Knock-down of both eIF4E1 and eIF4E2 genes confers broad-spectrum resistance against potyviruses in tomato

Background

The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance.

Methodology/Principal Findings

To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2.

Conclusion/Significance

These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato-potyvirus interactions.     

Crystal structure of a minimal eIF4E-Cup complex reveals a general mechanism of eIF4E regulation in translational repression

Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. Here, we present the 2.8 Å resolution crystal structure of a minimal eIF4E–Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E–eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation.     

Potyviral resistance derived from cultivars of Phaseolus vulgaris carrying bc-3 is associated with the homozygotic presence of a mutated eIF4E allele

Eukaryotic translation initiation factors (eIFs) play a central role in potyviral infection. Accordingly, mutations in the gene encoding eIF4E have been identified as a source of recessive resistance in several plant species. In common bean, Phaseolus vulgaris , four recessive genes, bc-1 , bc-2 , bc-3 and bc-u , have been proposed to control resistance to the potyviruses Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus . In order to identify molecular entities for these genes, we cloned and sequenced P. vulgaris homologues of genes encoding the eIF proteins eIF4E, eIF(iso)4E and nCBP. Bean genotypes reported to carry bc-3 resistance were found specifically to carry non-silent mutations at codons 53, 65, 76 and 111 in eIF4E . This set of mutations closely resembled a pattern of eIF4E mutations determining potyvirus resistance in other plant species. The segregation of BCMV resistance and eIF4E genotype was subsequently analysed in an F₂ population derived from the P. vulgaris all-susceptible genotype and a genotype carrying bc-3 . F₂ plants homozygous for the eIF4E mutant allele were found to display at least the same level of resistance to BCMV as the parental resistant genotype. At 6 weeks after inoculation, all F₂ plants found to be BCMV negative by enzyme-linked immunosorbent assay were found to be homozygous for the mutant eIF4E allele. In F₃ plants homozygous for the mutated allele, virus resistance was subsequently found to be stably maintained. In conclusion, allelic eIF4E appears to be associated with a major component of potyvirus resistance present in bc-3 genotypes of bean.     

Cap-binding activity of an eIF4E homolog from Leishmania

All eukaryotic mRNAs possess a 5'-cap (m(7)GpppN) that is recognized by a family of cap-binding proteins. These participate in various processes, such as RNA transport and stabilization, as well as in assembly of the translation initiation complex. The 5'-cap of trypanosomatids is complex; in addition to 7-methyl guanosine, it includes unique modifications on the first four transcribed nucleotides, and is thus denoted cap-4. Here we analyze a cap-binding protein of Leishmania, in an attempt to understand the structural features that promote its binding to this unusual cap. LeishIF4E-1, a homolog of eIF4E, contains the conserved cap-binding pocket, similar to its mouse counterpart. The mouse eIF4E has a higher K(as) for all cap analogs tested, as compared with LeishIF4E-1. However, whereas the mouse eIF4E shows a fivefold higher affinity for m(7)GTP than for a chemically synthesized cap-4 structure, LeishIF4E-1 shows similar affinities for both ligands. A sequence alignment shows that LeishIF4E-1 lacks the region that parallels the C terminus in the murine eIF4E. Truncation of this region in the mouse protein reduces the difference that is observed between its binding to m(7)GTP and cap-4, prior to this deletion. We hypothesize that variations in the structure of LeishIF4E-1, possibly also the absence of a region that is homologous to the C terminus of the mouse protein, promote its ability to interact with the cap-4 structure. LeishIF4E-1 is distributed in the cytoplasm, but its function is not clear yet, because it cannot substitute the mammalian eIF4E in a rabbit reticulocyte in vitro translation system.     

An eIF4E allele confers resistance to an uncapped and non-polyadenylated RNA virus in melon

The characterization of natural recessive resistance genes and virus-resistant mutants of Arabidopsis have implicated translation initiation factors of the 4E family [eIF4E and eIF(iso)4E] as susceptibility factors required for virus multiplication and resistance expression. To date, viruses controlled by these genes mainly belong to the family Potyviridae. Melon necrotic spot virus (MNSV) belongs to the family Tombusviridae (genus Carmovirus) and is an uncapped and non-polyadenylated RNA virus. In melon, nsv-mediated resistance is a natural source of recessive resistance against all strains of MNSV except MNSV-264. Analyses of chimeras between non-resistance-breaking and resistance-breaking strains have shown that the avirulence determinant maps to the 3'-untranslated region (3'-UTR) of the viral genome. Using a combination of positional cloning and microsynteny analysis between Arabidopsis thaliana and melon, we genetically and physically delimited the nsv locus to a single bacterial artificial chromosome clone and identified the melon eukaryotic translation initiation factor 4E (Cm-eIF4E) as a candidate gene. Complementation analysis using a biolistic transient expression assay, confirmed Cm-eIF4E as the product of nsv. A single amino acid change at position 228 of the protein led to the resistance to MNSV. Protein expression and cap-binding analysis showed that Cm-eIF4E encoded by a resistant plant was not affected in it's cap-binding activity. The Agrobacterium-mediated transient expression of the susceptibility allele of Cm-eIF4E in Nicotiana benthamiana enhanced MNSV-264 accumulation. Based on these results, a model to explain melon resistance to MNSV is proposed. These data, and data from other authors, suggest that translation initiation factors of the eIF4E family are universal determinants of plant susceptibility to RNA viruses.     

Tracking a refined eIF4E-binding motif reveals Angel1 as a new partner of eIF4E

The initiation factor 4E (eIF4E) is implicated in most of the crucial steps of the mRNA life cycle and is recognized as a pivotal protein in gene regulation. Many of these roles are mediated by its interaction with specific proteins generally known as eIF4E-interacting partners (4E-IPs), such as eIF4G and 4E-BP. To screen for new 4E-IPs, we developed a novel approach based on structural, in silico and biochemical analyses. We identified the protein Angel1, a member of the CCR4 deadenylase family. Immunoprecipitation experiments provided evidence that Angel1 is able to interact in vitro and in vivo with eIF4E. Point mutation variants of Angel1 demonstrated that the interaction of Angel1 with eIF4E is mediated through a consensus eIF4E-binding motif. Immunofluorescence and cell fractionation experiments showed that Angel1 is confined to the endoplasmic reticulum and Golgi apparatus, where it partially co-localizes with eIF4E and eIF4G, but not with 4E-BP. Furthermore, manipulating Angel1 levels in living cells had no effect on global translation rates, suggesting that the protein has a more specific function. Taken together, our results illustrate that we developed a powerful method for identifying new eIF4E partners and open new perspectives for understanding eIF4E-specific regulation.     

Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands

The activity of the eukaryotic translation initiation factor eIF4E is modulated through conformational response to its ligands. For example, eIF4G and eIF4E-binding proteins (4E-BPs) modulate cap affinity, and thus physiological activity of eIF4E, by binding a site distal to the 7-methylguanosine cap-binding site. Further, cap binding substantially modulates eIF4E's affinity for eIF4G and the 4E-BPs. To date, only cap-bound eIF4E structures were reported. In the absence of structural information on the apo form, the molecular underpinnings of this conformational response mechanism cannot be established. We report here the first cap-free eIF4E structure. Apo-eIF4E exhibits structural differences in the cap-binding site and dorsal surface relative to cap-eIF4E. Analysis of structure and dynamics of apo-eIF4E, and changes observed upon ligand binding, reveal a molecular basis for eIF4E's conformational response to these ligands. In particular, alterations in the S4-H4 loop, distal to either the cap or eIF4G binding sites, appear key to modulating these effects. Mutation in this loop mimics these effects. Overall, our studies have important implications for the regulation of eIF4E.     

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.     

Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation

Feeding promotes protein accretion in skeletal muscle through a stimulation of the mRNA translation initiation phase of protein synthesis either secondarily to nutrient-induced rises in insulin or owing to direct effects of nutrients themselves. The present set of experiments establishes the effects of meal feeding on potential signal transduction pathways that may be important in accelerating mRNA translation initiation. Gastrocnemius muscle from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled before, during, and after the meal. Meal feeding enhanced the assembly of the active eIF4G.eIF4E complex, which returned to basal levels within 3 h of removal of food. The increased assembly of the active eIF4G.eIF4E complex was associated with a marked 10-fold rise in phosphorylation of eIF4G(Ser(1108)) and a decreased assembly of inactive 4E-BP1.eIF4E complex. The reduced assembly of 4E-BP1.eIF4E complex was associated with a 75-fold increase in phosphorylation of 4E-BP1 in the gamma-form during feeding. Phosphorylation of S6K1 on Ser(789) was increased by meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of mammalian target of rapamycin (mTOR) on Ser(2448) or Ser(2481), an upstream kinase responsible for phosphorylating both S6K1 and 4E-BP1, was increased at all times during meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of PKB, an upstream kinase responsible for phosphorylating mTOR, was elevated only after 0.5 h of meal feeding for Thr(308), whereas phosphorylation Ser(473) was significantly elevated at only 0.5 and 1 h after initiation of feeding. We conclude from these studies that meal feeding stimulates two signal pathways in skeletal muscle that lead to elevated eIF4G.eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4E-BP1 with eIF4E.     

eIF4E is a central node of an RNA regulon that governs cellular proliferation

This study demonstrates that the eukaryotic translation initiation factor eIF4E is a critical node in an RNA regulon that impacts nearly every stage of cell cycle progression. Specifically, eIF4E coordinately promotes the messenger RNA (mRNA) export of several genes involved in the cell cycle. A common feature of these mRNAs is a structurally conserved, approximately 50-nucleotide element in the 3' untranslated region denoted as an eIF4E sensitivity element. This element is sufficient for localization of capped mRNAs to eIF4E nuclear bodies, formation of eIF4E-specific ribonucleoproteins in the nucleus, and eIF4E-dependent mRNA export. The roles of eIF4E in translation and mRNA export are distinct, as they rely on different mRNA elements. Furthermore, eIF4E-dependent mRNA export is independent of ongoing RNA or protein synthesis. Unlike the NXF1-mediated export of bulk mRNAs, eIF4E-dependent mRNA export is CRM1 dependent. Finally, the growth-suppressive promyelocytic leukemia protein (PML) inhibits this RNA regulon. These data provide novel perspectives into the proliferative and oncogenic properties of eIF4E.     

Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins

mRNA 5'-cap recognition by the eukaryotic translation initiation factor eIF4E has been exhaustively characterized with the aid of a novel fluorometric, time-synchronized titration method, and X-ray crystallography. The association constant values of recombinant eIF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m(7)GTP (approximately 1.1 x 10(8) M(-1)). The affinity of the cap analogues for eIF4E correlates with their ability to inhibit in vitro translation. The association constants yield contributions of non-covalent interactions involving single structural elements of the cap to the free energy of binding, giving a reliable starting point to rational drug design. The free energy of 7-methylguanine stacking and hydrogen bonding (-4.9 kcal/mol) is separate from the energies of phosphate chain interactions (-3.0, -1.9, -0.9 kcal/mol for alpha, beta, gamma phosphates, respectively), supporting two-step mechanism of the binding. The negatively charged phosphate groups of the cap act as a molecular anchor, enabling further formation of the intermolecular contacts within the cap-binding slot. Stabilization of the stacked Trp102/m(7)G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostatically steered eIF4E-cap association is accompanied by additional hydration of the complex by approximately 65 water molecules, and by ionic equilibria shift. Temperature dependence reveals the enthalpy-driven and entropy-opposed character of the m(7)GTP-eIF4E binding, which results from dominant charge-related interactions (DeltaH degrees =-17.8 kcal/mol, DeltaS degrees= -23.6 cal/mol K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to eIF4E, all the association constants were approximately 10(7) M(-1), in decreasing order: eIF4GI>4E-BP1>eIF4GII approximately 4E-BP1(P-Ser65) approximately 4E-BP1(P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to eIF4E. Enhancement of the eIF4E affinity for cap occurs after binding to eIF4G peptides.     

The domains of yeast eIF4G,eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects

Translation initiation in eukaryotes starts with the recognition of the mRNA 5′-cap by eIF4F, a hetero-trimeric complex of eIF4E, the cap-binding protein, eIF4A, a DEAD-box helicase, and eIF4G, a scaffold protein. eIF4G comprises eIF4E- and eIF4A-binding domains (4E-BD, 4A-BD) and three RNA-binding regions (RNA1–RNA3), and interacts with eIF4A, eIF4E, and with the mRNA. Within the eIF4F complex, the helicase activity of eIF4A is increased. We showed previously that RNA3 of eIF4G is important for the stimulation of the eIF4A conformational cycle and its ATPase and helicase activities. Here, we dissect the interplay between the eIF4G domains and the role of the eIF4E/cap interaction in eIF4A activation. We show that RNA2 leads to an increase in the fraction of eIF4A in the closed state, an increased RNA affinity, and faster RNA unwinding. This stimulatory effect is partially reduced when the 4E-BD is present. eIF4E binding to the 4E-BD then further inhibits the helicase activity and closing of eIF4A, but does not affect the RNA-stimulated ATPase activity of eIF4A. The 5′-cap renders the functional interaction of mRNA with eIF4A less efficient. Overall, the activity of eIF4A at the 5′-cap is thus fine-tuned by a delicately balanced network of stimulatory and inhibitory interactions.     

Stopped-flow kinetic analysis of eIF4E and phosphorylated eIF4E binding to cap analogs and capped oligoribonucleotides: evidence for a one-step binding mechanism

Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5' -terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m(7)GpppG, the rate constant of association, k(on), was dependent on ionic strength, decreasing progressively up to 350 mm KCl, but the rate constant of dissociation, k(off), was independent of ionic strength. Phosphorylation of eIF4E decreased k(on) by 2.1-2.3-fold at 50-100 mm KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mm. Contrary to published evidence, eIF4E phosphorylation had no effect on k(off). Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single- to double-exponential as the eIF4E concentration was increased. However, measuring k(off) for dissociation of a pre-formed eIF4E.m(7)GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in k(off). This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.     

Crystal structure of an activation intermediate of cathepsin E

Cathepsin E is an intracellular, non-lysosomal aspartic protease expressed in a variety of cells and tissues. The protease has proposed physiological roles in antigen presentation by the MHC class II system, in the biogenesis of the vasoconstrictor peptide endothelin, and in neurodegeneration associated with brain ischemia and aging. Cathepsin E is the only A1 aspartic protease that exists as a homodimer with a disulfide bridge linking the two monomers. Like many other aspartic proteases, it is synthesized as a zymogen which is catalytically inactive towards its natural substrates at neutral pH and which auto-activates in an acidic environment. Here we report the crystal structure of an activation intermediate of human cathepsin E at 2.35A resolution. The overall structure follows the general fold of aspartic proteases of the A1 family, and the intermediate shares many features with the intermediate 2 on the proposed activation pathway of aspartic proteases like pepsin C and cathepsin D. The pro-sequence is cleaved from the protease and remains stably associated with the mature enzyme by forming the outermost sixth strand of the interdomain beta-sheet. However, different from these other aspartic proteases the pro-sequence of cathepsin E remains intact after cleavage from the mature enzyme. In addition, the active site of cathepsin E in the crystal is occupied by N-terminal amino acid residues of the mature protease in the non-primed binding site and by an artificial N-terminal extension of the pro-sequence from a neighboring molecule in the primed site. The crystal structure of the cathepsin E/pro-sequence complex, therefore, provides further insight into the activation mechanism of aspartic proteases.     

Eukaryotic initiation factor 4E (eIF4E): A recap of the cap-binding protein

Eukaryotic initiation factor 4E (eIF4E), a fundamental effector and rate limiting element of protein synthesis, binds the 7-methylguanosine cap at the 5′ end of eukaryotic messenger RNA (mRNA) specifically as a constituent of eIF4F translation initiation complex thus facilitating the recruitment of mRNA to the ribosomes. This review focusses on the engagement of signals contributing to growth factor originated maxim and their role in the activation of eIF4E to achieve a collective influence on cellular growth, with a key focus on conjuring vital processes like protein synthesis. The review invites considerable interest in elevating the appeal of eIF4E beyond its role in regulating translation viz a viz cancer genesis, attributed to its phosphorylation state that improves the prospect for the growth of the cancerous cell. This review highlights the latest studies that have envisioned to target these pathways and ultimately the translational machinery for therapeutic intervention. The review also brings forward the prospect of eIF4E to act as a converging juncture for signaling pathways like mTOR/PI3K and Mnk/MAPK to promote tumorigenesis.     

The Cap-binding protein eIF4E promotes folding of a functional domain of yeast translation initiation factor eIF4G1

The association of eucaryotic translation initiation factor eIF4G with the cap-binding protein eIF4E establishes a critical link between the mRNA and the ribosome during translation initiation. This association requires a conserved seven amino acid peptide within eIF4G that binds to eIF4E. Here we report that a 98-amino acid fragment of S. cerevisiae eIF4G1 that contains this eIF4E binding peptide undergoes an unfolded to folded transition upon binding to eIF4E. The folding of the eIF4G1 domain was evidenced by the eIF4E-dependent changes in its protease sensitivity and (1)H-(15)N HSQC NMR spectrum. Analysis of a series of charge-to-alanine mutations throughout the essential 55.4-kDa core of yeast eIF4G1 also revealed substitutions within this 98-amino acid region that led to reduced eIF4E binding in vivo and in vitro. These data suggest that the association of yeast eIF4E with eIF4G1 leads to the formation of a structured domain within eIF4G1 that could serve as a specific site for interactions with other components of the translational apparatus. They also suggest that the stability of the native eIF4E-eIF4G complex is determined by amino acid residues outside of the conserved seven-residue consensus sequence.     

Participation of eIF4F complex in Junin virus infection: blockage of eIF4E does not impair virus replication

Translation efficiency of viral mRNAs is a key factor defining both cytopathogenicity and virulence of viruses, which are entirely dependent on the cellular translation machinery to synthesize their proteins. This dependence has led them to develop different translational reprogramming strategies to ensure viral mRNAs can effectively compete with cellular mRNAs. Junin virus (JUNV) is a member of the family Arenaviridae, whose mRNAs are capped but not polyadenylated. In this work we evaluated the relevance to JUNV replication of the main components of the eIF4F complex: eIF4A, eIF4GI and eIF4E. We found the viral nucleoprotein (N) of JUNV colocalized with eIF4A and eIF4GI but not with eIF4E. Moreover, N could be immunoprecipitated in association with eIF4A and eIF4GI but not with eIF4E. Accordingly, functional impairment of eIF4A as well as eIF4GI reduced JUNV multiplication. By contrast, inhibition of eIF4E did not show a significant effect on JUNV protein synthesis. A similar situation was observed for another two members of arenaviruses: Tacaribe (TCRV) and Pichinde (PICV) viruses. Finally, the nucleoproteins of JUNV, TCRV and PICV were able to interact with 7 methyl‐guanosine (cap), suggesting that the independence of JUNV multiplication on eIF4E, the cap‐binding protein, may be due to the replacement of this factor by N protein.     

The interaction of eIF4E with 4E-BP1 is an induced fit to a completely disordered protein.

4E binding protein 1 (4E-BP1) inhibits translation by binding to the initiation factor eIF4E and is mostly or completely unstructured in both free and bound states. We wished to determine whether the free protein has local structure that could be involved in eIF4E binding. Assignments were obtained using double and triple resonance NMR methods. Residues 4-10, 43-46, and 56-65 could not be assigned, primarily because of a high degree of 1H and 15N chemical shift overlap. Steady-state ¿1H¿-15N NOEs were measured for 45 residues in the assigned regions. Except for the two C-terminal residues, the NOEs were between -0.77 and - 1.14, indicating a high level of flexibility. Furthermore, the ¿1H¿-15N NOE spectrum recorded with presaturation contained no strong positive signals, making it likely that no other residues have positive or smaller negative NOEs. This implies that 4E-BP1 has no regions of local order in the absence of eIF4E. The interaction therefore appears to be an induced fit to a completely disordered protein molecule.