Turnip mosaic virus VPg interacts with Arabidopsis thaliana eIF(iso)4E and inhibits in vitro translation

The interaction between turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) and Arabidopsis thaliana eukaryotic initiation factor (iso)4E (eIF(iso)4E) was investigated to address the influence of potyviral VPg on host cellular translational initiation. Affinity chromatographic analysis showed that the region comprising amino acids 62-70 of VPg is important for the interaction with eIF(iso)4E. In vitro translation analysis showed that the addition of VPg significantly inhibited translation of capped RNA in eIF(iso)4E-reconstituted wheat germ extract. This result indicates that VPg inhibits cap-dependent translational initiation via binding to eIF(iso)4E. The inhibition by VPg of in vitro translation of RNA with wheat germ extract did not depend on RNase activity. Our present results may indicate that excess VPg produced at the encapsidation stage shuts off cap-dependent translational initiation in host cells by inhibiting complex formation between eIF(iso)4E and cellular mRNAs.     

Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity

The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m(7)GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thaliana as well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m(7)GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.     

Transgenic Brassica rapa plants over‐expressing eIF(iso)4E variants show broad‐spectrum Turnip mosaic virus (TuMV) resistance

The protein–protein interaction between VPg (viral protein genome‐linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad‐spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge‐based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap‐binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap‐binding pockets, and mutated. Yeast two‐hybrid assay and co‐immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E‐knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild‐type were over‐expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T₁ and T₂ transformants demonstrated that the over‐expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge‐based approaches for the engineering of broad‐spectrum resistance in Chinese cabbage.     

Potyvirus terminal protein VPg, effector of host eukaryotic initiation factor eIF4E

Potyvirus RNA contains at the 5' end a covalently linked virus-encoded protein VPg, which is required for virus infectivity. This role has been attributed to VPg interaction with the eukaryotic translation initiation factor eIF4E, a cap-binding protein. We characterized the dissociation constants for the interaction of the potato virus Y VPg with different plant eIF4Es and its isoforms and mapped the eIF(iso)4E attachment region on VPg. VPg/eIF4E interaction results in the inhibition of cell-free protein synthesis, and we show that it stems from the liberation of the cap moiety from the complex with eIF4E. Since VPg does not attach the cap, it appears that VPg induces changes in the eIF4E structure, diminishing its affinity to the cap. We show here that the initiation complex scaffold protein eIF(iso)4G increases VPg interaction with eIF(iso)4E. These data together suggest similar cap and VPg interactions with eIF4E and characterize VPg as a novel eIF4E-binding protein, which inhibits host protein synthesis at a very early stage of the initiation complex formation through the inhibition of cap attachment to the initiation factor eIF4E.     

Complex formation between recombinant protein VPg of potato virus Y and heterologous eukaryotic translation initiation factors eIF4E

Genomes of some positive-strand RNA viruses do not contain cap-structure, but instead their 5'-end is covalently linked to a viral protein called VPg. Complex formation between VPg and cellular translation initiation factors (eIFs) has been extensively studied in the context of the model of this complex involvement in virus mRNA translation initiation and cellular protein translation shut down in infected cells. The potato virus (PVY) VPg was expressed in bacterial and baculovirus systems in order to investigate its binding capacity to wheat eIF4E and its isoform. Both purified recombinant eIF4E and eIF(iso)4E were identified in vitro as binding partners of the purified recombinant VPg by using affinity chromatography, as well in vivo by coexpressing of recombinant VPg and eIFs in insect cells with following complex purification using affinity chromatography. Besides it was shown that PVY VPg also formed a complex with endogenous insect eIF4E in vivo. PVY VPg interaction with eIF4E of wheat (non permissive plant for PVY), and also with so evolutionary distant partner as insect eIF4E suggests the conservation of general structural features of eIF4E implicated in the formation of the complex with VPg.     

Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.     

The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses

An Arabidopsis thaliana line bearing a transposon insertion in the gene coding for the isozyme form of the plant-specific cap-binding protein, eukaryotic initiation factor (iso) 4E (eIF (iso) 4E), has been isolated. This mutant line completely lacks both eIF(iso)4E mRNA and protein, but was found to have a phenotype and fertility indistinguishable from wild-type plants under standard laboratory conditions. In contrast, the amount of the related eIF4E protein was found to increase in seedling extracts. Furthermore, polysome analysis shows that the mRNA encoding eIF4E was being translated at increased levels. Given the known interaction between cap-binding proteins and potyviral genome-linked proteins (VPg), this plant line was challenged with two potyviruses, Turnip mosaic virus (TuMV) and Lettuce mosaic virus (LMV) and was found resistant to both, but not to the Nepovirus, Tomato black ring virus (TBRV) and the Cucumovirus, Cucumber mosaic virus (CMV). Together with previous data showing that the VPg-eIF4E interaction is necessary for virus infectivity and upregulates genome amplification, this shows that the eIF4E proteins are specifically recruited for the replication cycle of potyviruses.     

Potyvirus genome-linked protein, VPg, directly affects wheat germ in vitro translation: interactions with translation initiation factors eIF4F and eIFiso4F

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nm, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nm compared with 108 nm). Kinetic studies of eIF4F and eIFiso4F with VPg show approximately 2.6-fold faster association for eIFiso4F.VPg as compared with eIF4F.VPg. The dissociation rate was approximately 2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F.VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.     

Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses

Arabidopsis thaliana plants with mutations in the genes encoding eukaryotic initiation factor (eIF4E) or isoform of eIF4E (eIF(iso)4E) were tested for susceptibility to Clover yellow vein virus (ClYVV), a member of the genus Potyvirus. ClYVV accumulated in both inoculated and upper uninoculated leaves of mutant plants lacking eIF(iso)4E, but not in mutant plants lacking eIF4E. In contrast, Turnip mosaic virus (TuMV), another member of the genus Potyvirus, multiplied in mutant plants lacking eIF4E but not in mutant plants lacking eIF(iso)4E. These results suggest the selective involvement of members of the eIF4E family in infection by potyviruses.     

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.     

Visualization of the interaction between the precursors of VPg, the viral protein linked to the genome of turnip mosaic virus, and the translation eukaryotic initiation factor iso 4E in Planta

The RNA genome of Turnip mosaic virus is covalently linked at its 5' end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication.     

Phosphorylation states of translational initiation factors affect mRNA cap binding in wheat

Phosphorylation of eukaryotic translational initiation factors (eIFs) has been shown to be an important means of regulating protein synthesis. Plant initiation factors undergo phosphorylation/dephosphorylation under a variety of stress and growth conditions. We have shown that recombinant wheat cap-binding protein, eIF(iso)4E, produced from E. coli can be phosphorylated in vitro. Phosphorylation of eIF(iso)4E has effects on m(7)G cap-binding affinity similar to those of phosphorylation of mammalian eIF4E even though eIF(iso)4E lacks an amino acid that can be phosphorylated at the residue corresponding to Ser-209, the phosphorylation site in mammalian eIF4E. The cap-binding affinity was reduced 1.2-2.6-fold when eIF(iso)4E was phosphorylated. The in vitro phosphorylation site for wheat eIF(iso)4E was identified as Ser-207. Addition of eIF(iso)4G and eIF4B that had also been phosphorylated in vitro further reduced cap-binding affinity. Temperature-dependent studies showed that DeltaH(degrees) was favorable for cap binding regardless of the phosphorylation state of the initiation factors. The entropy, however, was unfavorable (negative) except when eIF(iso)4E was phosphorylated and interacting with eIF(iso)4G. Phosphorylation may modulate not only cap-binding activity, but other functions of eukaryotic initiation factors as well.     

Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for eIF(iso)4E during potyvirus infection

The Arabidopsis thaliana-potyvirus system was developed to identify compatibility and incompatibility factors involved during infection and disease caused by positive-strand RNA viruses. Several Arabidopsis mutants with increased susceptibility to Tobacco etch potyvirus (TEV) were isolated previously, revealing a virus-specific resistance system in the phloem. In this study, Arabidopsis mutants with decreased susceptibility to Turnip mosaic potyvirus (TuMV) were isolated. Three independent mutants that conferred immunity to TuMV were isolated and assigned to the same complementation group. These mutants were also immune or near-immune to TEV but were susceptible to an unrelated virus. The locus associated with decreased susceptibility was named loss-of-susceptibility to potyviruses 1 (lsp1). The LSP1 locus was isolated by map-based cloning and was identified as the gene encoding translation factor eIF(iso)4E, one of several known Arabidopsis isoforms that has cap binding activity. eIF4E and eIF(iso)4E from different plant species were shown previously to interact with the genome-linked protein (VPg) of TEV and TuMV, respectively. Models to explain the roles of eIF(iso)4E during virus infection are presented.     

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.     

Poly(A)-binding protein increases the binding affinity and kinetic rates of interaction of viral protein linked to genome with translation initiation factors eIFiso4F and eIFiso4F·4B complex

VPg of turnip mosaic virus (TuMV) was previously shown to interact with translation initiation factor eIFiso4F and play an important role in mRNA translation [Khan, M. A., et al. (2008) J. Biol. Chem.283, 1340-1349]. VPg competed with cap analogue for eIFiso4F binding and competitively inhibited cap-dependent translation and enhanced cap-independent translation to give viral RNA a significant competitive advantage. To gain further insight into the cap-independent process of initiation of protein synthesis, we examined the effect of PABP and/or eIF4B on the equilibrium and kinetics of binding of VPg to eIFiso4F. Equilibrium data showed the addition of PABP and/or eIF4B to eIFiso4F increased the binding affinity for VPg (K(d) = 24.3 ± 1.6 nM) as compared to that with eIFiso4F alone (K(d) = 81.3 ± 0.2.4 nM). Thermodynamic parameters showed that binding of VPg to eIFiso4F was enthalpy-driven and entropy-favorable with the addition of PABP and/or eIF4B. PABP and eIF4B decreased the entropic contribution by 67% for binding of VPg to eIFiso4F. The decrease in entropy involved in the formation of the eIFiso4F·4B·PABP-VPg complex suggested weakened hydrophobic interactions for complex formation and an overall conformational change. The kinetic studies of eIFiso4F with VPg in the presence of PABP and eIF4B show 3-fold faster association (k(2) = 182 ± 9.0 s(-1)) compared to that with eIFiso4F alone (k(2) = 69.0 ± 1.5 s(-1)) . The dissociation rate was 3-fold slower (k(-2) = 6.5 ± 0.43 s(-1)) for eIFiso4F with VPg in the presence of PABP and eIF4B (k(-2) = 19.0 ± 0.9 s(-1)). The addition of PABP and eIF4B decreased the activation energy of eIFiso4F with VPg from 81.0 ± 3.0 to 44.0 ± 2.4 kJ/mol. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves to sequester initiation factors.     

The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment

The positive-strand RNA genomes of caliciviruses are not capped, but are instead covalently linked at their 5' ends to a viral protein called VPg. The lack of a cap structure typical of eukaryotic mRNA and absence of an internal ribosomal entry site suggest that VPg may function in translation initiation on calicivirus RNA. This hypothesis was tested by analyzing binding of Norwalk virus VPg to translation initiation factors. The eIF3d subunit of eIF3 was identified as a binding partner of VPg by yeast two-hybrid analysis. VPg bound to purified mammalian eIF3 and to eIF3 in mammalian cell lysates. To test the effects of the VPg- eIF3 interaction on translation, VPg was added to cell-free translation reactions programmed with either capped reporter RNA, an RNA containing an EMCV internal ribosomal entry site (IRES) or an RNA with a cricket paralysis virus IRES. VPg inhibited translation of all reporter RNAs in a dose-dependent manner. Together, the data suggest that VPg may play a role in initiating translation on calicivirus RNA through unique protein-protein interactions with the translation machinery.     

Natural variation and functional analyses provide evidence for co-evolution between plant eIF4E and potyviral VPg

Amino acid substitutions in the eukaryotic translation initiation factor 4E (eIF4E) result in recessive resistance to potyviruses in a range of plant species, including Capsicum spp. Correspondingly, amino acid changes in the central part of the viral genome-linked protein (VPg) are responsible for the potyvirus's ability to overcome eIF4E-mediated resistance. A key observation was that physical interaction between eIF4E and the VPg is required for viral infection, and eIF4E mutations that cause resistance prevent VPg binding and inhibit the viral cycle. In this study, polymorphism analysis of the pvr2-eIF4E coding sequence in a worldwide sample of 25 C. annuum accessions identified 10 allelic variants with exclusively non-synonymous variations clustered in two surface loops of eIF4E. Resistance and genetic complementation assays demonstrated that pvr2 variants, each with signature amino acid changes, corresponded to potyvirus resistance alleles. Systematic analysis of the interactions between eIF4E proteins encoded by the 10 pvr2 alleles and VPgs of virulent and avirulent potato virus Y (PVY) and tobacco etch virus (TEV) strains demonstrated that resistance phenotypes arose from disruption of the interaction between eIF4E and VPg, and that viral adaptation to eIF4E-mediated resistance resulted from restored interaction with the resistance protein. Complementation of an eIF4E knockout yeast strain by C. annuum eIF4E proteins further shows that amino acid changes did not impede essential eIF4E functions. Altogether, these results argue in favour of a co-evolutionary 'arms race' between Capsicum eIF4E and potyviral VPg.     

Caliciviruses differ in their functional requirements for eIF4F components

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.     

Eukaryotic translation initiation factor 4F architectural alterations accompany translation initiation factor redistribution in poxvirus-infected cells

Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly.