Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro

Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A(pro)). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L(pro) specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A(pro) in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L(pro) in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L(pro) and rhinovirus 2A(pro) enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.     

The adenovirus tripartite leader may eliminate the requirement for cap-binding protein complex during translation initiation.

The adenovirus tripartite leader is a 200-nucleotide 5' noncoding region that is found on all late viral mRNAs. This segment is required for preferential translation of viral mRNAs at late times during infection. Most tripartite leader-containing mRNAs appear to exhibit little if any requirement for intact cap-binding protein complex, a property previously established only for uncapped poliovirus mRNAs and capped mRNAs with minimal secondary structure. The tripartite leader also permits the translation of mRNAs in poliovirus-infected cells in the apparent absence of active cap-binding protein complex and does not require any adenovirus gene products for this activity. The preferential translation of viral late mRNAs may involve this unusual property.     

Relationship of eukaryotic initiation factor 3 to poliovirus-induced p220 cleavage activity.

The cleavage of the p220 subunit of eukaryotic initiation factor 4F (eIF-4F) that is induced by the poliovirus protease 2A has been shown previously to require another translation initiation factor, eIF-3. The role of eIF-3 in this cleavage reaction, however, is not known. An antiserum was raised against human eIF-3 and used to analyze the eIF-3 subunit composition in poliovirus-infected and uninfected HeLa cells and after incubation of eIF-3 in vitro with viral 2A protease. No evidence for 2Apro-dependent cleavage of any eIF-3 subunit was detected. Infected cells contain an activity that catalyzes the cleavage of p220 to a specific set of cleavage products. This activity is thought to be an activated form of a latent cellular protease. The p220-specific cleavage activity was partially purified. It was resolved from eIF-3 by both gel filtration and anion-exchange chromatography. Neither intact eIF-3 nor any detectable subunits of eIF-3 were found to copurify with the p220-specific cleavage activity. The latter activity behaves as a protein of 55,000 to 60,000 molecular weight and is inhibited by alkylating agents and metals, which indicates the presence of essential thiol groups. When this activity was incubated with partially purified p220, cleavage occurred only in the presence of eIF-3. Thus, eIF-3 appears to play a role in the p220 cleavage cascade which is subsequent to the 2Apro-induced activation of the p220-specific protease.     

The p220 component of eukaryotic initiation factor 4F is a substrate for multiple calcium-dependent enzymes

Eukaryotic initiation factor 4F (eIF-4F) is a multisubunit protein that functions in the first step of the binding of capped mRNAs to the small ribosomal subunit. Its largest polypeptide component, p220, is cleaved following poliovirus infection. This is thought to inactivate eIF-4F function, thereby preventing cap-dependent initiation of translation of cellular mRNAs. In this report, we show that p220 in extracts of uninfected HeLa cells is specifically lost in the presence of calcium. The responsible activities have been partially purified and identified as the calcium-dependent, neutral, cysteine proteases calpains I and II. In addition, a third calcium-dependent activity was resolved from the calpains and also results in the loss of p220. This activity has properties similar to a transglutaminase and copurifies with tissue transglutaminase through several chromatographic steps. None of these calcium-dependent activities appears to mediate p220 cleavage in poliovirus-infected cells.     

Malignant transformation by the eukaryotic translation initiation factor 3 subunit p48 (eIF3e)

Several components of translation, e.g. eIF4E and PKR, are implicated in cancer. The e-subunit (p48) of mammalian initiation factor 3 is encoded by the Int6 gene, a common site for integration of the mouse mammary tumor virus genome, leading to the production of a truncated eukaryotic initiation factor-3e (eIF3e). Stable expression of a truncated eIF3e in NIH 3T3 cells causes malignant transformation by four criteria: foci formation; anchorage independent growth; accelerated growth; and lack of contact inhibition. Stable expression of full-length eIF3e does not cause transformation. The truncated eIF3e also inhibits the onset of apoptosis caused by serum starvation.     

Cleavage of eukaryotic translation initiation factor 4GII correlates with translation inhibition during apoptosis

Eukaryotic translation initiation factor 4G (eIF4G), which has two homologs known as eIF4GI and eIF4GII, functions in a complex (eIF4F) which binds to the 5' cap structure of cellular mRNAs and facilitates binding of capped mRNA to 40S ribosomal subunits. Disruption of this complex in enterovirus-infected cells through eIF4G cleavage is known to block this step of translation initiation, thus leading to a drastic inhibition of cap-dependent translation. Here, we show that like eIF4GI, the newly identified homolog eIF4GII is cleaved during apoptosis in HeLa cells and can serve as a substrate for caspase 3. Proteolysis of both eIF4GI and eIF4GII occurs with similar kinetics and coincides with the profound translation inhibition observed in cisplatin-treated HeLa cells. Both eIF4GI and eIF4GII can be cleaved by caspase 3 with similar efficiency in vitro, however, eIF4GII is processed into additional fragments which destroy its core central domain and likely contributes to the shutoff of translation observed in apoptosis. Cell Death and Differentiation (2000) 7, 1234 - 1243.     

Cap-independent translation conferred by the 5' leader of tobacco etch virus is eukaryotic initiation factor 4G dependent

The 5' leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.     

Phosphorylation of maize eukaryotic translation initiation factor on Ser2 by catalytic subunit CK2

Alignment of eukaryotic translation initiation factor 5A (eIF5A) sequences has shown, for plants, in contrast to most other eukaryotes, the presence of N-terminal serine residue (Ser2) which could be phosphorylated by CK2. Using point directed mutagenesis, we demonstrate here that in recombinant maize ZmeIF5Awt Ser2 is exclusively phosphorylated by catalytic subunit of CK2 (CK2α), whereas its mutated variant Ser2Ala is not phosphorylated. To shed light on the physiological significance of this Ser2 phosphorylation, transient expression of fluorescence-labeled proteins was performed in maize protoplast. Wild-type ZmeIF5A was distributed evenly between nucleus and cytoplasm, but the replacement of Ser2 by aspartic acid, which mimics the phosphorylated serine, influences its intracellular localization. We postulate that phosphorylation of Ser2 in maize eIF5A, and most probably in other plant cells, plays a role in specific regulation of nuclear export of eIF5A-bound mRNAs.     

End-to-end annealing of plant microtubules by the p86 subunit of eukaryotic initiation factor-(iso)4F.

The p86 subunit of eukaryotic initiation factor-(iso)4F from wheat germ exhibits saturable and substoichiometric binding to maize microtubules, induces microtubule bundling in vitro, and is colocalized or closely associated with cortical microtubule bundles in maize root cells, indicating its function as a microtubule-associated protein (MAP). The effects of p86 on the growth of short, taxol-stabilized maize microtubules were investigated. Pure microtubules underwent a gradual length redistribution, an increase in mean length, and a decrease in number concentration consistent with an end-to-end annealing mechanism of microtubule growth. Saturating p86 enhanced the microtubule length distribution and produced significantly longer and fewer microtubules than the control, indicating a facilitation of annealing by p86. Confirmation of endwise annealing rather than of dynamic instability as the mechanism for microtubule growth was made using mammalian MAP2, which also promoted the redistribution of length, increase in mean length, and decrease in number concentration of taxol-stabilized maize microtubules. Enhancement of microtubule growth occurred concomitant with bundling by p86, indicating that an alignment of microtubules in bundles facilitated endwise annealing kinetics. The results demonstrate that nonfacile plant microtubules can spontaneously elongate by endwise annealing and that MAPs enhance the rate of annealing. The p86 subunit of eukaryotic initiation factor-(iso)4F may be an important regulator of microtubule dynamics in plant cells.     

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.     

Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220.

The poliovirus proteinase 2A is autocatalytically released from the poliovirus polyprotein by cotranslational cleavage at its own amino terminus, resulting in separation of structural and nonstructural protein precursors. Cleavage is a prerequisite for further processing of the structural protein precursor and consequently for poliovirus encapsidation. A second function of 2Apro is in the rapid shutoff of host cell protein synthesis that occurs upon infection with poliovirus. This is associated with proteolytic cleavage of the p220 component of eukaryotic initiation factor eIF-4F, which is induced but not directly catalyzed by 2Apro. We introduced single-amino-acid substitutions in the 2Apro-coding region of larger poliovirus precursors that were subsequently translated in vitro and thus demonstrated that His-20, Asp-38, and Cys-109 (which constitute the putative catalytic triad) are essential for, and that His-117 is an important determinant of, the autocatalytic activity of 2Apro. This is consistent with the proposal that 2Apro is structurally related to a subclass of trypsinlike serine proteinases. Moreover, 2Apro containing a Cys109Ser substitution retained a small but significant autocatalytic activity. Cleavage of p220 was not induced by those mutants that had reduced proteolytic activity, indicating that the cellular factor that cleaves p220 is probably activated by 2Apro-catalyzed proteolytic cleavage.     

Effect of eukaryotic initiation factor 4F on AUG selection in a bicistronic mRNA

Artificial bicistronic mRNAs based on rabbit beta-globin and bacterial chloramphenicol acetyltransferase protein-coding sequences were tested for translation activity in a mouse astrocytoma cell-free extract. This cell extract exhibited an apparent preference for 5'-distal or internal initiation over 5'-proximal ("first AUG") initiation. 5'-Distal initiation appeared to be 5'-cap independent, suggesting that nonstandard initiation was responsible. This conclusion was based on a lack of inhibition of internal initiation by added cap analog and insensitivity of internal initiation to the presence or absence of a 5'-cap structure. Exogenous reticulocyte initiation factors were tested for effect on 5'-proximal initiation. The only factor with a significant effect was found to be eukaryotic initiation factor 4F, or the cap-binding protein. Addition of this factor promoted 5'-end initiation as evident by a general increase in 5'-proximal open reading frame (ORF) product relative to 5'-distal ORF product. The relative expression of 5'-proximal to 5'-distal ORFs in bicistronic or multicistronic mRNAs may very well be dependent on activity levels of eukaryotic initiation factor 4F and possibly other mRNA-dependent initiation factors.     

Generation of multiple isoforms of eukaryotic translation initiation factor 4GI by use of alternate translation initiation codons

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5' end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5' sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5' untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

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.     

Mouse p56 blocks a distinct function of eukaryotic initiation factor 3 in translation initiation

Members of the p56 family of mammalian proteins are strongly induced in virus-infected cells and in cells treated with interferons or double-stranded RNA. Previously, we have reported that human p56 inhibits initiation of translation by binding to the "e" subunit of eukaryotic initiation factor 3 (eIF3) and subsequently interfering with the eIF3/eIF2.GTP.Met-tRNAi (ternary complex) interaction. Here we report that mouse p56 also interferes with eIF3 functions and inhibits translation. However, the murine protein binds to the "c" subunit, not the "e" subunit, of eIF3. Consequently, it has only a marginal effect on eIF3.ternary complex interaction. Instead, the major inhibitory effect of mouse p56 is manifested at a different step of translation initiation, namely the binding of eIF4F to the 40 S ribosomal subunit.eIF3.ternary complex. Thus, mouse and human p56 proteins block different functions of eIF3 by binding to its different subunits.     

Eap1p, a novel eukaryotic translation initiation factor 4E-associated protein in Saccharomyces cerevisiae

Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m(7)G(5')ppp(5')N, where N is any nucleotide] present at the 5' termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of the EAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.     

Characterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae

Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAi and mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis. Glucose repression of TIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+ affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity.     

Interaction of eukaryotic initiation factor eIF-4B with a picornavirus internal translation initiation site.

We studied the interaction of cellular proteins with the internal ribosome entry site (IRES) of foot-and-mouth disease virus by UV cross-linking and observed specific binding of a 80-kDa protein contained in cytosolic HeLa cell extract and in rabbit reticulocyte lysate. Binding of the protein was dependent on the presence of ATP. Immunoprecipitation with eIF-4B antiserum revealed that the protein is identical to the initiation factor eIF-4B. Deletions in the 3' part, but not in the 5' part, of the IRES interfered with UV cross-linking, indicating that the binding site of eIF-4B is located close to the end of the element. Attempts to separate ribosome-associated from non-ribosome-associated protein fractions of cytosolic cell extracts led to the loss of cross-linking activity. This finding suggests that additional protein factors contribute to this interaction of eIF-4B with the IRES of foot-and-mouth disease virus.