2 × 105

RESULTS AND DISCUSSION Duggar and Armstrong44 are credited with the discovery of type 1 ribosome inactivating proteins when they observed that a protein, isolated from extracts of Phytolacca americana, bears a potent antiviral activity, and inhibiting the transmission of plant and animal viruses. Yet, for many years PAP’s antiviral mechanism remained poorly understood. It was long suspected that a direct interaction of PAP with viral RNA (or DNA) might be an alternative mechanism that does not depend solely on the ribosomal inactivation. PAP and its isoforms cause a concentration-dependent depurination of genomic HIV-1,45−47 herpes simplex virus (HSV),48 poliovirus,49 influenza virus,50 BMV,51 and lymphocytic choriomeningitis virus (LCMV),52 among others, thus displaying a very broad spectrum of antiviral activity.19 eIFiso4F Increases PAP’s Specificity Constant for Uncapped FLTEV RNA. To examine how eIFiso4F affects the rates at which PAP depurinates uncapped TEV RNA, oligonucleotide RNA that mimics the SRL of large rRNA, and capped cellular luciferase RNA, standard quantification of adenine in the discontinuous assay format was performed.30 Analysis of the fractions with the HPLC indicated that the TEV RNA depurination by PAP was virtually 80% complete after 5

min, whereas the SRL RNA after 4 min, and that of luciferase RNA after 7 min (Figure 1A). To establish PAP’s catalytic constant for the depurination of uncapped TEV RNA, and then compare the rate of depurination to that of the SRL RNA and capped luciferase RNA, in the presence and absence of eIFiso4F, the RNA concentrations were varied. The progress of the reactions was monitored by the appearance of a UVabsorbing product at the saturating conditions. Experimental depurination rates were then plotted against the corresponding RNA concentrations, resulting in Michaelis−Menten type behavior (Figure 1B). The presence of the eIFiso4F increased PAP’s specificity constant (kcat/Km) for the TEV RNA (7.6 × 105 versus 88 × 105) nearly 12-fold. PAP’s specificity for the SRL RNA just about doubled (2.5 × 105 versus 4.8 × 105) in the presence of the eIF, while the specificity for capped luciferase RNA was marginally lowered by one-and-a-half fold (3.4 × 105 versus 2.2 × 105) (Table 1). Statistically, the presence of eIFiso4F has no significant effect on the depurination of the SRL or capped luciferase RNA. As negative controls, non-functional S1-3 TEV RNA and poly(C) RNA were employed, showing no effect of RNA depurination by PAP in the absence (or presence) of eIFiso4F (Figure 1). These data demonstrate that the eIFiso4F increases depurination of C

DOI: 10.1021/acs.biochem.7b00598 Biochemistry XXXX, XXX, XXX−XXX

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changes for the binding of PAP to eIFiso4G, in the absence and presence of eIFiso4E. Fluorescence intensity versus time data were fitted by nonlinear regression analysis30,31,42 as a singleexponential, as described under Materials and Methods; a double-exponential did not improve the data fitting. The presence of eIFiso4E increased the rate constant (kon) of PAP binding to eIFiso4G by nearly 2-fold (PAP-eIFiso4G, kon = 149.1 ± 9.0 s−1; PAP-eIFiso4F, kon = 292.1 ± 11.5 s−1) at 25 °C (Figure 2 and Table 2). The residuals representing the

viral RNA as compared to nonviral control RNAs, supporting our earlier proposed idea of eIFs directing depurination of viral RNA as a defense mechanism against viral infection. A valid question to ask would be if PAP depurinates both ribosomal and viral RNA, how does the pokeweed plant prevent its own death? Being a potent cellular toxin, PAP is exported out of the cell once it is synthesized and localized within the cell matrix.53 It is hypothesized that PAP gains access into the cytoplasm as the pathogen enters the cell, thus promoting its activity by impairing ribosomes.20 A recent study showed that PAP is able to form a homodimer complex in the cytosol of the pokeweed plant, while its monomeric form is formed predominantly outside the cell, the apoplast.54 The PAP homodimer was shown to be much less active on rRNA in comparison to the monomeric PAP. PAP dimerization involves an active site Tyr123; mutations of this aromatic residue prevent dimerization of PAP in vivo, supporting the biological role of homodimerization as a mechanism to limit ribosomal depurination.54 In recent years, a small viral protein (VPg), linked to the genome of TuMV was shown to inhibit PAP’s activity.30,55−57 It is hypothesized that VPg functions as a viral evolutionary adaptation, among other roles, aiding the virus in overcoming plant defense mechanisms.55,56 eIFiso4E Increases Kinetic Rate of PAP Binding to eIFiso4G. To examine how eIFiso4E affects the kinetic rate of PAP-eIFiso4G binding, stopped-flow fluorescence measurements were performed. Study of reaction rates is an important tool in investigating the chemical mechanism of PAP catalysis (RNA depurination). Knowledge of the dynamic properties of PAP catalysis is a prerequisite for the design of targeted antiviral agents. Figure 2 shows the stopped-flow fluorescence

Table 2. Effects of eIFiso4E on the Kinetic Parameters of PAP Binding to eIFiso4G complex

kon (μM−1·s−1)

koff (s−1)

Kd (nM)

PAP-eIFiso4G PAP-eIFiso4Fa

149 ± 9.0 292 ± 11.5

15.8 ± 5.2 22.7 ± 6.6

106 ± 4.5b 77.7 ± 9.0b

a

1:1 complex of eIFiso4E and eIFiso4G was used. bValues for the dissociation constants were calculated from Kd = koff/kon.

deviations between the calculated and the experimental data indicate that the single-exponential function fits the points over the entire time course of the measurements. Experiments were conducted using a high concentration of PAP and limited concentrations of eIFiso4G and eIFiso4F (eIFiso4E and eIFiso4G, 1:1 ratio) to ensure that the combination of PAP with eIFs was pseudo-first order. For the following one-step reaction mechanism: kon

PAP + elFiso4G XooY PAP − elFiso4G koff

where kon and koff are the rates of association and dissociation, respectively, for the interactions of PAP with eIFiso4G, the observed rate constant (kobs) is predicted to be a linear function of PAP concentration: kobs = kon·[PAP] + koff. The observed rate constant increased linearly with an increase in PAP concentration (Figure 3). The plot shown in Figure 3 was used to obtain values of kon (slope) and koff from the slope and the y axes intercept, respectively (Table 2). Concentration dependent reaction rates suggest a simple one-step association mechanism between PAP and eIFiso4G. The dissociation constant was calculated from the following expression: Kd = koff/kon, as 106.0 ± 4.5 nM for PAP-eIFiso4G binding and 77.7 ± 9.0 nM for PAP-eIFiso4F binding, indicating that the presence of eIFiso4E has increased PAP’s affinity for eIFiso4G by nearly 26.7%. PAP and eIFiso4E Bind Anthraniloyl-m7GTP Cap Analogue Competitively. Competitive binding of PAP and eIFiso4E to m7G cap analogue was determined by employing a fluorescent cap analogue, ant-m 7 GTP (anthraniloylm7GTP).35,36 These competitive substitution reactions were performed at a constant 100 nM ant-m7GTP concentration, monitoring the fluorescence changes of the analogue and increasing amounts of eIFiso4E, in the absence and presence of PAP (Figure 4). The ant-m7GTP was a suitable choice for our competition studies because the excitation (λex = 332 nm) and emission (λem = 420 nm) maxima of this extrinsic fluorophore differs greatly from native protein fluorescence (λex = 280 nm and λem = 332 nm); it was previously demonstrated that this fluorescent probe neither greatly affect interactions of capbinding proteins, nor perturbs the equilibrium of the fluorophore (Kd for PAP-ant-m7GTP interactions, 41.7 nM;56 essentially it is the same as reported previously for PAPm7GTP, 43.3 nM).26 Lineweaver−Burk plots (Figure 4 inset)

Figure 2. Stopped-flow fluorescence changes for the interaction of PAP with eIFiso4G and eIFiso4F. The curves represent a single exponential fit to the fluorescence data points. Residuals for the fits are shown in the lower panels. A solution of 500 nM (250 nM after mixing) eIFiso4G or eIFiso4F (125 nM of eIFiso4E and eIFiso4G each) was mixed with 2000 nM (1000 nM after mixing) PAP. The experimental conditions are described under Materials and Methods. D

DOI: 10.1021/acs.biochem.7b00598 Biochemistry XXXX, XXX, XXX−XXX

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Figure 3. PAP binds eIFiso4G and eIFiso4F in a one-step, bimolecular reaction. Varying PAP concentrations (50, 100, 250, 500, and 1000 nM final) were mixed with 50 nM (final) of (A) eIFiso4G and (B) eIFiso4F (1:1 complex of eIFiso4E and eIFiso4G) in 20 mM Tris-HCl, pH 7.6, 1 mM DTT, and 100 mM KCl at 25 °C. Excitation and emission wavelength were 494 and 518 nm, respectively. (C) The observed rate constant for the fluorescence change of PAP-eIFiso4G (−blue triangles−) and PAP-eIFiso4F (−light red diamond−) is plotted as a function of increasing PAP concentrations. Data points in the plot of kobs versus concentration were obtained from three independent experiments; the average values of the experimental data are reported. Extrapolation of the slope to the y-axes provides koff.

°C) according to the following equation: Kobs = Km × (1 + [I]/ Ki),40 where Kobs is the observed equilibrium constant in the presence of eIFiso4E (114.9 nM), Km is the equilibrium constant for PAP-ant-m7GTP interactions in the absence of eIFiso4E (42.51 nM), [I] is the final concentration of eIFiso4E (200 nM), and Ki is the inhibitions constant for the interactions. A mechanism to determine how PAP inhibits the translation of capped RNA viruses has been proposed by Hudak et al.,22 where PAP directly binds to the 5′-cap of capped viral RNA and depurinates the RNA downstream from the cap moiety. In these studies, PAP was able to distinguish between capped and uncapped transcripts, and specifically targeted capped RNA for depurination.22 PAP-m7GTP cap analogue interactions were characterized using fluorescence spectroscopy.26 However, many plant and animal positive strand RNA viruses do not include the 5′-cap structure in their genomes,58 and it was shown that PAP does not depurinate every capped RNA (e.g., alfalfa mosaic virus (AMV)), while exhibiting inhibitory effects on the replication of certain uncapped viruses (e.g., TMV and BMV, but not satellite panicum mosaic virus (SPMV) or tomato bushy stunt virus (TBSV)).23 We have demonstrated previously that PAP depurinates uncapped FLTEV RNA,24 but does not depurinate any of the truncated TEV mutants lacking an internal ribosome entry site (IRES). Inarguably, the mechanism of PAP’s selectivity for viral RNA is complex and entails factors other than the 5′-cap moiety.

Figure 4. Effects of varying PAP concentrations on eIFiso4E binding to ant-m 7GTP analogue. Fluorescence titrations of constant concentrations of ant-m7GTP (100 nM) with eIFiso4E were carried out in the presence of varying PAP concentrations. PAP concentrations shown are 0 nM (−dark brown circles−), 100 nM (−light purple triangles−), 200 nM (−pink squares−). Results were analyzed quantitatively and presented as Lineweaver−Burk plots (inset). All data were collected at 25 °C in titration buffer (20 mM HEPES-KOH, pH 7.5, 100 mM KCl, 1 mM MgCl2, 1 mM DTT), as described under Materials and Methods.

meet at the y-axis intercept, suggesting a competitive ligand binding between PAP and eIFiso4E for the cap analogue. Inhibition constant Ki was calculated to be 117.6 ± 8.4 nM (25 E

DOI: 10.1021/acs.biochem.7b00598 Biochemistry XXXX, XXX, XXX−XXX

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Figure 5. Protein copurification assay employing immobilized γ-aminophenyl-m7GTP (C10-spacer)-agarose column. Co-purified proteins were eluted from the column and subjected to SDS-PAGE analysis, with subsequent Coomassie Blue staining. (A) Protein samples prior to loading onto the column. (B) Controls: Top panel, PAP was loaded onto the column; then, m7GTP gradient (from 0 to 1250 nM) was applied; middle panel, eIFiso4E was loaded onto the column; then, m7GTP gradient (from 0 to 1250 nM) was applied; Bottom panel, eIFiso4G was loaded onto the column; then, m7GTP gradient (from 0 to 1250 nM) was applied. (C) PAP was loaded onto the column; then, (1) eIFiso4E gradient (from 0 to 1250 nM) was applied (first elution), followed by (2) m7GTP gradient (from 0 from 1250 nM) (second elution); (D) PAP was loaded onto the column; then, (1) eIFiso4G gradient (from 0 to 1250 nM) was applied (first elution), followed by (2) m7GTP gradient (from 0 to 1250 nM) (second elution); (E) PAP was loaded onto the column; then, (1) eIFiso4G gradient (from 0 to 1250 nM) was applied (first elution), followed by (2) eIFiso4E gradient (from 0 to 1250 nM) (second elution), then, followed by (3) m7GTP gradient (from 0 to 1250 nM) (third elution).

Co-Purification Assay Reveals that PAP, eIFiso4G, and eIFiso4E Form a Ternary Complex. PAP binds to eukaryotic translation initiation factors eIF4G and eIFiso4G;25 the binding of PAP to m7GTP-sepharose does not diminish eIFiso4G-PAP interactions, thus illustrating that PAP simultaneously forms a complex with eIFiso4G and the cap moiety. A model for PAP interaction with initiation factors and capped mRNA has been proposed.26 To further understand the nature of PAP−eIFs interactions, and to determine whether PAP, eIFiso4G, and eIFiso4E are able to form a stable ternary complex, affinity protein copurification assay, employing m7GpppG-agarose (Figure 5),41 was performed. The cap-binding nature of PAP and eIFiso4E proteins and their simultaneous affinity for eIFiso4G scaffolding protein results in a gradual elution of a resin-bound PAP and eIFiso4E, once a 5 mL gradient of 2 μM m7GTP cap analogue in buffer B-100 is applied to the column. Figure 5A displays preco-purified protein samples (eIFiso4E, 26 kDa; PAP, 29 kDa; eIFiso4G, 86 kDa), prior to their application onto the column (∼40 μL of protein samples were loaded in each of the gel lanes). Figure 5B presents controls for the assay: (i) PAP elutes gradually from the cap-binding resin (1 mL of a settled bead volume), when a 0.5 mL incremental gradient of the increasing m7GTP in buffer B-100 (from 0 to 1250 nM m7GTP final concentration) is applied to the column (Figure 5B top panel); (ii) similarly, eIFiso4E elutes gradually from the resin once an identical m7GTP-containing buffer B-100 gradient is implemented (Figure 5B middle panel); (iii) contrariwise, eIFiso4G displays no affinity for the cap-binding resin, eluting with the buffer B-100 flow through, and/or the m7GTP gradient. Further, protein binary and ternary complexes were confirmed when a differential elution is administered (Figure 5C bottom panel). Figure 5C demonstrates: (i) gradual displacement of a resin-bound PAP as the increasing amounts of the eIFiso4E protein being applied to the column (500 μL of buffer B-100 containing 0, 50, 100, 250, 500, 1000, and 1250

nM of eIFiso4E) (Figure 5C top panel), followed by a gradual elution of the eIFiso4E from the column when the m7GTP gradient is administered in the second elution step (500 μL of buffer B-100 containing 0, 50, 100, 250, 500, 1000, and 1,250 nM of m7GTP) (Figure 5C bottom panel). Figure 5D shows that a resin-bound PAP forms a binary complex with the eIFiso4G when the scaffolding protein is added to the column (eIFiso4G begins to elute from the column when the protein becomes excessive) (Figure 5D top panel), and gradual displacement of the PAP-eIFiso4G binary complex from the column once incremental amounts of m7GTP are applied (both PAP and eIFiso4G proteins elute from the column) (Figure 5D bottom panel). Figure 5E demonstrates that a resin-bound PAP is able to form a binary complex with the eIFiso4G (Figure 5E top inset), and simultaneously, a ternary complex with the eIFiso4E protein (Figure 5E middle panel) when the eIFiso4E is added to the column (addition of the eIFiso4E concentrations greater than 1000 nM results in the displacement of the ternary complex from the resin once the eIFiso4E becomes excessive). In the third elution step, gradual elution of the three proteins is observed when the incremental amounts of m7GTP are administered to the column (500 μL of buffer B-100 containing 0, 50, 100, 250, 500, 1000, and 1250 nM of m7GTP) (Figure 5E bottom panel). Fluorescence Resonance Energy Transfer (FRET) Studies of the Protein Ternary Complex. To further gain insight into PAP-eIFiso4G-eIFiso4E ternary complex organization and to determine the proximity of the bound proteins in the ternary complex, FRET was employed, adopting extrinsic fluorophore-labeled proteins (eIFiso4E-488,, eIFiso4G-555, and PAP-647). Relative fluorescence intensity of different donor and acceptor combinations (Table S1 of the Supporting Information) were used to calculate FRET efficiency (E) and the average distances between the fluorophores (r) in the binary and ternary protein complexes (Table 3). Fluorescence F

DOI: 10.1021/acs.biochem.7b00598 Biochemistry XXXX, XXX, XXX−XXX

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and eIFiso4E to eIFiso4G was determined by utilizing fluorescein-labeled eIFiso4G (λex = 495 nm and λem = 516 nm). PAP and eIFiso4E are cap-binding proteins; a control titration of PAP with eIFiso4E was performed to be certain that PAP is not just a sticky protein, binding to other proteins indiscriminately (Figure S2 of the Supporting Information). The competitive titration reactions were performed at a constant 100 nM concentration of eIFiso4G with increasing amounts of PAP, while monitoring the fluorescence changes of the labeled eIFiso4G, in the absence and presence of varying amounts of eIFiso4E (Figure 6). PAP competes with eIFiso4E

Table 3. FRET Efficiencies and Fluorophore Distances between eIFiso4E, eIFiso4G, and PAP protein(s) (λex = 488 nm) eIFiso4E‑488−eIFiso4G‑555 eIFiso4G‑555−PAP‑647 eIFiso4E‑488−PAP‑647 eIFiso4E‑488−eIFiso4G‑555− PAP‑647

FRET efficiency, E (%) 88 ± 6.4 73 ± 8.2 67 ± 8.8 E1(488−555) = 86 ± 7.1 E2(555−647) = 91 ± 11.3

fluorophore distance, r (Å) 50.2 ± 3.4 43.2 ± 2.8 49.8 ± 3.1 r1(488−555) = 51.0 ± 4.2 r2(555−647) = 34.7 ± 3.8

signal intensity of the unlabeled native proteins and their complexes (at 332 nm) was determined to be negligible, and therefore was integrated into the background correction. All samples were excited with a blue laser of 488 nm wavelength, unless otherwise stated. Excitation of a single donor (eIFiso4E488)) at 488 nm shows a prominent energy emission at 519 nm (6.11 × 106 a.u.) (weak residual emission energy was detected at 565 nm due to an unsymmetrical lagging spectrum of the emitting fluorophore). When the eIFiso 4E-488(donor) was incubated with the eIFiso4G-555 (acceptor), the two proteins formed a binary complex (eIFiso4F), placing both fluorophores in proximity to each other and allowing for the emission energy of the 488 dye to be transferred to the 555 dye (eIFiso 4E488donor only, FD = 6.1 × 106 a.u.; eIFiso4E-488-eIFiso4G-555 binary complex, FDA = 7.3 × 105 a.u.). An appropriate correction factor was introduced, accounting for the energy emission: a direct result from the acceptor fluorophore excitation. Spectra containing multiple elements were decomposed into individual spectral components, and the area under each spectrum was integrated; spectral overlap integral was calculated by the FluorEssence software (Horiba Scientific). FRET efficiency was estimated at 88 ± 6.4%, and the average distance between the eIFiso 4E-488and the eIFiso4G-555 was calculated as 50.2 ± 3.4 Å (Table 3). Further, the eIFiso4G-555 (donor) was combined with the PAP-647 (acceptor); the pair was excited at 555 nm, and the fluorescence intensity of the acceptor fluorophore was measured at 655 nm (eIFiso4G-555 donor only, FD = 5.2 × 105 a.u.; eIFiso4G-555-PAP-647 binary complex, FDA = 1.35 × 105 a.u.) (Table S1 of the Supporting Information). FRET efficiency was estimated at 73 ± 8.2%, and the average distance between the eIFiso4G-555 and PAP-647 pair was calculated as 43.2 ± 2.8 Å (Table 3). Combination of the eIFiso 4E-488and PAP-647 proteins, resulted in a small change of fluorescence intensity, yielding 67 ± 8.8% FRET efficiency, and an average distance between the eIFiso4E-PAP pair was estimated at 49.8 ± 3.1 Å (Table 3). When the eIFiso4E-488, eIFiso4G-555, and PAP-647 were combined simultaneously, the three proteins formed a stable ternary complex, where in tandem FRET was observed: i.e., excitation of the eIFiso 4E-488at 488 nm (donor 1), allowed for sufficient energy to be transferred to the eIFso4G-555 (acceptor-1), which, in turn, emitted its absorbed energy, and caused the excitation of the PAP-647 (acceptor 2). FRET efficiency was estimated at 86 ± 7.1% from the eIFiso 4E-488 to the eIFiso4G555, following by 91 ± 11.3% FRET from the eIFiso4G-555 to the PAP-647. Average distance between the eIFiso 4E-488 and the eIFiso4G-555 pair was estimated at 51.0 ± 4.2 Å, and 34.7 ± 3.8 Å between the eIFiso4G-555 and the PAP-647 pair (Table 3). PAP and eIFiso4E Bind eIFiso4G Competitively at Distinct and Different Sites. Competitive binding of PAP

Figure 6. eIFiso4E competes with PAP for eIFiso4G binding. The fluorescence intensity of fluorescein-labeled eIFiso4G (100 nM) was monitored when the protein was titrated with increasing amounts of PAP, in the absence and presence of various eIFiso4E concentrations (0 nM, −brown diamonds−; 50 nM, −orange circles−; 100 nM, −yellow triangles−) at 25 °C. The excitation and emission wavelengths of the fluorophore were 495 and 516 nm, respectively. The solid lines are the fitted curves. The inset shows Lineweaver−Burk plots for competition of eIFiso4E and PAP for two distinct sites on eIFiso4G. Data points were fitted using least-square analysis.

for eIFiso4G binding, despite the fact that the binding sites are different. The competitive behavior of these two proteins is supported by the previously proposed model26 and a protein copurification assay. Lineweaver−Burk plots (Figure 6 inset) display parallel trend lines, indicative of noncompetitive or uncompetitive (anticompetitive) type of ligand binding behavior between PAP and eIFiso4E for eIFiso4G. This suggests that PAP binds eIFiso4G at a site distinct from the eIFiso4E binding site, and the two binding sites are structurally different from each other. Binding affinity of PAP for eIFiso4G (Kd = 113.8 ± 8.7 at 25 °C) was reduced in the presence of stoichiometric ratios of eIFiso4E (100 nM; Kd = 218.5 ± 15.4 nM at 25 °C). Under cellular conditions the binding affinity of plant eIFiso4E for eIFiso4G is quite strong (subnanomolar Kd = 0.080 ± 0.002 for a native eIFiso4F binary complex and ∼10 nM for a mixed eIFiso4E-eIFiso4G complex),59 suggesting that once formed, these complexes do not dissociate easily, unlike the mammalian system with eIF4E binding proteins (PBs) that sequester eIF4E from eIF4G.59 Western blot analyses of cellular extracts show that the eIFiso4E to eIFiso4G ratio is uniform G

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binding studies, and the group does not perturb binding equilibrium,26 allowing us to use this extrinsic fluorophore (λex = 495 nm and λem = 516 nm) to study changes in PAP tertiary structure as it binds to eIFiso4G. After inner-filter effect corrections, we have observed a total 61% quench in the fluorescein-labeled PAP fluorescence when eIFiso4G was added at the highest molar ratio. Figure 7 (top inset) presents the corresponding Scatchard plot for the interactions. The slope and the intercept of the straight line, obtained from the plot Q/ [eIFiso4G] × 10−6 vs Q, has provided the binding capacity (n) of the labeled PAP for eIFiso4G. The dissociation constant (Kd) was obtained using a nonlinear least-squares analysis.34,35 The binding capacity for the PAP-eIFiso4G complex was determined as 1.04 ± 0.03, indicating that there is one binding site on PAP for eIFiso4G. The thermodynamics of PAP-eIFiso4G interactions show that the affinity of PAP for eIFiso4G has decreased as the temperature gradually increased (Kd = 56.9 ± 2.5 nM at 10 °C versus 113.8 ± 8.7 nM at 25 °C) (Figure 7 bottom inset and Table 4). A van’t Hoff plot of −ln Keq versus the reciprocal of the absolute temperature (1/T) was used to calculate the enthalpy (ΔH°) and the entropy (ΔS°) (Figure 7 and Table 5). The values of ΔH° and ΔS° were obtained from the intercept and the slope, respectively (correlation coefficient of >0.94). The van’t Hoff analyses showed that eIFiso4G binding to PAP is enthalpy-driven (ΔH° = −32.1 ± 0.7 kJ/mol) and entropy favored (ΔS° = +25.8 ± 2.3 J/Kmol), leading to negative favorable ΔG° (−39.8 ± 1.4 kJ/mol) (Table 5). The TΔS van’t Hoff component contributes 19.3% to the overall value of ΔG° at 25 °C. The relatively large, favorable, entropic contribution to the PAP−eIFiso4G binding suggests that hydrophobic residues become less solvent exposed in the combined binary complex. The energy values are relatively insensitive to the temperature range examined. The relative invariance of the free energy on temperature (similar order of magnitude) is important and fits PAP biological function as a defense mechanism, which needs to be able to exert its antiviral effect under unpredictable conditions.

(1:1), and neither of the eIFs exist in much greater excess than the other.59 PAP Binding to eIFiso4G is Enthalpically-Driven and Entropically Favored. To understand the forces that drive PAP−eIF interactions, and in the interim to be able to modulate them, binding isotherms for the interactions of fluorescein-labeled PAP with eIFiso4G were acquired by performing direct fluorescence titrations at various temperatures (e.g., 10, 20, 15, and 25 °C) (Figure 7). Since both

Figure 7. (A) Temperature dependence of fluorescein-labeled PAP− eIFiso4G interactions. The normalized fluorescence values (λex = 495 nm and λem = 516 nm) for the fraction of the bound ligand (ΔF/ ΔFmax) are plotted versus eIFiso4G concentration at 10 °C (−yellow triangles−), 15 °C (−purple squares−), 20 °C (−pink circles−), and 25 °C (−green triangles−). Fluorescein-labeled PAP concentration was 500 nM in titration buffer. The curves were fit to obtain dissociation constants (Kd) as described under Materials and Methods. (B) Scatchard plots showing one binding site on PAP for eIFiso4G. The slope and the intercept of the straight line, obtained from the plot Q/[eIFiso4G] × 10−6 versus Q, provided the binding capacity (n) for the above proteins at various temperatures. Q is the fractional quench of fluorescence in titration and n for these interactions was determined as 1.04 ± 0.03. (C) van’t Hoff plot for the interactions of fluoresceinlabeled PAP with eIFiso4G.

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CONCLUSION Our findings further support the notion that eIFs may play an additional role that limits the toxicity to cells synthesizing PAP (Figure 8). Finally, we conclude that eIFs further promote the biological function of PAP as an antiviral agent by escalating PAP’s specificity for the uncapped viral RNA, and its rate of depurination over that of the rate of rRNA and capped mRNA depurination. The transient gene expression system employing Arabidopsis mesophyll protoplasts have demonstrated an important and versatile tool for performing cell-based experiments using molecular, biochemical and cellular, genetic, genomic and proteomic approaches to analyze functions of diverse signaling pathways and cellular machineries.60−62 In the interim, transient expression in Arabidopsis mesophyll protoplast (TEAMP) will be investigated to show the relevancy of the findings in vivo, and examine PAP-eIF protein−protein

proteins, PAP and eIFiso4G, have moderate intrinsic fluorescence, we employed the fluorescein labeling to in our studies. The fluorescein group has been successfully used in

Table 4. Equilibrium Dissociation Constants for PAP−eIFiso4G and PAP−eIFiso4F Interactionsa equilibrium dissociation constant, Kd, (nM)

a

complex

10 °C

15 °C

20 °C

25 °C

PAP−eIFiso4G PAP−eIFiso4Fb

56.90 ± 2.5 ND

64.31 ± 2.7 ND

79.84 ± 5.2 ND

113.8 ± 8.7 54.3 ± 1.1

ND is not determined. b1:1 complex of eIFiso4E and eIFiso4G was used. H

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Biochemistry Table 5. Thermodynamic Parameters for the Interactions of PAP with eIFiso4Ga complex

enthalpy (ΔH°) kJ·mol−1

entropy (ΔS°) J·K‑1·mol−1

Gibbs free energy (ΔG°) kJ·mol−1

PAP−eIFiso4G

−32.1 ± 0.7

+25.8 ± 2.3

−39.8 ± 1.4

ΔH° and ΔS° values were determined from van’t Hoff plot and dissociation constants. However, ΔG° value were calculated at 25 °C using the equation ΔG° = −RT ln Keq.

a

Figure 8. Mechanism of PAP interaction with rRNA, cellular mRNA, and uncapped viral RNA.

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interaction analysis by biomolecular fluorescence complementation (BiFC), firefly luciferase complementation (FLC), and subcellular localization of PAP−eIF supramolecular complexes.63−66 Perhaps it would be possible to modulate these PAP−eIFs interactions in a way that selectively advances PAP’s specificity for the uncapped viral RNA. One method of such modulation is to engineer PAP with exclusive association to the cellular eIF complex, leading to the selective targeting of PAP− eIF to depurinate uncapped viral RNA, while avoiding apoptosis in cells or altering expression of the PAP−eIF complex, leading to greater specificity for viral RNA of that of rRNA or cellular mRNA. In doing so, PAP−eIFs conjugates may provide an anchor for the development of effective antiviral agents for the prevention of plant and animal diseases caused by uncapped RNA viruses.

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AUTHOR INFORMATION

Corresponding Author

*Address: Department of Sciences, John Jay College of Criminal Justice, the City University of New York, 524 West 59th Street, New York, New York 10019. Telephone: (646) 557-4640. Fax: (212) 621-3739. E-mail: adomashevskiy@jjay. cuny.edu. ORCID

Artem V. Domashevskiy: 0000-0001-7511-8191 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mr. Jason A. Domashevskiy and Ms. Suzanne Sherbell for their critical review of the manuscript. We wish to acknowledge our deceased friend, a great mentor, and a wonderful colleague, Dr. Diana E. Friedland (Department of Sciences, John Jay College of Criminal Justice, CUNY, NY, USA), for both her tangible contribution towards this research and for her inspiration regarding this project. The recombinant wheat eIFiso4E and eIFiso4G plasmids were gifted by Dr. Karen S. Browning (Department of Molecular Biosciences, University of Texas at Austin, TX, USA), the FLTEV RNA leader sequence and pLUC0 plasmid by Dr. Daniel R. Gallie (Department of Biochemistry, University of California, Riverside, CA, USA). Mass spectrometry analysis was performed by Dr. Brandon Fowler (Department of Chemistry, Columbia University, NY, USA). We are grateful to Dr. Lawrence Kobilinsky, Chair of the Department of Sciences at John Jay College, and the Office for the Advancement of Research (OAR), for their financial contributions towards this publication.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00598. (1) Supporting Information and methods (pp S2−S6); (2) Supplemental Table S1: Fluorescence intensity of donor or/and acceptor pairs of proteins (p S7); (3) Supplemental Figure S1: Structure of SRL, oligonucleotide RNA, FLTEV RNA, and S1-3 TEV RNA (p S8); Supplemental Figure S2: Control titration of fluoresceinlabeled PAP with eIFiso4F (p S9); Supplemental Figure S3: Structural formula of γ-aminophenyl-m7GTP, immobilized to agarose (p S10); Supplemental Figure S4: Spectral profile of Alexa Fluor-labeled proteins (p S11); Supplemental Figure S5: Mass spectra of anthraniloyl-7methylguanosine triphosphate (Ant-m7GTP) (p S12); and Supplemental Figure S6: N6-Ethenoadenine standard curve and HPLC chromatogram showing fluorescence intensity of the adenines released from PAP substrates (p S13); (4) supplemental references (p S14). (PDF)

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ABBREVIATIONS PAP, pokeweed antiviral protein; RIP, ribosome inactivating protein; eIF, eukaryotic translation initiation factor; FRET, fluorescence resonance energy transfer; ESI, electrospray I

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(15) Battelli, M. G. (2004) Cytotoxicity and toxicity to animals and humans of ribosome-inactivating proteins. Mini-Rev. Med. Chem. 4, 513−521. (16) Narayanan, S., Surendranath, K., Bora, N., Surolia, A., and Karande, A. A. (2005) Ribosome inactivating proteins and apoptosis. FEBS Lett. 579, 1324−1331. (17) Tesh, V. L. (2011) The induction of apoptosis by Shiga toxins and ricin. Curr. Top. Microbiol. Immunol. 357, 137−178. (18) Irvin, J. D. (1975) Purification and partial characterization of the antiviral protein from Phytolacca americana which inhibits eukaryotic protein synthesis. Arch. Biochem. Biophys. 169, 522−528. (19) Domashevskiy, A. V., and Goss, D. J. (2015) Pokeweed Antiviral Protein, a Ribosome Inactivating Protein: Activity, Inhibition and Prospects. Toxins 7, 274−298. (20) Bonness, M. S., Ready, M. P., Irvin, J. D., and Mabry, T. J. (1994) Pokeweed antiviral protein inactivates pokeweed ribosomes; implications for the antiviral mechanism. Plant J. 5, 173−183. (21) Parikh, B. A., and Tumer, N. E. (2004) Antiviral activity of ribosome inactivating proteins in medicine. Mini-Rev. Med. Chem. 4, 523−543. (22) Hudak, K. A., Wang, P., and Tumer, N. E. (2000) A novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped RNA template. RNA 6, 369−380. (23) Vivanco, J. M., and Tumer, N. E. (2003) Translation Inhibition of Capped and Uncapped Viral RNAs Mediated by RibosomeInactivating Proteins. Phytopathology 93, 588−595. (24) Domashevskiy, A. V., and Cheng, S. Y. (2015) Thermodynamic Analysis of Binding and Enzymatic Properties of Pokeweed Antiviral Protein (PAP) toward Tobacco Etch Virus (TEV) RNA. J. Nat. Sci. 1, e82. (25) Wang, M., and Hudak, K. A. (2006) A novel interaction of pokeweed antiviral protein with translation initiation factors 4G and iso4G: a potential indirect mechanism to access viral RNAs. Nucleic Acids Res. 34, 1174−1181. (26) Baldwin, A. E., Khan, M. A., Tumer, N. E., Goss, D. J., and Friedland, D. E. (2009) Characterization of pokeweed antiviral protein binding to mRNA cap analogs: competition with nucleotides and enhancement by translation initiation factor iso4G. Biochim. Biophys. Acta, Gene Regul. Mech. 1789, 109−116. (27) Aitbakieva, V. R., and Domashevskiy, A. V. (2016) Insights into the Molecular Antiviral Mechanism of Pokeweed Antiviral Protein from Phytolacca americana. Biochem. Pharmacol. (Los Angeles, CA, U. S.) 5, 210. (28) Zeenko, V., and Gallie, D. R. (2005) Cap-independent translation of tobacco etch virus is conferred by an RNA pseudoknot in the 5′-leader. J. Biol. Chem. 280, 26813−26824. (29) de Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani, S. (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725−737. (30) Domashevskiy, A. V., Miyoshi, and Goss, D. J. (2012) Inhibition of pokeweed antiviral protein (PAP) by turnip mosaic virus genomelinked protein (VPg). J. Biol. Chem. 287, 29729−29738. (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 virus with wheat germ translation initiation factors eIFiso4E and eIFiso4F. J. Biol. Chem. 281, 28002−28010. (32) van Heerden, A., and Browning, K. S. (1994) Expression in Escherichia coli of the two subunits of the isozyme form of wheat germ protein synthesis initiation factor 4F. Purification of the subunits and formation of an enzymatically active complex. J. Biol. Chem. 269, 17454−17457. (33) Ray, S., Yumak, H., Domashevskiy, A., Khan, M. A., Gallie, D. R., and Goss, D. J. (2006) Tobacco etch virus mRNA preferentially binds wheat germ eukaryotic initiation factor (eIF) 4G rather than eIFiso4G. J. Biol. Chem. 281, 35826−35834. (34) Mayberry, L. K., Dennis, M. D., Leah Allen, M., Ruud Nitka, K., Murphy, P. A., Campbell, L., and Browning, K. S. (2007) Expression and purification of recombinant wheat translation initiation factors

ionization; MS, mass spectrometry; SRL, sarcin/ricin loop; BMV, brome mosaic virus; TEV, tobacco mosaic virus; m7G, 7methyl guanosine; TE, translational enhancer; IRES, internal ribosome entry site; m7GTP, 7-methylguanosine triphosphate; ant-m7GTP, anthraniloyl-7-methylguanosine triphosphate; Kd, dissociation constant; NHS-fluorescein, N-hydroxysuccinamide fluorescein; TMV, tobacco mosaic virus; HIV, human immunodeficiency virus; HSV, herpes simplex virus; LCMV, lymphocytic chorimeningitis virus; AMV, alfalfa mosaic virus; SPMV, satellite panicus mosaic virus; TBSV, tomato bushy stunt virus; Tris-HCl, tris-(hydroxymethyl)aminomethane hydrochloride; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; PVP, polyvinylpyrrolidone; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; MES, 2-(Nmorpholino)ethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; BSA, bovine serum albumin; DEPC, diethylpyrocarbonate; TLC, thin-layer chromatography; DMSO, dimethyl sulfoxide

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DOI: 10.1021/acs.biochem.7b00598 Biochemistry XXXX, XXX, XXX−XXX