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.     

Deaggregation of eIF4E induced by mRNA 5' cap binding

All eukaryotic mRNAs contain a 5' terminal cap structure, which consists of 7-methylguanosine linked by a 5-5' triphosphate bridge to the first transcribed nucleoside (m7GpppN). Specific recognition of the cap by the eukaryotic initiation factor eIF4E plays a key role in regulation of translation initiation as a rate-limiting step. Using dynamic light scattering (DLS), the apo-form of murine eIF4E (33-217) was shown to aggregate. After addition of m7G7P, progressive deaggregation with the time of incubation in the presence of the cap analogue has been observed.     

Identification of the cap binding protein of influenza virus.

The presence of a cap binding protein in influenza virus PR8 has recently been demonstrated by photoaffinity labelling with the cap-analogue (gamma [3 2P]-[4-(benzoylphenyl)methylamido]-7-methylguanosine 5'-triphosphate). This paper describes the identification of the labelled protein using two-dimensional gel electrophoresis. The protein is shown to be PB2, the smaller of the two basic P proteins in the polymerase complex.     

The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue

The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5' end. It was previously reported that VPg binds specifically to eIF4E, the mRNAcap-binding protein of the eukaryotic translation initiation complex. We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV). The cap analogue m7GDP and VPg bind to eIF4E at two distinct sites with similar affinity (K(d) = 0.3 microm). A deeper examination of the interaction pathway showed that the binding of one ligand induces a decrease in the affinity for the other by a factor of 15. GST pull-down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation. Our data suggest that eIF4G recruitment by VPg is indirectly mediated through VPg-eIF4E association. The strength of interaction between eIF4E and pep4G, the eIF4E-binding domain on eIF4G, was increased significantly by VPg. Taken together these quantitative data show that VPg is an efficient modulator of eIF4E biochemical functions.     

Synthesis and evaluation of potential inhibitors of eIF4E cap binding to 7-methyl GTP

Cap-dependent translation is initiated by the binding of eIF4E to capped mRNA (m(7)GpppN). We have prepared a small library of 7-methyl guanosine nucleoside and nucleotide analogs and evaluated their ability to inhibit eIF4E binding to 7-methyl GTP with a competitive eIF4E binding immunoassay. 5'-H-Phosphonate derivatives in which the 2'- and 3'-riboside hydroxyls were tethered together by an isopropylidene group were shown to be a new class of inhibitors of eIF4E binding to capped mRNA.     

A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1

Modulation of interactions among proteins is an important mechanism for regulating both the subcellular location and the function of proteins. An example of the importance of protein-protein interaction is the reversible association of eukaryotic initiation factor eIF4E with the eIF4E binding proteins 4E-BP1 and eIF4G. When bound to 4E-BP1, eIF4E cannot bind to eIF4G to form the active eIF4F complex, an event that is required for the binding of mRNA to the ribosome. Thus, association of eIF4E with 4E-BP1 represses mRNA translation by preventing the binding of mRNA to the ribosome. Previous studies have measured the amount of 4E-BP1 or eIF4G bound to eIF4E by either affinity chromatography or immunoprecipitation of eIF4E followed by Western blot analysis for quantitation of 4E-BP1 and eIF4G. Both of these techniques have significant limitations. In the present study, we describe a microtiter plate-based assay for quantitation of the amount of 4E-BP1 and eIF4G bound to eIF4E that obviates many of the limitations of the earlier approaches. It also has the advantage that absolute amounts of the individual proteins can be easily estimated. The approach should be applicable to the study of a wide variety of protein-protein interactions.     

Poisson–Boltzmann model analysis of binding mRNA cap analogues to the translation initiation factor eIF4E

The electrostatic free energy of binding of two analogues of the 5′-mRNA cap, differing in size and electric charge, to the wild type and mutated eukaryotic initiation factor eIF4E was computed using the finite difference solutions to the Poisson–Boltzmann equation. Two definitions of the solute–solvent dielectric boundary were used: van der Waals model, solvent exclusion (SE) model. The computed electrostatic energies were supplemented by estimations of the non polar and entropic contributions. A comparison with experimental data for the investigated systems was done. It appears that the SE model with additional contribution fits experimental findings better than the van der Waals model does.     

Expression of a synthetic gene for human cap binding protein (human IF-4E) in Escherichia coli and fluorescence studies on interaction with mRNA cap structure analogues

An artificial gene coding for the human cap binding protein (hCBP: human IF-4E) was chemically synthesized and expressed in Escherichia coli under the control of a trp promoter. The DNA duplex of 662 bp was designed and constructed from 44 oligodeoxynucleotide fragments of typically 30 nucleotides in length. Although the hCBP gene was not directly expressed in E. coli HB101, we succeeded in its high-level expression as a fusion protein connected with a portion of human growth hormone through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) that contains the recognition sequence for a site-specific protease alpha-thrombin. Upon induction with 3-indoleacrylic acid, the fusion protein accumulated with a yield of about 20% of the total proteins of the host cell. Upon the treatment of the fusion protein with alpha-thrombin, which recognizes the sequence "Val-Pro-Arg," specific proteolysis at the fused junction occurred efficiently. In this system, nonspecific digestion by alpha-thrombin was not marked. About 15 mg of recombinant hCBP was obtained from a 1-liter culture. Association constants between the recombinant hCBP and mRNA cap structure analogues were determined by fluorescence spectroscopy. The values obtained for the m7GpppA, m7GTP, and m7GMP were almost the same as those reported for the IF-4E isolated from human erythrocyte cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of eIF4E attenuates its interaction with mRNA 5' cap analogs by electrostatic repulsion: intein-mediated protein ligation strategy to obtain phosphorylated protein

Phosphorylation of the eukaryotic initiation factor eIF4E in response to mitogenic stimuli and cytokines is implicated in the regulation of the initiation step of translation. It still remains unclear how the phosphorylation of eIF4E regulates the translation. To address this problem, we applied a unique technique in protein engineering, intein-mediated protein ligation, to synthesize eIF4E, which is selectively phosphorylated at Ser 209. Using selectively chosen synthetic cap analogs, we compared quantitatively the cap affinity for phosphorylated and unphosphorylated eIF4E by a fluorometric time-synchronized titration method. A 1.5- to 4.5-fold reduction of the cap affinity for phosphorylated eIF4E was observed, depending on the negative charge of the 5'-to-5' phosphate chains as well as the presence of a longer tetraribonucleotide strand. Possible implications for understanding the regulation of eIF4E functioning, cap complex formation, and stability, are discussed.     

Discrimination between mono- and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E

Primitive eukaryotes like Caenorhabditis elegans produce mRNAs capped with either m(7)GTP or m(3)(2,2,7)GTP. Caenorhabditis elegans also expresses five isoforms of the cap-binding protein eIF4E. Some isoforms (e.g. IFE-3) bind to m(7)GTP-Sepharose exclusively, whereas others (e.g. IFE-5) bind to both m(7)GTP- and m(3)(2,2,7)GTP-Sepharose. To examine specificity differences, we devised molecular models of the tertiary structures of IFE-3 and IFE-5, based on the known structure of mouse eIF4E-1. We then substituted amino acid sequences of IFE-5 with homologous sequences from IFE-3. As few as two changes (N64Y/V65L) converted the cap specificity of IFE-5 to essentially that of IFE-3. Molecular dynamics simulations suggested that the width and depth of the cap-binding cavity were larger in IFE-5 than in IFE-3 or the N64Y/V65L variant, supporting a model in which IFE-3 discriminates against m(3)(2,2,7)GTP by steric hindrance. Furthermore, the affinity of IFE-5 (but not IFE-3) for m(3)(2,2,7)GTP was reversibly increased when thiol reagents were removed. This was correlated with the formation of a disulfide bond between Cys-122 and Cys-126. Thus, translation of m(3)(2,2,7)GTP-capped mRNAs may be regulated by intracellular redox state.     

Thermodynamics of mRNA 5' cap binding by eukaryotic translation initiation factor eIF4E

Translation of mRNA in eukaryotes begins with specific recognition of the 5' cap structure by the highly conserved protein, eIF4E. The thermodynamics of eIF4E interaction with nine chemical cap analogues has been studied by means of emission spectroscopy. High-sensitivity measurements of intrinsic protein fluorescence quenching upon cap binding provided equilibrium association constants in the temperature range of 279 to 314 K. A van't Hoff analysis yielded the negative binding enthalpies for the entire cap analogue series, -16.6 to -81 kJ mol(-1), and the entropies covering the range of +40.3 to -136 J mol(-1) K(-1) at 293 K. The main enthalpic contributions come from interactions of the phosphate chains and positively charged amino acids and the cation-pi stacking of 7-methylguanine with tryptophans. A nontrivial, statistically important isothermal enthalpy-entropy compensation has been detected (T(c) = 399 +/- 24 K), which points to significant fluctuations of apo-eIF4E and indicates that the cap-binding microstate lies 9.66 +/- 1.7 kJ mol(-1) below the mean energy of all available conformational states. For five cap analogues, large and positive heat capacity changes have been found. The values of DeltaC(p) degrees correlate with the free energies of eIF4E binding due to stiffening of the protein upon interaction with cap analogues. At biological temperatures, binding of the natural caps has both favorable enthalpy and favorable entropy. Thermodynamic coupling of cap-eIF4E association to intramolecular self-stacking of dinucleotide cap analogues strongly influences the enthalpies and entropies of the binding, but has a negligible effect on the resultant DeltaG degrees and DeltaC(p) degrees values.     

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.     

Binding preference of eIF4E for 4E-binding protein isoform and function of eIF4E N-terminal flexible region for interaction, studied by SPR analysis

To investigate the binding preference of eIF4E for the three eIF4E-binding isoforms (4E-BP1-3) and the function of N-terminal flexible region of eIF4E for their interactions, the binding parameters of recombinant full-length and N-terminal residues-deleted eIF4Es with 4E-BP1-3 were investigated by the surface plasmon resonance (SPR) analysis. Consequently, it was clarified that 4E-BP2 exhibits the highest binding affinity for both m7GTP-bound and -unbound full-length eIF4Es when compared with 4E-BP1 and 4E-BP3. This is primarily due to the difference among their dissociation rates, because their association rates are almost the same. Interestingly, the deletion of the 33 N-terminal residues of eIF4E increased its binding affinities for 4E-BP1 and 4E-BP2 markedly, whereas such a change was not observed by at least the N-terminal deletion up to 26 residues. In contrast, the binding parameters of 4E-BP3 were hardly influenced by N-terminal deletion up to 33 residues. From the comparison of the amino acid sequences of 4E-BP1-3, the present result indicates the importance of N-terminal flexible region of eIF4E for the suppressive binding with 4E-BP1 and 2, together with the possible contribution of N-terminal sequence of 4E-BP isoform to the regulative binding to eIF4E.     

Thermodynamics of 7-methylguanosine cation stacking with tryptophan upon mRNA 5' cap binding to translation factor eIF4E

All eukaryotic nuclear transcribed mRNAs possess the cap structure, consisting of 7-methylguanosine linked by the 5'-5' triphosphate bridge to the first nucleoside. The goal of the present study is to dissect the enthalpy and entropy changes of association of the mRNA 5' cap with eIF4E into contributions originating from the interaction of 7-methylguanosine with tryptophan. The model results are discussed in the context of the thermodynamic parameters for the association of eIF4E with synthetic cap analogues.     

Mutational analysis of the DEAD-box RNA helicase eIF4AII characterizes its interaction with transformation suppressor Pdcd4 and eIF4GI

Eukaryotic initiation factor (eIF) 4A unwinds secondary and tertiary structures in the 5'-untranslated region of mRNA, permitting translation initiation. Programmed cell death 4 (Pdcd4) is a novel transformation suppressor and eIF4A-binding partner that inhibits eIF4A helicase activity and translation. To elucidate the regions of eIF4A that are functionally significant in binding to Pdcd4, we generated point mutations of eIF4A. Two-hybrid analysis revealed that five eIF4A mutants completely lost binding to Pdcd4 while four eIF4A mutants retained wild-type levels of binding. The residues that, when mutated, inactivated Pdcd4 binding specified ATP binding, ATP hydrolysis, or RNA binding. With the exception of the Q-motif mutant eIF4AP56L, the eIF4A mutants inactivated for Pdcd4 binding were inactivated for binding to eIF4G (GM, GC, or both) and for enhancing translation. Several eIF4A mutants showing wild-type level binding to Pdcd4 were also inactivated for binding to eIF4G and for enhancing translation. Thus, significant dissociation of eIF4A's Pdcd4- and eIF4G-binding regions appears to occur. Because three of the four eIF4A mutants that retained Pdcd4 binding also suppressed translation activity in a dominant-negative manner, the structure that defines the Pdcd4-binding domain of eIF4A may be necessary but is insufficient for translation. A structural homology model of eIF4A shows regions important for binding to Pdcd4 and/or eIF4G lying on the perimeters of the hinge area of eIF4A. A competition experiment revealed that Pdcd4 competes with C-terminal eIF4G for binding to eIF4A. In summary, the Pdcd4-binding domains on eIF4A impact both binding to eIF4G and translation initiation in cells.     

Positive heat capacity change upon specific binding of translation initiation factor eIF4E to mRNA 5' cap

Specific recognition of the mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry were used to examine the thermodynamics of eIF4E binding to a cap-analogue, 7-methylGpppG. A van't Hoff plot revealed nonlinearity characterized by an unexpected, large positive molar heat capacity change (DeltaC(degree)(p) = +1.92 +/- 0.93 kJ.mol(-1).K(-1)), which was confirmed by direct ITC measurements (DeltaC(degree)(p) = +1.941 +/- 0.059 kJ.mol(-1).K(-1)). This unique result appears to come from an extensive additional hydration upon binding and charge-related interactions within the binding site. As a consequence of the positive DeltaC(degree)(p), the nature of the thermodynamic driving force changes with increasing temperature, from enthalpy-driven and entropy-opposed, through enthalpy- and entropy-driven in the range of biological temperatures, into entropy-driven and enthalpy-opposed. Comparison of the van't Hoff and calorimetric enthalpy values provided proof for the ligand protonation at N(1) upon binding, which is required for tight stabilization of the cap-eIF4E complex. Intramolecular self-stacking of the dinucleotide cap-analogue was analyzed to reveal the influence of this coupled process on the thermodynamic parameters of the eIF4E-mRNA 5' cap interaction. The temperature-dependent change in the conformation of 7-methylGpppG shifts significantly the intrinsic DeltaH(degree)(0) = -72.9 +/- 4.2 kJ.mol(-1) and DeltaS(degree)(0) = -116 +/- 58 J.mol(-1).K(-1) of binding to the less negative resultant values, by DeltaH(degree)(sst) = +9.76 +/- 1.15 kJ.mol(-1) and DeltaS(degree)(sst) = +24.8 +/- 2.1 J.mol(-1).K(-1) (at 293 K), while the corresponding DeltaC(degree)(p)(sst) = -0.0743 +/- 0.0083 kJ.mol(-1).K(-1) is negligible in comparison with the total DeltaC(degree)(p) .     

Kinetics of binding the mRNA cap analogues to the translation initiation factor eIF4E under second-order reaction conditions

The kinetics of binding of five analogues of the 5'-mRNA cap, differing in size and electric charge, to the eukaryotic initiation factor eIF4E, at 20 degrees C, pH 7.2, and ionic strength of 150 mM, were measured, after mixing solutions of comparable concentrations of the reagents, in a stopped-flow spectrofluorimeter. The registered stopped-flow signals were fitted using an efficient software package, called Dyna Fit, based on a numerical solution of the kinetic rate equations for assumed reaction mechanisms. One-, two-, and three-step binding models were considered. The quality of fits for these models were compared using two statistical criteria: Akaike's Information Criterion and Bayesian Information Criterion. Based on resulting probabilities of the models, it was concluded that for all investigated ligands a one-step binding model has essentially no support in the experimental observations. Our conclusions were also analysed from the perspective of kinetic transients obtained for cap-eIF4E systems under the so called pseudo-first order reaction condition, which result in the linear correlation of the observed association rate constant with ligand concentration. The existence of such a linear correlation is usually considered as proof of a one-step binding mechanism. The kinetic and optical parameters, derived from fitting a two-step cap-binding model with the DynaFit, were used to simulate kinetic transients under pseudo-first order reaction conditions. It appeared that the observed association rate constants derived from these simulated transients are also linearly correlated with the ligand concentration. This indicated that these linear dependencies are not sufficient to conclude a one-step binding.     

Regulation of human eIF4E by 4E-BP1: binding analysis using surface plasmon resonance

The mitochondria play a pivotal role in regulating glucose-induced insulin secretion in the pancreatic beta cell. We have recently demonstrated that glutamate derived from mitochondria participates directly in the stimulation of insulin exocytosis. In the present study, mitochondria isolated from the beta cell line INS-1E generated glutamate when incubated with the tricarboxylic acid cycle intermediate succinate. The generation of glutamate correlated with stimulated mitochondrial activity monitored as oxygen consumption and was inhibited by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Glutamate is formed by the mitochondrial enzyme glutamate dehydrogenase from alpha-ketoglutarate. Transient overexpression of glutamate dehydrogenase in INS-1E cells resulted in potentiation of glucose-stimulated hormone secretion without affecting basal release. These results further point to glutamate as an intracellular messenger playing a key role in the control of insulin exocytosis.     

Charge distribution in 7-methylguanine regarding cation-pi interaction with protein factor eIF4E

Electric charge distribution in mRNA 5' cap terminus has been exhaustively characterized in respect to the affinity for cap-binding proteins. Formation of the stacked configuration of positively charged 7-methylguanine in between two aromatic amino acid rings, known as sandwich cation-pi stacking, is thought to be prerequisite for the specific recognition of the cap by eukaryotic initiation factor eIF4E; i.e., discrimination between the cap and nucleotides without the methyl group at N(7). Nuclear magnetic resonance spectroscopy of (15)N/(13)C-double-labeled 7-methylguanosine 5'-triphosphate and 7-methylguanosine, as well as their unsubstituted counterparts, GTP and guanosine, yielded characteristic changes of the electron-mediated spin-spin couplings and chemical shifts due to the methylation at N(7). The experimentally measured changes of the nuclear magnetic resonance parameters have been analyzed in respect to the electric charge distribution calculated by means of quantum chemical methods, and interpreted in terms of new proposed positive charge localization in the 7-methylguanine five-member ring.     

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.