Structural studies of the eIF4E–VPg complex reveal a direct competition for capped RNA: Implications for

Luciana Coutinho de Oliveiraa,1,2, Laurent Volpona,1, Amanda K. Rahardjoa, Michael J. Osbornea, Biljana Culjkovic-Kraljacica, Christian Trahanb, Marlene Oeffingerb,c,d, Benjamin H. Kwoka, and Katherine L. B. Bordena,3

aInstitute of Research in Immunology and Cancer, Department of Pathology and Cell Biology, Université de Montréal, Pavilion Marcelle-Coutu, Chemin Polytechnique, Montréal, QC H3T 1J4, Canada; bDepartment for Systems Biology, Institut de Recherches Cliniques de Montréal, Montréal, QC H2W 1R7, Canada; cDépartement de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3T 1J4, Canada; and dDivision of Experimental Medicine, McGill University, Montréal, QC H3A 1A3, Canada

Edited by Lynne E. Maquat, University of Rochester School of Medicine and Dentistry, Rochester, NY, and approved October 16, 2019 (received for review March 19, 2019) Viruses have transformed our understanding of mammalian RNA of eIF4E can be targeted in patients to provide clinical benefit, processing, including facilitating the discovery of the methyl-7- highlighting its critical importance. guanosine (m7G)caponthe5′ end of RNAs. The m7Gcapisrequired Viruses have paved the way for our understanding of many for RNAs to bind the eIF4E aspects of host-cell RNA processing, including m7G capping. and associate with the translation machinery across plant and ani- Indeed, studies into cytoplasmic polyhedrosis virus (CPV) infec- mal kingdoms. The potyvirus-derived viral genome-linked tion in silkworm and vaccinia virus (VV) in mammalian cells were (VPg) is covalently bound to the 5′ end of viral genomic RNA (gRNA) critical for the elucidation of the m7G cap structure over 40 y ago and associates with host eIF4E for successful infection. Divergent (3, 14, 15). Here, we exploited unusual features of potyvirus bio- models to explain these observations proposed either an un- chemistry to unearth unknown strategies that can be implemented known mode of eIF4E engagement or a competition of VPg for to engage eIF4E. Potyviruses are members of the picorna-like 7 the m G cap-binding site. To dissect these possibilities, we resolved plant viruses. Their infection of mainstay crops has devastating the structure of VPg, revealing a previously unknown 3-dimensional economic consequences (16). Genetic studies revealed that poty- BIOCHEMISTRY – (3D) fold, and characterized the VPg eIF4E complex using NMR and viruses require host-cell translation machinery to replicate, and biophysical techniques. VPg directly bound the cap-binding site of 7 specifically, these reports have associated the potyviral protein eIF4E and competed for m G cap analog binding. In human cells, VPg genome linked (viral genome-linked protein [VPg]) with host plant inhibited eIF4E-dependent RNA export, translation, and oncogenic eIF4E (17–21). Indeed, mutations in plant eIF4E are associated transformation. Moreover, VPg formed trimeric complexes with eIF4E–eIF4G, eIF4E bound VPg–luciferase RNA conjugates, and these VPg–RNA conjugates were templates for translation. Infor- Significance matic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a sim- RNA processing including covalent modifications (e.g., the ad- 7 ilar manner to VPg, demonstrating that this form of engagement is dition of the methyl-7-guanosine [m G] “cap” on the 5′ end of relevant beyond potyviruses. In all, we revealed an unprecedented transcripts) centrally influences the proteome. For example, 7 modality for control and engagement of eIF4E and show that VPg– eIF4E recruits RNAs for translation by binding the m G cap. RNA conjugates functionally engage eIF4E. As such, potyvirus VPg eIF4E is engaged and controlled by the binding of factors to its 7 provides a unique model system to interrogate eIF4E. dorsal surface while leaving its m G cap-binding site free for RNA recruitment. Here, we unexpectedly found that a small VPg | m7 cap | potyvirus | translation | eIF4E viral protein, viral genome-linked protein (VPg), directly binds the cap-binding site of eIF4E, indicating that eIF4E can addi- tionally be controlled through direct competition with its cap- he eukaryotic translation initiation factor eIF4E plays important binding site. Furthermore, VPg–RNA conjugates also bind eIF4E Troles in posttranscriptional control in plant and animals (1). Its 7 and are templates for translation, suggesting that VPg may association with the methyl-7-guanosine (m G) “cap” on the 5′ end 7 substitute for the m G cap during infection. of RNAs allows eIF4E to recruit transcripts to the RNA processing 7 machinery (2). To date, the m G cap is generally accepted as the Author contributions: L.C.d.O., L.V., M.J.O., B.C.-K., C.T., M.O., B.H.K., and K.L.B.B. de- universal 5′ adaptor for RNAs in eukaryotes (3), with the exception signed research; L.C.d.O., L.V., A.K.R., M.J.O., B.C.-K., C.T., and K.L.B.B. performed re- i 3 search; L.V., M.J.O., B.C.-K., B.H.K., and K.L.B.B. contributed new reagents/analytic tools; of ( ) the structurally related m G cap, which is also used by L.C.d.O., L.V., A.K.R., M.J.O., B.C.-K., C.T., and K.L.B.B. analyzed data; and L.C.d.O., L.V., nematodes (4), and (ii) with lower-frequency, nicotinamide ade- M.J.O., B.C.-K., C.T., M.O., and K.L.B.B. wrote the paper. nine dinucleotide (NAD) and related analogs that destabilize The authors declare no competing interest. transcripts and thus, are probably not involved in active translation This article is a PNAS Direct Submission. 7 (5). Through its m G cap-binding activity, eIF4E recruits specific Published under the PNAS license. transcripts to the translation machinery in the cytoplasm and Data deposition: The NMR, atomic coordinates, chemical shifts, and restraints reported in promotes the nuclear export of selected RNAs from the nucleus (6, the paper have been deposited in the Biological Magnetic Resonance Data Bank (http:// www.bmrb.wisc.edu/; accession no. 27506) and the Protein Data Bank (https://www.rcsb. 7). Both activities contribute to modulation of the proteome and in org; ID code 6NFW). mammals, to its oncogenic activity (6, 7). For instance, eIF4E is 1L.C.d.O. and L.V. contributed equally to this work. dysregulated in many human cancers (6). In humans, targeting 2Present address: NMX Research and Solutions Inc., Laval, QC H7V 5B7, Canada. eIF4E with a cap competitor, the guanosine analog ribavirin, 3To whom correspondence may be addressed. Email: [email protected]. impairs its biochemical activities correlating with clinical re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sponses in early-phase trials in leukemia, prostate, head, and 1073/pnas.1904752116/-/DCSupplemental. neck cancers among others (8–13). Thus, the cap-binding activity

www.pnas.org/cgi/doi/10.1073/pnas.1904752116 PNAS Latest Articles | 1of10 Downloaded by guest on September 27, 2021 with potyviral resistance (18–20). VPgs exist in other virus families, constructs and purified these from the soluble fraction with the such as poliovirus (22). The VPg designation is based on the co- quality of the confirmed by SDS/PAGE (Sodium Dodecyl valent linkage of viral RNA to VPg. For the case of potyviruses, Sulphate-PolyAcrylamide Gel Electrophoresis) (Fig. 1C) (34), the 5′ end of the genomic RNA (gRNA) is covalently attached to NMR (Fig. 1D and SI Appendix, Fig. S2), and mass spectrometry the hydroxyl of tyrosine 64 (potato virus Y [PVY] numbering) (23– (MS) (SI Appendix,Fig.S3A). We recently reported the NMR 25). The genetic interaction between VPg and eIF4E is only assignments for a VPg construct in which the first 37 residues were reported for potyviruses (20), while other virus families typically removed to improve stability (VPgΔ37) (34). Here, the 3- use these RNA conjugates for replication (22). Consistent with dimensional (3D) solution structure of this construct was de- this, potyviral VPgs only show significant with termined by using an automated procedure for iterative nuclear each other and not with VPgs from other families (SI Appendix, Overhauser effect (NOE) assignment using CYANA (35). The Fig. S1). structure of VPg is shown in Fig. 1 A and B and SI Appendix, Fig. While genetic studies linked VPg and eIF4E, conclusions from S3B, and the structural statistics are in SI Appendix,TableS1.The biochemical studies were highly divergent, leaving the mecha- rmsd for the ordered regions was 0.68 Å for the backbone atoms nism as to how VPg coopts eIF4E activity unsettled. Some groups of the top 20 structures. reported that PVY VPg binds m7Gcap–eIF4E–eIF4G, forming The VPg structure is unlike the previously proposed models. a quaternary complex, which suggests that VPg utilizes a novel VPgΔ37 adopts a well-folded core as well as 2 substantial un- surface on eIF4E for binding and thereby, engaging its activity (17, structured regions at the N and C termini (residues 38 to 70) and a 26). Supporting this model, mutation of the cap-binding site in flexible loop between β4- and β5-strands (residues 145 to 165) (Fig. wheat eIF4E (W123A, W102 in human eIF4E) did not reduce the 1A and SI Appendix,Fig.S3B). We note that VPg is not an ability of eIF4E to bind VPg but did reduce binding to the 7- intrinsically disordered protein. Specifically, there were many long- methylguanosine diphosphate (m7GDP) cap analog. This sug- range NOEs, indicating a tertiary structure (SI Appendix,Table gested that VPg bound to a part of eIF4E not previously known to S1), and furthermore, values for the 15N-1H heteronuclear NOE be involved in its control or engagement (26). By contrast, another and chemical shift index also indicated the presence of struc- study provided evidence that PVY VPg directly competes for the tured elements (34). The VPg structure is composed of a 5- cap-binding site on plant eIF4E (21). Other reports suggested that stranded β-sheet with 2 consecutive α-helices between β-strands PVY VPg interacts with the eIF4F (eIF4E–eIF4G–eIF4A/B) 2and3(Fig.1A). Our structure of PVY VPg does not resemble complex to stimulate cap-independent internal ribosomal entry VPg structures from other virus families, consistent with the lack site (IRES)-mediated translation, but the exact eIF4F compo- of sequence conservation observed (SI Appendix,Fig.S1A). Residue nent required was not ascertained, and whether IRES-mediated Y64, which is covalently attached to gRNA during infection (23–25), translation is relevant to potyviruses is not clear (27). Just as the is located within the flexible N terminus but close to the folded mode of binding to eIF4E is controversial, there are divergent domain, which starts at residue 72 (Fig. 1A and SI Appendix,Fig. models regarding the molecular basis for the VPg–eIF4E in- S1B). Inspection of the structure revealed that there were no ele- teraction. One study proposed that residues 41 to 93 of PVY VPg ments within VPg that possessed any structural similarity to reported were used for binding to eIF4E (21), while others reported that eIF4E-binding motifs (e.g., the helical turn describing the eIF4E residues on a predicted long amphipathic helix spanning residues consensus motif [YXXXXLΦ, where X is any residue and Φ is any 90 to 125 were required (28). Efforts to solve the VPg structure hydrophobic] or RING (really interesting new ) domains [6]). had been to date unsuccessful. Modeling efforts yielded disparate solutions, including a helical bundle (29), a long amphipathic helix Elucidation of the eIF4E Binding Site on VPg. We used NMR and for the VPg-binding site for eIF4E (28), and a model based on the pulldown studies to garner information regarding the eIF4E– FOK1a kinase structure, which included a β-sheet core with flex- VPg complex structure (see below). We selected human eIF4E ible helices (30). Adding to the confusion, multiple biophysical for these studies because of the extensive knowledge accumu- studies proposed that VPg is an intrinsically disordered protein lated regarding its structure, allosteric effects of ligands, and dy- (21, 30–33). namics (36–38). Importantly, human cap-bound eIF4E is highly While the mechanistic underpinnings of the interplay between homologous in sequence (SI Appendix,Fig.S4A) and structure (SI VPg and eIF4E remains controversial, it is clear that eIF4E is Appendix,Fig.S4B) to the 3 cap-bound eIF4E plant structures required for infection and that there is a genetic interaction be- solved (rmsd ∼ 0.7 Å for 175 atom pairs). Both human and melon tween eIF4E and VPg. In this way, VPg provides a unique model eIF4E structures were available in the apo- and cap-bound forms as system to interrogate the modalities required for the engagement well as ternary complexes with eIF4G (38, 39). These structures are and control of eIF4E. No structure of any potyvirus protein exist. highly homologous, indicating that these were conserved across Here, we report the structure of the potyvirus protein, VPg, and kingdoms. The large positive surface used to bind the phosphates of characterized the VPg–eIF4E complex using high-resolution NMR the m7G cap in the unoccupied cap-binding site was conserved and biophysical methods. We demonstrated that VPg binds the across kingdoms (SI Appendix,Fig.S4C). Given the overall simi- cap-binding site of eIF4E and forms trimeric complexes with larity between structures coupled with the deeper structural un- eIF4E and eIF4G; furthermore, we demonstrated that VPg–RNA derstanding of human eIF4E, we studied the VPg–human eIF4E conjugates directly bind eIF4E and were templates for translation complex to ascertain how VPg engaged and/or con-trolled eIF4E. consistent with the requirement of potyviruses for VPg–eIF4E We identified the VPgΔ37 and eIF4E interface using a com- interactions for their lifecycle. Furthermore, the structural simi- bination of NMR-based strategies and mutagenesis. We used cap- larities of VPg to human proteins suggest that this modality for free eIF4E as a starting point for these studies. First, using 50 μM engagement of eIF4E could be conserved across kingdoms. 15N-,13C-, or specifically labeled Ile, Leu and Val methyl groups (ILV) VPgΔ37, we monitored the effects of addition of unlabeled Results and Discussion eIF4E (150 μM) on signal broadening and chemical shift pertur- VPg Adopts a Previously Unknown Protein Fold. We determined the bation (CSP) of the amide nitrogens, methyl carbons, and carbonyl structure of PVY VPg protein in order to understand its mo- groups using the 1H-15N HSQC (heteronuclear single quantum lecular relationship with eIF4E and thus, its role in RNA re- coherence), the methyl region of the 1H-13Cspectra,ortheH/C cruitment to the host-cell translation machinery. We studied PVY projection of the 3D-HNCO experiment, respectively (Fig. 2 A VPg, since it is the archetypal potyvirus and thus, representative of and B and SI Appendix,Fig.S5). We observed substantial broad- the family (SI Appendix,Fig.S1B). We generated full-length and ening for residues located in the loop E108-G119 of VPgΔ37, and truncated forms of PVY VPg protein using bacterial expression in particular, for carbonyls of E108, R109, and Q116 and amides of

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1904752116 Coutinho de Oliveira et al. Downloaded by guest on September 27, 2021 AC (kDa) 100 75 loop 1- 2 2 50 2 37 25 VPg FL 4 20 3 1 1 3 4 VPg 37 5 1 15 VPg 62 2 2 1 5 10 Ct Ct 180o D 105 VPg 37 Nt 110 B Nt

115 loop 1- 2 120 N (ppm) 15

125

130 180o 10.5 9.09.510.0 8.5 8.0 7.5 7.0 6.5 1H (ppm)

Fig. 1. Structure of the PVY VPg protein. (A) Cartoon representation of the closest to the average structure in the ensemble for VPgΔ37. The family of best 20 structures is shown in SI Appendix, Fig. S3B. Residues starting at F60 shown as residues 37 to 70 are disordered. (B) Surface rendering of VPg structure. Red indicates negative charged area, blue indicates positive, and white are hydrophobic. (C) SDS/PAGE gel of full-length (FL) and VPg truncation constructs used for NMR analysis; molecular mass markers are shown. (D) 1H-15N HSQC spectrum of VPgΔ37. BIOCHEMISTRY R109, D111, I113, and M115-G119, including the side-chain amide substantial broadening was detected around the cap-binding site, of Q116 (Fig. 2 C and E). Methyl carbons in the region V103- particularly for backbone residues in the F48-L60 loop (Fig. 3C) L118 also exhibited broadening (Fig. 2B) in addition to residues which also contains W56, one of the tryptophan residues that binds neighboring this loop (L80 and L166). The flexible regions of the m7G cap. Other proton amides around the cap-binding pocket VPgΔ37 (i.e., the large loop [N145-E165] and both N [S38-E70] of eIF4E were also broadened (i.e., the K95 and the H200). As and C termini [A182-E188]) were not altered by eIF4E, indicating shown for VPgΔ37 (Fig. 2D), the empty spaces indicate residues that they are unlikely to be involved in the interaction. Consistent that were not quantified due to spectral overlap (Fig. 3C). Con- with this observation, deletion of the first 62 residues in VPg did sistently, we also observed carbon chemical shifts for methyl not impair binding to eIF4E (SI Appendix,Fig.S3C and E). groups located directly behind the phosphate-binding region of Second, we used transferred cross-saturation (TCS) experiments the cap-binding site of eIF4E (I63 and L85) (SI Appendix,Fig.S7). (40) to directly detect through space interactions between eIF4E Given that these changes were concentrated in the cap-binding and VPg. In this experiment, the labeled VPg was in 4-fold excess site, we individually mutated 2 parts of this site: the m7G moiety- relative to unlabeled eIF4E (90 μM eIF4E, 360 μMVPg).Con- binding region, which includes W56, and the phosphate-binding sistent with the above NMR data, we observed substantial re- region, which includes R157, K159, and K162 that form a ductions in signal intensity of the amide side chain of residue positively charged patch. The R157E/K159E/K162E triple mutation Q116 and to a lesser extent, of the backbone amides of D111 and (4ETrMut) substantially reduced the interaction of eIF4E with E114 (Fig. 2D), indicating that these residues were close in space VPgΔ37 as did the W56A mutation (SI Appendix,Figs.S8A–C to eIF4E. We then used mutagenesis to validate the NMR data. and S9). These findings were confirmed by glutathione S-trans- VPgΔ37 mutants M115A/Q116A and D111K/E114K/Q116K had ferase (GST) pulldown experiments using murine eIF4E, which is weaker affinity for eIF4E compared with wild-type VPgΔ37 as only 4 residues different from human eIF4E (SI Appendix,Fig. observed by increased intensities of the NMR signals (SI Appendix, S8E). Importantly, none of these mutants altered the overall fold Fig. S6). These spectra were acquired under identical concentra- of eIF4E as assessed by HSQC and CD (SI Appendix,Fig.S8A– tions and ratios; thus, differences in intensities in comparing D). Importantly, W56 and R157/K159/K162 comprise one end spectra directly reflect affinity. To support the delineation of the of the cap-binding site and are relatively close in space in both binding surface, we also mutated residues outside of the predicted the apo- and cap-bound eIF4E forms (<11 Å). We note that the binding site and examined their impact. Consistently, these mu- W102A mutation, allied to W123A in wheat, barely affects the tations (E86K/E87K, D92K, or E98K/E102K) did not significantly ability of eIF4E to bind VPg (SI Appendix,Fig.S9). In apo- impact the VPg–eIF4E interaction (SI Appendix,Fig.S6D). None eIF4E, W102 is at the other end of the cap-binding site, while of the mutants described above disrupted the VPg structure as upon cap binding, this residue moves to be in close proximity observed by NMR or circular dichroism (CD) (SI Appendix,Fig. with W56, R157, K159, and K162 (38). In all, our NMR data S3D). Altogether, TCS, CSP, signal broadening, and mutational strongly indicate that the VPg-binding site on eIF4E is clustered studies indicate that the α1–α2 loop forms the surface on VPg that in the region of the cap-binding site, including W56, R157, K159, binds to eIF4E. and K162.

Mapping of the VPg Binding Site on eIF4E. We used the same The VPg–eIF4E Complex. We used the above NMR and mutagenesis strategy to determine the binding site of VPgΔ37 on eIF4E. We data to generate a model of the eIF4E–VPgΔ37 complex using monitored spectral perturbations of 50 μM 15N- or 13C-eIF4E the restraint-driven docking program HADDOCK (41). We note samples as a function of unlabeled VPgΔ37 addition to a maxi- that the intermediate NMR exchange regimen observed for the mum of 150 μM eIF4E (Fig. 3 and SI Appendix,Fig.S7). The most complex meant that it was impossible to observe NOEs due to

Coutinho de Oliveira et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 27, 2021 and the buried surface area for the complex was ∼1,960 Å2 (Fig. A 15N-VPg 4A). In this complex, W56 is buried in a hydrophobic pocket 15 N-VPg / eIF4E formed by M107, V108, M115, and L118 in the helix–loop–helix structure of VPg. Also, the positively charged residues (R157 and K162) in the phosphate-binding pocket of eIF4E are facing D111 and D112 of VPg (Fig. 4B). To independently validate our model, we performed cross- linking mass spectrometry (XL-MS) on cross-linked heterodimers that were isolated by size exclusion chromatography. According to our model, only 3 lysines of VPg are located near the interface of the heterodimer (K105, K106, and K138), and hence, a low number of cross-links between both proteins was expected. Fur- thermore, most of the cross-linker was absorbed by intramolecular cross-link between VPg K105-K106 (extracted ion chromatogram BC [XIC] values in SI Appendix,TableS2), thus contributing greatly to I96-1 a reduction of interprotein cross-link occurrences between VPg and eIF4E. Nevertheless, consistent with our HADDOCK model, M115 the most frequently observed cross-link (15 occurrences), which G119 L118 also had the highest score (5.33), bridged K106 of VPgΔ37 with Q116 I113 V108 K159 of eIF4E, for which the Euclidean distance measured by the L80 Xwalk software (42) was found to be 16.0 Å (SI Appendix,Table E109 S2). This most frequent cross-link was consistent with the magni- L166 tude of line broadening in the different HSQCs, TCS, and muta- L141 S101 genesis data defining the binding sites for both eIF4E and VPg. V103 The second most frequent interprotein cross-link only occurred I96 4 times and was found between eIF4E–K192 and VPg–K47, the V108 W132 L118-1 latter of which is in a highly flexible region of VPg. The presence of L80 H Nt L166 this cross-link is consistent with previous studies that showed that L141 the N terminus of VPg was involved in binding to eIF4E (21). – Δ – Ct Finally, a cross-link between eIF4E K192 and VPg 37 K138 was observedbutonlywith2occurrences.Asobservedintheapo form of eIF4E, the K192-containing loop and a fortiori, its side D VPg/eIF4E vs VPg chain are flexible (Protein Data Bank [PDB] ID code 2GPQ), 1.0 positioning these 2 lysines between 25 and 37 Å apart (calcu- 0.8 lated by the Xwalk software). Thus, in some conformations, these 0.6 lysines would be accessible. Taken together, the XL-MS data sup- 0.4 port our HADDOCK model based on NMR and mutagenesis

Intensity ratio 0.2 restraints. – 0.0 The VPg eIF4E complex presented here provides a molecular 70 80 90 100 110 120 130 140 150 160 170 180 basis for understanding the genetic studies of eIF4E and VPg. The E 1.0 positions of eIF4E mutants that impart resistance to potyvirus 0.8 0.6 0.4 0.2 AB Intensity ratio 0.0 70 80 90 100 110 120 130 140 150 160 170 180 residue number

Fig. 2. The binding surface on VPg used to interact with eIF4E. (A) 1H-15N HSQC of 50 μM 15N-labeled VPg in the absence (red) or presence (blue) of a 3-molar excess of unlabeled eIF4E. (B) Constant time 1H-13C HSQC spectrum of ILV-labeled VPg (50 μM) in the absence (red) or presence of 2-molar excess eIF4E (blue). (C) VPg residues are perturbed by eIF4E binding. Light blue indi- cates 1H CSP or broadening, dark blue indicates 1H methyl CSP, and yellow C indicates broadening from the 1H-13C projection of the 3D HNCO spectra (SI Appendix,Fig.S5). (D) Per residue plot of backbone amide line broadening of 15N-labeled VPgΔ37 in response to binding of eIF4E (extracted from A). Resi- dues that undergo line broadening below the dashed line are in cyan. Note that empty spaces correspond to residues that overlap, residues that are not assigned, or Proline residues. (E)Plotoftheintensityratiosofthecross-peaksin the TCS experiment for the backbone (blue) and asparagine/glutamine side- chain (orange) resonances for VPg. Fig. 3. Interaction surface used by eIF4E to bind VPg. (A) 1H-15NHSQCof 15N-labeled eIF4E (50 μM) in the absence (red) or presence (blue) of 3-molar excess broadening of signals for residues at the interface. The resulting VPg. (B) Broadening and CSPs mapped onto the apo-eIF4E structure (PDB ID code HADDOCK calculation led to a solution where 150 of 200 struc- 2GPQ) depicted as light blue balls. (C) Per residue plot of backbone amide line tures converged to the same eIF4E–VPgΔ37 complex. Of these broadening of 50 μM eIF4E in response to binding of VPgΔ37 (extracted from A). 150 structures, the backbone rmsd for the complex was ∼0.8 Å, Residues that undergo line broadening below the dashed line are in cyan.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1904752116 Coutinho de Oliveira et al. Downloaded by guest on September 27, 2021 eIF4E the small ribosomal subunit in order to initiate translation (47, 48). AB – Ct Q116 M115 To determine whether eIF4E VPg complexes bind eIF4G, we eIF4E Δ W56 carried out NMR experiments where VPg 37 was added to Nt 15N-labeled eIF4E in complex with an eIF4G peptide containing the L118 consensus binding site (4Gp). Both VPgΔ37 and 4Gp were used in V108 μ μ VPg a 3-fold molar excess (150 M) compared with eIF4E (50 M); M107 4Gp binds on the dorsal surface of eIF4E (SI Appendix,Fig. Ct K162 S11D), a region distal to the cap-binding and VPg-binding pocket W56 D111 VPg Nt R157 D112 in eIF4E. Interaction of both partners affects eIF4E’s signals dif- (E70) ferently in the HSQC, serving as reporters for binding. Indeed, on VPgΔ37 addition, most signals were broadened (SI Appendix, Fig. CD4E B F VPg S11 and ), while 4Gp addition induces peak shifting only for 1.0 VPg residues in the proximity to its binding site on eIF4E. In particular, 4E cap addition of 4Gp perturbed residues W73, Y34, I35, K36, L75, 0.5 and L137 on the dorsal surface of eIF4E as expected (SI Ap- pendix,Fig.S11A and E and Fig. 5C), which were not altered 0.0 by VPgΔ37 binding. Overall, on addition of 4Gp to the eIF4E– Normalized  [F] 01 23VPgΔ37 complex, we observed 2 phenomena (SI Appendix,Fig. eIF4E (M) S11 C and G): 1) signals remain broad, indicating that VPgΔ37 was still bound to eIF4E, and 2) residues close to the 4Gp-binding site Fig. 4. (A) Restraint-driven model of the VPg (green) and eIF4E (blue) com- (see above) were shifted similarly as in the eIF4E/4Gp complex. plex. Red highlights W56 from eIF4E involved in the association with VPg (SI Thus, our NMR data analysis revealed that eIF4E associates with a Appendix,Fig.S8C). (B) Close-up view of the complex highlighting the positive 4Gp and VPgΔ37 simultaneously. patch on eIF4E and its interaction with negative residues on VPg (rotation of 120° compared with A). This region was confirmed to bind eIF4E by mutation 7 15 m G Cap Analogs Compete for VPg Binding to eIF4E. Given that VPg (SI Appendix,Fig.S8B and E). (C) Overlay of the HSQC spectra of N-labeled 7 μ and the m G cap analogs bound overlapping surfaces on eIF4E, eIF4E (50 M) in the presence of 3-fold excess of unlabeled VPg (blue) and 7 Δ afteradditionof20-foldmolarexcessofm7GDP relative to eIF4E (orange), we explored whether the cap analog m GDP and VPg 37 competed 15 demonstrating that VPg and the cap compete for overlapping binding surfaces for binding of N-eIF4E by HSQC experiments. Addition of 7 on eIF4E. (D) eIF4E specifically binds to VPg with submicromolar affinity (∼0.3 20-fold excess m GDP to preformed VPgΔ37–eIF4E complexes BIOCHEMISTRY μM). Normalized change in fluorescence at emission of 484 nm (Δ[F]) as a (50 μM eIF4E, 150 μMVPgΔ37, 1 mM m7GDP)ledtothe function of eIF4E concentration for 0.5 μMVPgΔ37. Measurements were car- reemergence of eIF4E resonances but now in their m7GDP-bound ried out 3 independent times. positions (Fig. 4C and SI Appendix,Fig.S12A–C). This indicated that the m7GDP cap analog and VPgΔ37 competed for eIF4E on the same site. Importantly, VPgΔ37 did not bind to m7GDP cap infection in plants are mapped onto our eIF4E–VPg complex SI Appendix F itself as demonstrated by the observation that HSQC spectra of structure ( ,Fig.S8). These mutants are generally 15N-labeled VPg protein were unchanged in the presence or ab- located in the loop containing W56 (using human eIF4E amino sence of the m7GDP (SI Appendix,Fig.S13D). Thus, the m7GDP acid numbering) or in the nearby phosphate-binding site (43, 44), cap analog competed for VPg binding in vitro. This was confirmed which overlays with the proposed VPg-binding site. Interestingly, using the reverse titration in which we monitored changes to the the engineered wheat W123A mutant (or human W102A), which 15N transverse relaxation-optimized spectroscopy (TROSY) – SI Appendix did not disrupt the eIF4E VPg interaction (27) ( ,Fig. HSQC of 2H-15NVPg(50μM) on addition of unlabeled eIF4E S9), was found in the upper part of the cap-binding site and was (up to 175 μM) in the absence and presence of 1 mM m7GDP. not included in the experimentally defined eIF4E-binding surface 7 Residues at the proposed VPg-binding site (D111, I113, M115, used to interact with VPg, but it is clearly important for m Gcap Q116, L118, G119, and N121) were substantially broadened binding. Additionally, the VPg residues, which interacted with upon eIF4E addition to VPg (SI Appendix,Fig.S13A). How- eIF4E, were consistent with substitutions in this region being ever, in the presence of 1 mM m7GDP (SI Appendix,Fig.S13B critical for viral pathogenicity (S101-N121 of PVY–VPg) (SI Ap- and C), these peaks reappeared, indicating that m7GDP competes pendix,Fig.S8G)(43–45). for the same binding site on eIF4E as VPg. The same experiment We next determined whether VPgΔ37 bound plant eIF4E in a was performed with wheat eIF(iso)4E and yielded equivalent similar manner to human eIF4E. Previous biophysical studies results (SI Appendix,Fig.S10D). Thus, VPg utilized the cap- showed that wheat eIF(iso)4E bound to VPg, and hence, we binding site on both human and plant eIF4E for recognition focused on this homolog for our studies (27, 46). Given that there and competed for cap analogs. were no NMR assignments available for eIF(iso)4E, we exam- To gain a quantitative understanding of this interaction, we ined the interaction via 1H-15N HSQCs using 35 μM 15N-labeled determined the dissociation constants for eIF4E–VPg and eIF4E VPgΔ37 with unlabeled eIF(iso)4E (105 μM). We found that VPg cap analogs. Unfortunately, NMR titrations of the eIF4E–VPg used the same binding region (I113-N121) to bind eIF(iso)4E (SI complex via 1H-15N HSQC techniques resulted in disappearance Appendix B C ,Fig.S10 and ) as it used to associate with human of peaks, making determination of Kd values challenging for a eIF4E (Fig. 2), indicating that this was conserved across kingdoms. variety of technical reasons, including the inability to obtain in- Superimposing our homology model of wheat eIF(iso)4E onto the formation of the completely broadened state where binding would wheat eIF4E structure revealed that the orientation of the set of be saturated. Using isothermal calorimetry (ITC), we determined 7 positive residues (corresponding to the R157, K159, and K162) the Kd for the human eIF4E–m GDP cap analog. Consistent with and W56 (human numbering) was the same in plant and human previous results in phosphate buffer used in the above NMR eIF4Es (SI Appendix, Figs. S4 and S10A). Thus, the basic princi- experiments, we obtained a Kd of 0.37 ± 0.02 μM, which is ples for the VPg–eIF4E interaction seem evolutionarily conserved. consistent with previous literature reports for this interac- tion in phosphate buffer (49). When evaluating the Kd for the VPg Binds eIF4E in the Presence of an eIF4G Peptide. eIF4G is a eIF4E–VPgΔ37 interaction by ITC using the same buffer major platform protein in the translation initiation complex, conditions, we observed no heat release, suggesting that the recruiting not only eIF4E–RNA complexes but other cofactors to interaction is mainly entropic. Thus, we used a fluorescence

Coutinho de Oliveira et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 27, 2021 assay, whereby VPg was labeled with N-(iodoacetyl)-N′-(5- sulfo-1-naphtyl)ethylenediamine (IAEDANS) (33), an organic fluorophore that fluoresces at 490 nm, a region where the VPg and eIF4E proteins produced no signal. We observed a Kd of AB0.26 ± 0.07 μM(Fig.4D) and a Hill coefficient of ∼1.1, in- – inputs (10%) dicating a 1:1 ratio of VPg eIF4E complexes, consistent with our NMR studies. This affinity is very similar to literature values for wheat eIF4E and VPg (∼0.3 μM)(27).Thus,the 7 VPg RNActl RNase ctl VPg-RNAVPg-RNAVPg-RNA + RNase + Prot.K GSTGST-eIF4EVPgVPg-RNAGST +GST-eIF4E VPg-RNA + VPg-RNA affinities of eIF4E for VPg and the m G cap analog were very 50 37 VPg dimer 37 VPg dimer similar, consistent with the competition that we observed in our 25 25 VPg-RNA VPg-RNA above NMR studies. 20 20 VPg * 15 VPg RNase VPg–RNA Conjugates Directly Bind eIF4E and Were Templates for 10 GST-eIF4E Translation. Our above studies demonstrated that VPgΔ37 com- RNA 7 GST peted for m G cap binding to eIF4E and in this way, could in- GST loading terfere with host-cell translation. However, they also suggested Nt that VPgΔ37–RNA conjugates engaged eIF4E–eIF4G complexes eIF4E possibly to be recruited to the translation machinery. As a first step C Nt to test this possibility, we investigated whether RNA conjugation disrupted the interaction with eIF4E or alternatively, if VPg–RNA conjugates could directly bind to eIF4E. During infection, VPgΔ37 is conjugated through the side-chain hydroxyl group of Y64 to the 5′ end of the viral gRNA (23–25). Y64 is in a flexible region distal Ct eIF4G VPg to the eIF4E-binding site, consistent with the possibility that Ct VPgΔ37 interacts with eIF4E and RNA simultaneously (Fig. 4A). Unfortunately, potyvirus VPg–gRNA conjugates are not available in purified forms to directly investigate this with the viral gRNA. Using maleimide chemistry (50), we thus conjugated a 19-mer fragment of luciferase RNA onto VPgΔ37 (SI Appendix, Fig. Ct Nt S14A). In this case, we generated VPgΔ37 Y64C for conjugation to the 5′ end of the RNA. In this same construct, we mutated the Y64 only naturally occurring cysteine C150, which is found in the flexible loop, to alanine in order to ensure a single conjugation site for the protein. This protein was folded as the wild type and binds DEVPg-RNA conj eIF4E similarly to wild-type VPgΔ37 (SI Appendix,Fig.S14B). uncapped RNA The 19-mer RNA segment with a maleimide group on its 5′ end capped RNA capped RNA + VPg was then cross-linked to the sulfur side chain of C64 (SI Appendix, 2.5 ****** * Fig. S14A). We verified that the 19-mer RNA was conjugated 2.0 using SDS/PAGE and silver staining, where the VPgΔ37(C150A/ 1.5 Y64C)–RNA conjugate was ∼6 kDa larger as expected. We fur- VPg-RNAuncapped conjcapped RNAcapped RNA RNA+VPg 1.0 ther confirmed the presence of RNA by treating the conjugate -Luciferase 0.5 with RNase (ribonuclease), which resulted in a reduction in size 0.0 corresponding to the unmodified form of VPgΔ37(C150A/Y64C) (Fig. 5A). Similarly, treatment with proteinase K also disrupted the Fig. 5. (A) Silver-stained SDS/PAGE gel showing the conjugation between conjugate, leaving only the free 19-mer RNA band. Using eIF4E– VPgΔ37 (Y64C, C150A) and the 19-mer RNA with a 5′ maleimide group. No Δ – β GST pulldown, we observed that the VPg 37(C150A/Y64C) -mercaptoethanol or DTT (dithiothreitol) was present in order to preserve the B conjugate. VPg, the RNA fragment, and the conjugated form are shown (lanes RNA conjugate bound eIF4E but not the GST control (Fig. 5 ). 1, 2, and 4, respectively). Validation of the conjugate is shown by treatment Thus, VPg recruited RNA to eIF4E. with either RNase A (lane 5) or proteinase K (Prot.K; lane 6). The 19-mer RNA is Given the above findings, we examined the possibility that diffuse because of the percentage of acrylamide used. Position of the RNase A VPg–RNA conjugates were templates in in vitro translation as- protein is shown in lane 3 for comparison. We note the presence of dimeric says. To produce conjugates with full-length messenger RNAs forms through C64 in the absence of reductant. (B) Association of the VPg–RNA (mRNAs), we had to alter our strategy in order to conjugate full- conjugate with GST–eIF4E but not GST alone by western blot using an anti-His length luciferase RNAs (∼1,800 nucleotides) to VPgΔ37(C150A/ tag antibody (Upper). As expected, the VPg dimer also binds GST–eIF4E. An ∼ – Y64C), which yielded a species of 500 kDa. Using in vitro asterisk is shown to highlight a VPg RNA degradation product that occurred transcription, guanosine-5′-monophosphorothioate (GMPS) was during the GST pulldown. GST loading is shown below by western blot using incorporated into the 5′ end of luciferase transcripts and sub- an anti-GST antibody. The samples were run on the same gel, with unrelated ′ samples removed for clarity. (C) Model representing eIF4G (orange; PDB ID sequentlycoupledto2,2-pyridine disulfide using standard code 5T46) derived from the crystal structure of the eIF4E–eIF4G complex with methods (51, 52). A disulfide exchange reaction of the resulting the VPgΔ37 (green) and eIF4E (blue) as displayed in Fig. 4A.TheY64,whichis pyridyl-disulfide linkage on the 5′ end of the RNA was used covalently attached to gRNA during infection, is shown. Similarly, the VPg–RNA for conjugation to VPgΔ37(C150A/Y64C) (53). To monitor the conjugates made in vitro were also at the same position, C64 (in the text). (D) efficiency of conjugation, VPg–RNA conjugates were subjected to Western blot for Luciferase protein produced in in vitro translation reactions agarose gel electrophoresis due to their large size followed by using wheat germ lysates and different luciferase RNAs: conjugated to VPg SI Appendix – immunoblotting (54) for the His tag of VPg ( , Fig. (VPg RNA conj), uncapped, m7G-capped, and m7G-capped luciferase RNA in S14C). Unconjugated VPgΔ37(C150A/Y64C) is shown for com- the presence of 10 μM VPg protein (capped RNA + VPg protein). Loading of parison. As observed, all of VPgΔ37(C150A/Y64C) was conju- different luciferase RNAs was confirmed by qRT-PCR (SI Appendix,Fig.S14D). luciferase (E) Quantification of western blots for in vitro translation assays described in D. gatedtothe RNA, and no unconjugated RNA was Data were derived from 3 independent experiments. Mean intensities ± SDs are detected after the reaction. For comparison, we generated shown. P values are from Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001). luciferase transcripts using in vitro transcription without any

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1904752116 Coutinho de Oliveira et al. Downloaded by guest on September 27, 2021 modifications (referred to as uncapped) and also, generated luciferase A B 1.5 Vector ** capped transcripts using the VV-capping enzyme. VpG luciferase Equal amounts of each RNA, confirmed by qRT-PCR ** ** SI Appendix D 1.0 ( ,Fig.S14 ), were used as templates for in vitro Actin polysomes translation assays, and the production of luciferase protein was 0.5 monitored by western blot using an antiluciferase antibody with Vector the experiments carried out 3 independent times (Fig. 5 D and E). VPg

Cyt/Nuc mRNA Ratio Cyt/Nuc 0.0 We observed ∼2-fold more luciferase protein produced from m7G- cMyc Mcl-1 VEGF capped luciferase RNA templates than uncapped templates as GAPDH expected. The uncapped templates provided a lower bound for background translation, where it is well established that translation g C (kDa) VectorVP of uncapped RNAs occurs in in vitro systems but less efficiently 60 7 than when RNAs are m G capped (55). The levels of translation cMyc polysomes for VPg-capped luciferase transcripts were nearly identical to 40 7 m G-capped RNAs and ∼2-fold higher than observed for uncapped Vector 30 RNA (Fig. 5 D and E). This demonstrated that the VPg conju- VPg gation to the luciferase RNA did not interfere with its translation, 43 indicating that VPg–luciferase RNA conjugates were templates 25 for translation. Moreover, VPg–luciferase conjugates were trans- lated with the same efficiency as capped RNAs, suggesting that AG AG VPg could functionally substitute for the m7G cap. These obser- L D or-FL vations are consistent with our identification of VPg–eIF4E– ct Mcl1 polysomes (kDa) VPg-FLAGVe eIF4E-F eIF4G complexes (Fig. 6) and VPg–RNA–eIF4E complexes 200 eIF4G Vector (Fig. 5). We note that the dynamic range of our assay was limited 73 DDX3 VPg (2-fold between capped/VPg relative to uncapped RNA). Finally, 30 the addition of free VPg (i.e., not conjugated to the RNA) reduced eIF4E-FLAG 25 translation, consistent with our model of cap competition and Endo eIF4E previous reports (49). 30 eIF4E-FLAG Relative mRNA levels Relative mRNA levels Relative mRNA levels Relative mRNA levels Relative mRNA levels Relative 25 VPg-FLAG BIOCHEMISTRY VPg Suppressed Cap-Dependent eIF4E Activities in Cells. Next, we 15 4E-BP1 explored the impact of VPg on eIF4E activity in human cells to determine if the effects observed in vitro were recapitulated in E 60 cellulo. We postulated that VPg without RNA would potently Vector *** VPg inhibit cap-dependent activities of endogenous eIF4E in human 40

cells by competing for host-cell transcripts. To test this hy- A254 pothesis, we generated stable human osteosarcoma U2Os cell 20 lines expressing either full-length VPg–FLAG or vector controls. U2Os cells were selected based on their well-characterized eIF4E number Foci 0 activities (6, 56, 57). We investigated the effects of VPg on the 1 2 3 4 5 6 7 8 9 10 nuclear functions in mRNA export and cytoplasmic functions in Mono- Polysomes Pg translation, because both functions require the ability of eIF4E to somes Vector eIF4E bind the m7G cap (6, 7). eIF4E+V We first examined translational efficiency using polysomal Fig. 6. VPg represses eIF4E function in human cancer cells. (A)Polysome analysis in VPg–FLAG cells vs. vector controls. We monitored analyses of cells expressing VPg or vector controls indicates that VPg reduces translation and RNA export of 2 well-characterized target tran- translation efficiency of c-Myc and Mcl1 RNAs but not Actin,thenegative scripts of eIF4E: c-Myc and MCL1 (56, 58). VPg overexpression control, without altering the global polysome profile (Lower). (B) VPg inhibited did not alter the overall polysomal profile, indicating that it did not eIF4E-dependent mRNA export for targets RNAs. RNA levels were measured in nuclear and cytoplasmic fractions by qRT-PCR. While the increase in GAPDH interfere with the formation of (Fig. 6A). However, VPg MCL1 c-Myc was significant, it was so modest that it seems unlikely to be physiologically reduced translational efficiency for both and tran- relevant. P values are shown. (C) Western blot analysis of the effects of VPg scripts; in contrast, it did not alter the total RNA levels for either overexpression on eIF4E targets Mcl1 and cMyc. VPg–FLAG levels are given, and of these transcripts, and it did not affect the translation efficiency actin is provided as a loading control. Note that VPg does not lower endoge- of Actin, an RNA insensitive to eIF4E (Fig. 6A and SI Appendix, nous eIF4E protein levels. (D) FLAG immunoprecipitations from cells over- Fig. S15A). We next explored whether the RNA export activity of expressing VPg–FLAG, eIF4E–FLAG, or controls. Blots were probed as indicated. eIF4E, which is also cap dependent, was impaired in VPg–FLAG (E) VPg overexpression suppresses formation of foci in eIF4E–Myc over- < cells relative to vector controls (Fig. 6B). Cells were fractionated expressing cells. ANOVA (P 0.0009) was conducted. Experiments were carried out 3 independent times; means ± SDs are shown in A, B,andE (***P < 0.001). into nuclear and cytoplasmic compartments, and RNAs were quantified by qRT-PCR (quality of fractionation was verified by SI Appendix C – semi-qPCR) ( ,Fig.S15 ). VPg FLAG overexpression RNAs (59), likely because only a subset of these is sensitive to MCL1 impaired nuclear export of eIF4E target transcripts (e.g., eIF4E inhibition with other compensatory mechanisms coming and c-Myc)by∼2-fold but not eIF4E-independent transcripts, such into play, such as eIF3d-mediated cap-dependent translation for as GAPDH and VEGF RNAs. Again, total levels of RNA were not altered for any of the examined transcripts as expected (SI Ap- example (60). Thus, VPg inhibited activity of endogenous eIF4E. pendix,Fig.S15A). Consistent with VPg inhibiting both export and To further dissect the role of VPg in eIF4E-dependent trans- translation of these RNAs, protein levels for MCL1 and c-Myc lation in cells, we explored whether VPg, via eIF4E, also interacted D – were substantially decreased in VPg–FLAG cells relative to vec- with endogenous eIF4G (Fig. 6 ). We immunoprecipitated VPg tor controls, while β-Actin protein levels were unchanged (Fig. FLAG using an FLAG antibody and compared this with vector 6C). This is also consistent with previous studies in plants that or eIF4E–FLAG-expressing cells. We observed that VPg–FLAG showed that potyvirus infection inhibited translation of specific immunoprecipitated with endogenous eIF4E, endogenous eIF4G,

Coutinho de Oliveira et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 27, 2021 and the RNA helicase DDX3, which is part of the active trans- cap-binding site to prevent host-cell RNA association with eIF4E. lation complex, but not with 4E-BP1, the inhibitor of eIF4E. Thus, Our studies with the human EG5 protein suggest that this modality VPg engaged active translation initiation complexes. As expected, is not restricted to potyvirus but rather, is conserved across king- eIF4E–FLAG bound to eIF4G, DDX3, and 4E-BP1, while no doms. Based on these findings, VPg represents a previously proteins were found in the negative control FLAG immunopre- unknown class of inhibitor of human eIF4E, which could be cipitations from vector controls. This suggests that eIF4G–eIF4E– leveraged in future for therapeutic purposes. VPg–RNA complexes readily formed in human cells, which is Importantly, previous genetic studies demonstrated the re- consistent with in vitro translation assays, where addition of quirement for VPg–eIF4E interactions and thus, presumably, the VPgΔ37, when not conjugated to RNA, impeded translation of host-cell host translation machinery for successful viral infection. luciferase RNAs (Fig. 5 D and E). Taken together, VPg is able to Given our data, one possibility is that free VPg (not conjugated to impair host-cell RNA export and translation by competing with RNA) sequesters eIF4E–eIF4G complexes to allow for either eIF4E for host-cell m7G-capped RNAs as well as during infection IRES-mediated or some other form of translation to be engaged when,conjugatedtogRNA,VPgispositionedtorecruitgRNAto for VPg–gRNA conjugates. Another nonmutually exclusive pos- the translation machinery. sibilitybasedonourfindingsisthatVPg–gRNA conjugates are Given that VPg inhibited the mRNA export and translation recruited by eIF4E–eIF4G complexes to the translation machin- activities of eIF4E, both of which contribute to its oncogenic ery. In this model, VPg would substitute for the m7G RNA cap on potential and require cap binding (6, 7), we explored the possi- the gRNA, mediating a form of m7G cap-independent, eIF4E- bility that VPg inhibited foci formation in eIF4E-overexpressing dependent translation. In all, our studies demonstrated the exis- cells. In this case, stable U2Os cell lines were generated expressing tence of a fundamentally different form of cap competitor (VPg eIF4E–Myc, VPg–FLAG + eIF4E–Myc, or vector controls. As and EG5) than m7G cap analogs and identified an unanticipated expected, eIF4E–Myc increased foci formation ∼3-fold over vec- binding surface for proteins to engage eIF4E. Furthermore, our tor. VPg–FLAG + eIF4E–Myc cells produced ∼3-fold fewer foci findings suggest that VPg could substitute as the cap for potyvirus relative to eIF4E–Myc, comparable with vector controls (Fig. 6E gRNA, which in physical nature, is a significant departure from the and SI Appendix,Fig.S15B). While the initial focus of our VPg m7G cap first identified using CPV and VV over 40 y ago (3, 14, studies was to discover biochemical principles with regard to en- 15) or recently discovered adenosine nucleotides (5). Investigating gagement of eIF4E, it is clear that the unusual properties of VPg these possibilities will be interesting areas of future study. could be exploited in the future to inhibit the oncogenic activity of eIF4E in human cancer cells. Materials and Methods Protein Purification. Unlabeled and labeled constructs of PVY VPg encoding Human Homologs Based on Structural Similarity with VPg. Given that residues 38 to 188 were expressed and purified as described previously (34). potyvirus VPg did not adopt a fold like VPgs from other virus VPg mutants and deletion constructs (38 to 188 and 63 to 188) were gen- families (consistent with the lack of sequence homology) (SI Ap- erated using Quick Change mutagenesis (Bio Basic Inc.). eIF4E was expressed pendix,Fig.S1A) or like known eIF4E-binding partners, we used both in pET-28a for NMR and biophysics and in pGEX-6p1 for GST pulldown experiments (64). Wheat eIF(iso)4E was expressed in pET28a and purified the DALI server to ascertain if the VPg fold was similar to any similarly as human eIF4E. Human EG5 was overexpressed in a pGEX-6p1 others in the protein databank. The top 10 DALI hits included construct. All constructs were verified by sequencing. After overnight induc- human kinesin 5 family member KIF11/EG5 (top hit) and pro- tion at 20 °C, pelleted cells were resuspended in Tris Buffer (TB) buffer (10 mM

karyotic or yeast-derived ribosomal large subunit protein 1 com- Tris, pH 7.5, 250 mM KCl, 0.1% Triton X-100, 1 mM MgCl2, 1 mM DTT) and lysed plexed to 23S ribosomal RNA or a viral IRES (SI Appendix,Table using sonication (8 rounds of 10 s at high power using the Sonic Dismembrator S3). With regard to the top hit, VPg is about half the size of EG5, Model 500 from Fisher); the supernatant of the lysate was added to pre- where the homologous region includes the motor domain (SI Ap- equilibrated glutathione Sepharose 4B beads for affinity purification. After pendix,Fig.S16A). Given the structural homology, we investigated extensive washing with TB buffer containing 500 mM KCl, the protein was whether EG5 bound eIF4E. Using NMR, we found that EG5 di- cleaved overnight with the Prescission Protease in TB buffer, eluted, and fur- ther purified by gel filtration (Superdex 75). High levels of purity (>95%) were rectly bound eIF4E, leading to a similar pattern of spectral broad- obtained. ening observed for VPgΔ37 (SI Appendix,Fig.S16C and E). Like Δ 7 – VPg 37, excess m G cap competed for eIF4E in preformed EG5 NMR Spectroscopy. NMR experiments were performed on Bruker Avance III eIF4E complexes, leading to reemergence of cross-peaks at spectrometers running at 600 or 800 MHz equipped with 5-mm QCIP (quadruple cap-bound positions (SI Appendix,Fig.S16D). Furthermore, resonance probe with phosphorus) or 5-mm TCI (triple resonance inverse probe) 15 peak broadening of the N-labeled eIF4E TrMut was significantly cryoprobes, respectively. For structure determination, typical sample conditions reduced compared with wild-type eIF4E on addition of EG5 (SI were 0.4 to 0.5 mM VPg in 50 mM phosphate buffer, 150 mM NaCl, 1 mM DTT, Appendix,Fig.S16E), again supporting that both EG5 and VPg and 0.02% NaN3 with either 7 or 100% D2O (pH 7.5), and all spectra were ran at interact with the cap-binding surface on eIF4E. Interestingly, this 20 °C. 3D NOESY (NOE spectroscopy) spectra were acquired using nonuniform interaction could have important implications for newly identified sampling (NUS) with 30% Poisson Gap sampling (65). For NMR titrations, con- centrations for labeled and unlabeled proteins were 50 and 150 μM, respec- functions of EG5 (i.e., it interacted with the RNA-binding ZBP tively, and when the m7GDP cap analog was added, a 20-molar excess to eIF4E protein to traffic actin transcripts to modulate cell motility and was used. Spectra were processed with NMRPipe (66) and SMILE (67) and ana- additionally, was found to associate with and traffic ribosomes) lyzedwithSPARKY(68). (61–63). Thus, our studies into potyviruses revealed a previously un- known eIF4E partner protein EG5 and a means for human pro- HADDOCK-Derived Complex Structure of VpG–eIF4E. The HADDOCK2.2 webserver teins to engage eIF4E. (41) was used to generate restraint-driven docking for interaction be- tween eIF4E and VPg using the standard protocols with the VPg structure Conclusions reported here and eIF4E (PDB ID code 2GPQ). Default HADDOCK settings These studies leveraged previous plant genetic investigations to were used for the docking, generating a final 200 structures. The final reveal the existence of a fundamentally different modality to en- models were clustered based on the fraction of common contacts using a0.60cutoff. gage and control eIF4E. The traditional view is that eIF4E is only controlled or engaged by interactions with its dorsal surface as XL-MS. Cross-linked sample was prepared by mixing equimolar amounts of observed for eIF4G. The notion that eIF4E could be inhibited by eIF4E and His-tagged VPgΔ37 (0.15 μmol) with 1 mM DSS (4,4-Dimethyl-4- proteins competing for the cap-binding site was not previously silapentane-1-sulfonic acid). After a 15-min incubation at room temperature,

considered. Consistent with this, VPg impaired host-cell eIF4E- the reaction was stopped using NH4HCO3 and further purified by gel dependent RNA export and translation through binding the filtration chromatography (Superdex-75 column). Proteins were digested

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1904752116 Coutinho de Oliveira et al. Downloaded by guest on September 27, 2021 overnight at 37 °C using a 1:20 trypsin-to-protein ratio, and trypsin was in SI Appendix, Fig. S14E (53), and a detailed protocol is given in SI Appendix, inactivated with 0.5% trifluoroacetic acid. A series of samples (4 μg per well) Supplementary Methods. Validation of the conjugates was carried out using was loaded into a 2-mg sorbent 96-well plate Oasis MCX μElution plate formaldehyde-agarose gel electrophoresis followed by immunoblotting with (Waters), washed, and eluted according to SI Appendix, Table S4 to enrich anti-His antibodies. − for multiply charged peptides. All samples were loaded at 600 nL min 1 on a 17-cm × 75-μm inner diameter PicoFrit fused silica capillary column (New In Vitro Translation Assays. Wheat germ lysate system was used to assess the Objective) and packed in house with Jupiter 5 μm C18 300 Å (Phenomenex). relative translation of VPg–luciferase RNA conjugates, m7G-capped lucif- The column was mounted in an Easy-nLC II system (Proxeon Biosystems) and erase RNA, or uncapped luciferase RNA. More details are in SI Appendix, coupled to an Orbitrap Fusion mass spectrometer (ThermoFisher Scientific) Supplementary Methods. equipped with a Nanospray Flex Ion source (Proxeon Biosystems). MS raw files were analyzed using SIM-XL (69, 70). MS2 spectra were visually inspec- Cell Culture and Transfection. U2Os cells (ATCC) were maintained in 5% CO2 at ted, and theoretical surface-accessible solvent distances for each compiled 37 °C in DMEM (Dulbecco’s Modified Eagle Medium, Gibco BRL) supple- cross-link were measured using Xwalk (42). mented with 10% FBS (Fetal Bovine Serum) and 1% penicillin-streptomycin (Invitrogen). Transfections for stable cell lines were performed using Trans CD. CD spectra were collected on a Jasco-810 spectropolarimeter using a IT-LT1 Transfection Reagent (Mirus) as specified by the manufacturer and μ 0.1-cm quartz cuvette (Hellma) on pure proteins at 20 M at room temperature. selected in puromycin-containing medium (10 μg/mL) for pMSCVeIF4E–Myc Relative ellipticity was converted to mean residue molar ellipticity according and/or G418 (1 mg/mL) for 2Flag–VPg overexpressing cell lines. The identity to Fasman (71). of U2Os cell line has been authenticated using STR profiling (Montreal EpiTerapia Inc.). Fluorescence Spectroscopy. Cysteine residues were reduced by buffer- exchanging VPg into phosphate-buffered saline (PBS) buffer at pH 7.4 (con- Cellular Fractionation, mRNA Export Assay, and Polysomal Profiling. Nucleo- taining 50 μM tris(2-carboxyethyl)phosphine [TCEP] and purged with N )using 2 cytoplasmic fractionations for mRNA export assays and polysomal profiling an NAP-5 column (Fisher). VPg was then labeled by adding 10-molar excess of were done as previously described (56, 58, 72) and detailed in SI Appendix, the organic fluorophore IAEDANS dropwise while stirring. The reaction was Supplemental Methods. TRIzol reagent (ThermoFisher Scientific) was added left at 25 °C for 2 h in the dark and stopped by adding 10 mM DTT. Buffer to each fraction, and RNAs were extracted using a DirectZol RNA Miniprep exchange was performed over an NAP-5 column using a spin column (Amicon kit (Zymo Research), including deoxyribonuclease (DNase) treatment. RNAs 10-kDa MWCO [molecular cutoff]; Fisher) to remove unbound dye. Fluores- were reversed transcribed using M-MLV Reverse Transcriptase (ThermoFisher cence measurements were carried out as follows. Briefly, 0.5 μM labeled VPg Scientific). SensiFastSybr Lo-Rox Mix (Bioline) was used for qRT-PCR analyses was incubated with increasing concentrations of eIF4E (0 to 2.5 μM) in PBS by relative standard curve method (Applied Biosystems User Bulletin #2). buffer containing 1 mM DTT. Fluorescence measurements were performed in a 0.3 × 0.3-cm2 fluorescence cuvette (Hellma) using a Cary Eclipse Fluorescence

Spectrophotometer (Agilent Tech.). The degree of IAEDANS labeling was de- Anchorage-Dependent Foci Assays. Experiments were carried out as described BIOCHEMISTRY termined by measuring the absorbance at 336 nm using the extinction coef- previously. A total of 500 cells were seeded per 10-cm plate or 100 cells per well in ficients 5,700 M−1 cm−1. The IAEDANS-labeled VPg displayed a maximum 6-well plates for 14 d, and then, they were stained with Giemsa (Sigma-Aldrich). fluorescence intensity at 484 nm. The fluorescence intensity increased ∼45% on association of eIF4E. Binding isotherms were fit according to the Hill model. Immunoprecipitations. U2Os cells were fixed with 1% paraformaldehyde Measurements were carried out at least 3 independent times. (PFA) for 10 min at room temperature (RT) and quenched with 0.15 M glycine for 5 min at RT. Cells were than washed 3 times with cold 1× PBS, lysed by ITC. ITC was performed with a Microcal ITC200 calorimeter operating at 20 °C. sonication (4 rounds of 5 s at lowest power) in NucleoTrap (NT2) buffer (56), × The data were analyzed with MicroCal Origin software. The protein con- and centrifuged for 10 min at 12,000 g. Lysates were precleared with centration was 10 μM in the cell, while the m7GDP cap analog was 100 μMin Sephadex G beads (GE Healthcare) for 30 min at 4 °C, and 0.75 to 1 mg of μ the syringe. Experiments consisted of 16 injections of 2.5 μL at a rate of precleared lysates were used for immunoprecipitation with 7 to 10 g anti- 0.5 μLs−1 at 180-s intervals. The first injection peak was discarded from the Flag antibody (Sigma) overnight at 4 °C. After incubation, complexes were isotherm. The baseline was automatically generated by the MicroCal Origin washed 6 times with NT2 buffer, eluted by boiling in Tris(hydroxymethyl) package and corrected manually. The binding isotherm was fitted using the aminomethane (Tris) EDTA (ethylenediaminetetraacetic acid) containing 1% One Set of Sites model in the MicroCal Origin package. sodium dodecyl sulfate (SDS) and 12% β-mercaptoethanol, and analyzed by western blot. RNA Conjugation. VPgΔ37 was mutated in positions 64 (Y64C) and 150 (C150A) to present only 1 cysteine at the position where VPg is known to be Data Availability. The processed spectra and atomic coordinates for VPg were covalently attached to gRNA during infection. The sulfhydryl group of deposited into the PDB and the Biological Magnetic Resonance Data Bank (PDB ID C64 was conjugated with the maleimide functional group of a succinimidyl- code 6NFW and Biological Magnetic Resonance Data Bank accession no. 27506). 4-(N-maleimidomethyl)cyclohexane-1-carboxylate oligonucleotide (GeneLink) as shown in SI Appendix, Fig. S14A. The reaction was performed at room ACKNOWLEDGMENTS. We are grateful for helpful discussions and VPg and temperature with a 20-fold excess of the modified oligonucleotide. More wheat eIF(iso)4E constructs from Dr. Jadwiga Chroboczek (Université Greno- details are given in SI Appendix, Supplementary Methods. ble Alpes-Centre National de la Recherche Scientifique [UGA-CNRS]) and To generate RNA templates suitable for translation, full-length luciferase Dr. Karen Browning (University of Texas), respectively. We thank Jose Rafael RNA (∼1,800 nucleotides) was in vitro transcribed (using the T7 Megascript Kit; Dimayacyac (Institute of Research in Immunology and Cancer) and Dr. Jack Kornblatt (Concordia University) for technical assistance. We also thank Ambion) and 5′ end primed with GMPS (Axxora; Biolog Life Science Institute) ′ Dr. Tara Sprules at Quebec/Eastern Canada High-Field NMR facility for use (51, 52). GMPS-primed RNAs were than coupled to 2,2 -pyridine disulfide (SIGMA) of the 800-MHz NMR. K.L.B.B. acknowledges financial support from NIH ′ to produce pyridyl-disulfide linkage on the 5 end of RNAs. Disulfide ex- Grants R01 CA80728 and R01 CA98571, Canadian Institutes of Health Re- change chemistry between the thiol group from VpG and pyridyl-disulfide search (CIHR) Grant PJT159785, the Canada Research Chair in Molecular Bi- on the 5′ end on luciferase RNA was used to covalently attach RNA to pu- ology of the Cell Nucleus, and the Canadian Foundation for Innovation for rified His–VPg C150A/Y64C. A schematic diagram of these reactions is shown upgrades to the 600-MHz instrument.

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