Structural Insights into Parasite eIF4E Binding Specificity for m7G and m2,2,7G mRNA Caps

The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, a key step in translation initiation. Here we have characterized eIF4E from the human parasite Schistosoma mansoni. Schistosome mRNAs have either the typical monomethylguanosine (m⁷G) or a trimethylguanosine (m^2,2,7G) cap derived from spliced leader trans-splicing. Quantitative fluorescence titration analyses demonstrated that schistosome eIF4E has similar binding specificity for both caps. We present the first crystal structure of an eIF4E with similar binding specificity for m⁷G and m^2,2,7G caps. The eIF4E·m⁷GpppG structure demonstrates that the schistosome protein binds monomethyl cap in a manner similar to that of single specificity eIF4Es and exhibits a structure similar to other known eIF4Es. The structure suggests an alternate orientation of a conserved, key Glu-90 in the cap-binding pocket that may contribute to dual binding specificity and a position for mRNA bound to eIF4E consistent with biochemical data. Comparison of NMR chemical shift perturbations in schistosome eIF4E on binding m⁷GpppG and m^2,2,7GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m⁷G versus m^2,2,7G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m^2,2,7G cap.     

Structural basis for m7G-cap hypermethylation of small nuclear,small nucleolar and telomerase RNA by the dimethyltransferase TGS1

The 5′-cap of spliceosomal small nuclear RNAs, some small nucleolar RNAs and of telomerase RNA was found to be hypermethylated in vivo. The Trimethylguanosine Synthase 1 (TGS1) mediates this conversion of the 7-methylguanosine-cap to the 2,2,7-trimethylguanosine (m₃G)-cap during maturation of the RNPs. For mammalian UsnRNAs the generated m^2,2,7G-cap is one part of a bipartite import signal mediating the transport of the UsnRNP-core complex into the nucleus. In order to understand the structural organization of human TGS1 as well as substrate binding and recognition we solved the crystal structure of the active TGS1 methyltransferase domain containing both, the minimal substrate m⁷GTP and the reaction product S-adenosyl-l-homocysteine (AdoHcy). The methyltransferase of human TGS1 harbors the canonical class 1 methyltransferase fold as well as an unique N-terminal, α-helical domain of 40 amino acids, which is essential for m⁷G-cap binding and catalysis. The crystal structure of the substrate bound methyltransferase domain as well as mutagenesis studies provide insight into the catalytic mechanism of TGS1.     

Intrinsic RNA Binding by the Eukaryotic Initiation Factor 4F Depends on a Minimal RNA Length but Not on the m7G Cap

The eukaryotic initiation factor 4F (eIF4F) is thought to be the first factor to bind mRNA during 7-methylguanosine (m⁷G) cap-dependent translation initiation. The multipartite eIF4F contains the cap-binding protein eIF4E, and it is assumed that eIF4F binds mRNAs primarily at the 5′ m⁷G cap structure. We have analyzed equilibrium binding of rabbit eIF4F to a series of diverse RNAs and found no impact of the 5′-cap on the stability of eIF4F-RNA complexes. However, eIF4F preferentially and cooperatively binds to RNAs with a minimum length of ∼60 nucleotides in vitro. Furthermore, translation activity in rabbit reticulocyte lysate is strongly inhibited by RNAs exceeding this length, but not by shorter ones, consistent with the notion that eIF4F in its physiological environment preferentially binds longer RNAs, too. Collectively, our results indicate that intrinsic RNA binding by eIF4F depends on a minimal RNA length, rather than on cap recognition. The nonetheless essential m⁷G cap may either function at steps subsequent to eIF4F-RNA binding, or other factors facilitate preferential binding of eIF4F to the m⁷G cap.     

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m⁷G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m⁷G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.Cap-dependent translation initiation in eukaryotes is a complex process involving many factors and serves as the primary mechanism for eukaryotic translation (37, 44). The first step in the initiation process, recruitment of the m⁷G (7-methylguanosine)-capped mRNA to the ribosome, is widely considered the rate-limiting step. It begins with recognition of and binding to the m⁷G cap at the 5′ end of the mRNA by the eukaryotic translation initiation factor 4F (eIF4F) complex, which contains three proteins: eIF4E (a cap-binding protein), eIF4G (a scaffold protein with RNA binding sites), and eIF4A (an RNA helicase). eIF4G''s interaction with eIF3, itself a multisubunit complex that interacts with the 40S ribosome, facilitates the actual recruitment of capped RNA to the ribosome. With the help of several other initiation factors, the small ribosomal subunit scans the mRNA from 5′ to 3′ until a translation initiation codon (AUG) in appropriate context is identified and an 80S ribosomal complex is formed, after which the first peptide bond is formed, thus ending the initiation process (37, 44). The AUG context can play an important role in the efficiency of translation initiation (23, 44). The length, structure, and presence of AUGs or open reading frames in the mRNA 5′ untranslated region (UTR) can negatively affect cap-dependent translation and ribosomal scanning. In general, long and highly structured 5′ UTRs, as well as upstream AUGs leading to short open reading frames, can impede ribosome scanning and lead to reduced translation (23, 44). In addition, 5′ UTRs less than 10 nucleotides (nt) in length are thought to be too short to enable preinitiation complex assembly and scanning (24). Thus, several attributes of the mRNA 5′ UTR are known to negatively affect translation initiation, whereas only the AUG context and the absence of negative elements are known to have a positive effect on translation initiation (44).Two of the important mRNA features associated with cap-dependent translation, the cap and the 5′ UTR, are significantly altered by an RNA processing event known as spliced leader (SL) trans splicing (3, 8, 17, 26, 36, 47). This takes place in members of a diverse group of eukaryotic organisms, including some protozoa, sponges, cnidarians, chaetognaths, flatworms, nematodes, rotifers, crustaceans, and tunicates (17, 28, 39, 55, 56). In SL trans splicing, a separately transcribed small exon (16 to 51 nucleotides [nt]) with its own cap gets added to the 5′ end of pre-mRNAs. This produces mature mRNAs with a unique cap and a conserved sequence in the 5′ UTR. In metazoa, the m⁷G cap is replaced with a trimethylguanosine (TMG) cap (m^2,2,7GpppN) (27, 30, 46, 49). In nematodes, ∼70% of all mRNAs are trans spliced and therefore have a TMG cap and an SL (2). In general, eukaryotic eIF4E proteins do not effectively recognize the TMG cap (35). This raises the issues of how the translation machinery in trans-splicing metazoa effectively recognizes TMG-capped trans-spliced mRNAs, what role the SL sequence plays in translation initiation, and how the conserved translation initiation machinery has adapted to effectively translate trans-spliced mRNAs.Previous work has shown that efficient translation of TMG-capped messages in nematodes requires the SL sequence (22 nt) immediately downstream of the cap (5, 25, 29). In the current studies, we sought to understand the manner in which the SL enhanced the translation of TMG-capped mRNAs. Using a cell-free nematode in vitro translation system, we carried out mutational analyses that define the specific sequences in the SL that are required and sufficient for efficient translation of TMG-capped mRNAs. These analyses led to the discovery of a small, discrete stem-loop immediately adjacent to the TMG cap in trans-spliced messages required for efficient translation. Notably, the sequences involved in the base pairing of the stem are highly conserved in alternative SL sequences found in nematodes. We further show that the nematode eIF4E/G complex plays a major role in facilitating the SL enhancement of TMG-capped mRNA that likely occurs after the initial cap-binding step. The results demonstrate the importance of specific enhancing elements in the 5′ UTR and adaptation in the eIF4F complex necessary for optimal cap-dependent translation.     

The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote compartmentalized translation initiation of the HIV-1 genomic RNA

Here, we show a novel molecular mechanism promoted by the DEAD-box RNA helicase DDX3 for translation of the HIV-1 genomic RNA. This occurs through the adenosine triphosphate-dependent formation of a translation initiation complex that is assembled at the 5′ m⁷GTP cap of the HIV-1 mRNA. This is due to the property of DDX3 to substitute for the initiation factor eIF4E in the binding of the HIV-1 m⁷GTP 5′ cap structure where it nucleates the formation of a core DDX3/PABP/eIF4G trimeric complex on the HIV-1 genomic RNA. By using RNA fluorescence in situ hybridization coupled to indirect immunofluorescence, we further show that this viral ribonucleoprotein complex is addressed to compartmentalized cytoplasmic foci where the translation initiation complex is assembled.     

Ribavirin targets eIF4E dependent Akt survival signaling

The eukaryotic translation initiation factor eIF4E is dysregulated in many cancers. eIF4E, through its mRNA export and translation functions, combinatorially modulates the expression of genes involved in Akt dependent survival signaling. For these activities, eIF4E must bind the 7-methyl guanosine (m⁷G) cap moiety on the 5′-end of mRNAs. We demonstrate that a physical mimic of the m⁷G cap, ribavirin, inhibits eIF4E dependent Akt survival signaling. Specifically, ribavirin impairs eIF4E mediated Akt activation via inhibiting the production of an upstream activator of Akt, NBS1. Consequently, ribavirin impairs eIF4E dependent apoptotic rescue. A ribavirin analog with distinct physico-chemical properties, tiazofurin, does not impair eIF4E activity indicating that only analogs that mimic the m⁷G cap will inhibit eIF4E function. Ribavirin represents a first-in-class strategy to inhibit eIF4E dependent cancers, through competition for m⁷G cap binding. Thus, ribavirin coordinately impairs eIF4E dependent pathways and thereby, potently inhibits its biological effects.     

Structure-based mutational analysis of eIF4E in relation to sbm1 resistance to pea seed-borne mosaic virus in pea

Background

Pea encodes eukaryotic translation initiation factor eIF4E (eIF4E^S), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4E^R) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection.

Methodology/Principal Findings

To study structure-function relationships between pea eIF4E and PSbMV VPg, we obtained an X-ray structure for eIF4E^S bound to m⁷GTP. The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E. To assess indirectly the importance of key residues in binding to VPg and/or m⁷GTP, an extensive range of point mutants in eIF4E was tested for their ability to complement PSbMV multiplication in resistant pea tissues and for complementation of protein translation, and hence growth, in an eIF4E-defective yeast strain conditionally dependent upon ectopic expression of eIF4E. The mutants also dissected individual contributions from polymorphisms present in eIF4E^R and compared the impact of individual residues altered in orthologous resistance alleles from other crop species. The data showed that essential resistance determinants in eIF4E differed for different viruses although the critical region involved (possibly in VPg-binding) was conserved and partially overlapped with the m⁷GTP-binding region. This overlap resulted in coupled inhibition of virus multiplication and translation in the majority of cases, although the existence of a few mutants that uncoupled the two processes supported the view that the specific role of eIF4E in potyvirus infection may not be restricted to translation.

Conclusions/Significance

The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance. In addition to defining functional domains within the eIF4E structure, we identified eIF4E alleles with the potential to convey novel virus resistance phenotypes.     

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA: EFFECTS OF eIF4B AND POLY(A)-BINDING PROTEIN*

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k_on) and dissociation rate constant (k_off) for eIF4F binding to IRES were 59 ± 2.1 μm⁻¹ s⁻¹ and 12.9 ± 0.3 s⁻¹, respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F ∼3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.     

Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1

In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m7G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m7G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m3G)-cap. The import adaptor snurportin1 recognises the m3G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m3G-cap-binding domain of snurportin1 with bound m3GpppG at 2.4 A resolution, revealing a structural similarity to the mRNA-guanyly-transferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m7G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m3G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m7G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants.     

Backbone resonance assignment of the HEAT1-domain of the human eukaryotic translation initiation factor 4GI

Controlling translation during protein synthesis is crucial for cell proliferation and differentiation. Protein translation is orchestrated by an assembly of various protein components at the ribosomal subunits. The eukaryotic translation initiation factor 4G (eIF4G) plays an important role in the formation of the translation initiation complex eIF4F consisting of eIF4G, the ATP dependent RNA helicase eIF4A and the cap binding protein eIF4E. One of the functions of eIF4G is the enhancement of the activity of eIF4A facilitated mainly through binding to the HEAT1 domain of eIF4G. In order to understand the interaction of HEAT1 with eIF4A and other components during translation initiation backbone assignment is essential. Here we report the ¹H, ¹³C and ¹⁵N backbone assignment for the HEAT1 domain of human eIF4G isoform I (eIF4GI-HEAT1), the first of three HEAT domains of eIF4G (29 kDa) as a basis for the elucidation of its structure and interactions with its binding partners, necessary for understanding the mechanism of its biological function.     

Identification in the ancient protist Giardia lamblia of two eukaryotic translation initiation factor 4E homologues with distinctive functions

Eukaryotic translation initiation factor 4E (eIF4E) binds to the m(7)GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m(7)GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m(2,2,7)GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m(7)GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m(2,2,7)GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m(2,2,7)GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.     

A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m⁷G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.     

Cap-independent translation initiation of Apaf-1 mRNA based on a scanning mechanism is determined by some features of the secondary structure of its 5′ untranslated region

We have earlier shown that the 5′-untranslated region (5′ UTR) of the mRNA coding for activation factor of apoptotic peptidase 1 (Apaf-1) can direct translation in vivo by strictly 5′ end-dependent way even in the absence of m⁷G-cap. Dependence of translational efficiency on the cap availability for this mRNA turned out to be relatively low. In this study we demonstrate that this surprising phenomenon is determined the 5′-proximal part (domains I and II) of highly structured Apaf-1 5′ UTR. Remarkably, domain II by itself was able to reduce dependence of the mRNA on the cap on its transferring to a short 5′ UTR derived from a standard vector. We suggest that the low cap-dependence inherent to some cellular mRNAs may have an important physiological significance under those stress conditions when the function of cap-binding factor eIF4E is impaired.     

Effect of 5′-terminal structure and base composition on polyribonucleotide binding to ribosomes

Ribopolymers of variable base composition and 5′-terminal structure were synthesized with polynucleotide phosphorylase. Under primer-dependent conditions, m⁷GpppGmpC (m⁷G-cap)², its alkali-treated m⁷G ring-opened derivative, GpppGpC and ppGpC but not m⁷GpppGmpCp, m⁷GpppGm or GpppG were incorporated as 5′-termini. The ribopolymers were compared with reovirus mRNA, which contains m⁷G-cap, for their ability to form initiation complexes with wheat germ 40 S ribosomal subunits and 80 S ribosomes. The presence of 5′-terminal m⁷G was required for stable complex formation by some ribopolymers while for others binding was increased by two- to fourfold. The final level of binding observed was similar to that with reovirus mRNA. In addition to 5′-terminal m⁷G, the base composition of the ribopolymers markedly influenced binding. Some ribopolymers including m⁷G-cap (A)_n did not bind significantly; m⁷G-cap (U)_n formed 40 S complexes while m⁷G-cap (A,U)_n bound to 80 S ribosomes. The ribopolymer m⁷G-cap (A₂,U₂,G)_n directed protein synthesis as measured by amino acid incorporation into polypeptides, methionine tRNA association with 40 S complexes, and puromycin reactivity of 80 S-associated methionine and, like reovirus mRNA, its binding to ribosomes was inhibited by 7-methylguanosine 5′-monophosphate.     

Structure of a Viral Cap-independent Translation Element That Functions via High Affinity Binding to the eIF4E Subunit of eIF4F

RNAs of many positive strand RNA viruses lack a 5′ cap structure and instead rely on cap-independent translation elements (CITEs) to facilitate efficient translation initiation. The mechanisms by which these RNAs recruit ribosomes are poorly understood, and for many viruses the CITE is unknown. Here we identify the first CITE of an umbravirus in the 3′-untranslated region of pea enation mosaic virus RNA 2. Chemical and enzymatic probing of the ∼100-nucleotide PEMV RNA 2 CITE (PTE), and mutagenesis revealed that it forms a long, bulged helix that branches into two short stem-loops, with a possible pseudoknot interaction between a C-rich bulge at the branch point and a G-rich bulge in the main helix. The PTE inhibited translation in trans, and addition of eIF4F, but not eIFiso4F, restored translation. Filter binding assays revealed that the PTE binds eIF4F and its eIF4E subunit with high affinity. Tight binding required an intact cap-binding pocket in eIF4E. Among many PTE mutants, there was a strong correlation between PTE-eIF4E binding affinity and ability to stimulate cap-independent translation. We conclude that the PTE recruits eIF4F by binding eIF4E. The PTE represents a different class of translation enhancer element, as defined by its structure and ability to bind eIF4E in the absence of an m⁷G cap.Regulation of translation occurs primarily at the initiation step. This involves recognition of the 5′ m⁷G(5′)ppp(5′)N cap structure on the mRNA by initiation factors, which recruit the ribosome to the 5′-end of the mRNA (1–5). The 5′ cap structure and the poly(A) tail are necessary for efficient recruitment of initiation factors on eukaryotic mRNAs (3, 6–8). The cap is recognized by the eIF4E subunit of eukaryotic translation initiation factor complex eIF4F (or the eIFiso4E subunit of eIFiso4F in higher plants). The poly(A) tail is recognized by poly(A)-binding protein. In plants, eIF4F is a heterodimer consisting of eIF4E and eIF4G, the core scaffolding protein to which the other factors bind. eIF4A, an ATPase/RNA helicase, interacts with eIF4F but is not part of the eIF4F heterodimer (9, 10). For translation initiation, the purpose of eIF4E is to bring eIF4G to the capped mRNA. eIF4G then recruits the 43 S ternary ribosomal complex via interaction with eIF3.The RNAs of many positive sense RNA viruses contain a cap-independent translation element (CITE)³ that allows efficient translation in the absence of a 5′ cap structure (11–13). In animal viruses and some plant viruses, the CITE is an internal ribosome entry site (IRES) located upstream of the initiation codon. Most viral IRESes neither interact with nor require eIF4E, because they lack the m⁷GpppN structure, which, until this report, was thought to be necessary for mRNA to bind eIF4E with high affinity (3, 14). Translation initiation efficiency of mRNA is also influenced by the length of, and the degree of secondary structure in the 5′ leader (15–17).Many uncapped plant viral RNAs harbor a CITE in the 3′-UTR that confers highly efficient translation initiation at the 5′-end of the mRNA (18–22). These 3′ CITEs facilitate ribosome entry and apparently conventional scanning at the 5′-end of the mRNA (17, 23, 24). A variety of unrelated structures has been found to function as 3′ CITEs, suggesting that they recruit the ribosome by different interactions with initiation factors (13).The factors with which a plant CITE interacts to recruit the ribosome have been identified for only a potyvirus, a luteovirus, and a satellite RNA. The 143-nt 5′-UTR CITE of the potyvirus, tobacco etch virus is an IRES that functions by binding of its AU-rich pseudoknot structure with eIF4G (25). It binds eIF4G with up to 30-fold greater affinity than eIFiso4G and does not require eIF4E for IRES activity. In addition to RNA elements, the genome-linked viral protein (VPg) of potyviruses may participate in cap-independent translation initiation by interacting with the eIF4E and eIFiso4E subunits of eIF4F and eIFiso4F, respectively (26–31). In contrast, the 130-nt cap-independent translation enhancer domain (TED) in the 3′-UTR of satellite tobacco necrosis virus (STNV) RNA forms a long bulged stem-loop, which interacts strongly with both eIF4F and eIFiso4F and weakly with their eIF4E and eIFiso4E subunits (32), suggesting that the TED requires the full eIF4F or eIFiso4F for a biologically relevant interaction. Barley yellow dwarf luteovirus (BYDV) and several other viruses, have a different structure, called a BYDV-like CITE (BTE), in the 3′-UTR. The BTE is characterized by a 17-nt conserved sequence incorporated in a structure with a variable number of stem-loops radiating from a central junction (20, 33, 34). It requires and binds the eIF4G subunit of eIF4F and does not bind free eIF4E, eIFiso4E, or eIFiso4G, although eIF4E slightly enhances the BTE-eIF4G interaction (35). Other 3′ CITEs have been identified, but the host factors with which they interact are unknown.Here we describe unprecedented factor interactions of a CITE found in an umbravirus and a panicovirus. Umbraviruses show strong similarity to the Luteovirus and Dianthovirus genera in (i) the sequence of the replication genes encoded by ORFs 1 and 2, (ii) the predicted structure of the frameshift signals required for translation of the RNA-dependent RNA polymerase from ORF 2 (36, 37), (iii) the absence of a poly(A) tail, and (iv) the lack of a 5′ cap structure (37, 38). Umbraviruses are unique in that they encode no coat protein. For the umbravirus pea enation mosaic virus 2 (PEMV-2), the coat protein is provided by PEMV-1, an enamovirus (39). Uncapped PEMV-2 RNA (PEMV RNA 2), transcribed in vitro, is infectious in pea (Pisum sativa),⁴ indicating it must be translated cap-independently. The 3′-UTRs of some umbraviruses such as Tobacco bushy top virus and Groundnut rosette virus harbor sequences resembling BYDV-like CITEs (BTE).⁵ However, no BTE is apparent in the 3′-UTR of PEMV RNA 2. In this report we identify a different class of CITE in the 705-nt long 3′-UTR of PEMV RNA 2, determine its secondary structure, which may include an unusual pseudoknot, and we show that, unlike any other natural uncapped RNA, it has a high affinity for eIF4E, which is necessary to facilitate cap-independent translation.