Presence of the cap-binding protein in initiation factor preparations from poliovirus-infected HeLa cells.

Crude preparations of initiation factors from mock-infected and poliovirus-infected HeLa cells were analyzed for the presence of proteins which could be cross-linked to the 5' cap group of mRNA. A protein having an apparent molecular weight of 26,000, similar to the cap-binding protein in rabbit reticulocytes described by Sonenberg and Shatkin (Proc. Natl. Acad. Sci. U.S.A. 75:4843-4847, 1978), was found in the ribosomal salt wash from both uninfected and infected cells. Cross-linking of this polypeptide was inhibited by the cap analog m7GMP. In addition, cross-linking of a protein having an approximate molecular weight of 60,000 was similarly inhibited by cap analog. The smaller cap-binding protein fractionated in a 0 to 40% ammonium sulfate precipitate of ribosomal salt wash; the larger protein was found in the 40 to 70% ammonium sulfate fraction. Although the cap-binding proteins were present in both mock-infected and poliovirus-infected ribosomal salt wash, only preparations from uninfected HeLa cells were able to restore translation of capped vesicular stomatitis virus mRNA by extracts prepared from poliovirus-infected cells.     

Regulation of protein synthesis in vesicular stomatitis virus-infected mouse L-929 cells by decreased protein synthesis initiation factor 2 activity.

Infection of mouse L-cell spinner cultures by vesicular stomatitis virus (VSV) effected the selective translation of viral mRNA by 4h after viral adsorption. Cell-free systems prepared from mock- and VSV-infected cells reflected this phenomenon; protein synthesis was reduced in the virus-infected cell lysate by approximately 75% compared with the mock-infected (control) lysate. This effect appeared to be specific to protein synthesis initiation since (i) methionine incorporation into protein from an exogenous preparation of initiator methionyl-tRNA gave completely analogous results and (ii) the addition of a ribosomal salt wash (containing protein synthesis initiation factors) stimulated protein synthesis by the infected cell lysate but had no effect on protein synthesis by the control. Micrococcal nuclease-treated (initiation-dependent) VSV-infected cell lysates were not able to translate L-cell mRNA unless they were supplemented with a ribosomal salt wash; a salt wash from ribosomes from uninfected cells effected a quicker recovery than a salt wash from ribosomes from infected cells. When salt wash preparations from ribosomes from uninfected and infected cells were tested for initiation factor 2 (eIF-2)-dependent ternary complex capacity with added GTP and initiator methionyl-tRNA, we found that the two preparations contained equivalent levels of eIF-2. However, initiation complex formation by the factor from virus-infected cells proceeded at a reduced initial rate compared with the control. When the lysates were supplemented with a partially purified eIF-2 preparation, recovery of activity by the infected cell lysate was observed. Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.     

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.     

Specificity of initiation factor preparations from poliovirus-infected cells.

Crude initiation factor preparations from poliovirus-infected cells stimulated the translation of poliovirus RNA in vitro, but were inactive for the translation of host cell or vesicular stomatitis virus mRNA's. In contrast, similar preparations from either uninfected or vesicular stomatitis virus-infected cells supported the initiation of translation of host cell mRNA's and both viral mRNA's. These results reflect a specific alteration of some components(s) of the initiation factor preparation from poliovirus-infected cells which is consistent with the ability of the virus to inhibit the translation of host cell and vesicular stomatitis virus-directed protein synthesis.     

Activation of double-stranded RNA-activated protein kinase in HeLa cells after poliovirus infection does not result in increased phosphorylation of eucaryotic initiation factor-2.

Protein kinase activity in general is stimulated at least 5- to 10-fold in ribosomal salt wash preparations from poliovirus-infected HeLa cells compared with those from mock-infected cells. The stimulation of kinase activity is manifested by increased phosphorylation of ribosome-associated polypeptides having approximate molecular weights of 135,000, 120,000, 85,000, 68,000, 65,000, 40,000, 28,000, 25,000, and 21,000. The Mr 68,000 phosphoprotein is structurally identical to the interferon-induced, double-stranded RNA-activated protein kinase (P1) which phosphorylates the alpha subunit of eucaryotic initiation factor-2 (eIF-2). A similar protein of Mr 68,000 is more phosphorylated in poliovirus-infected cells than in mock-infected cells. Increased phosphorylation of P1 protein in poliovirus-infected cells, however, does not result in an increased phosphorylation of the alpha subunit of endogenous or exogenously added eIF-2, both in vitro and in vivo. These results suggest that a mechanism must exist in poliovirus-infected HeLa cells which prevents further phosphorylation of eIF-2 by the activated kinase.     

Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular system may affect poliovirus RNA synthesis. In conclusion, monensin and nigericin are potent inhibitors of poliovirus genome replication that prevent the shutoff of host translation by poliovirus while still permitting cleavage of p220.     

Localization of cap-binding protein in subcellular fractions of HeLa cells

The 26,000-M(r) cap-binding protein was analyzed by a cross-linking assay in cell fractions from uninfected and poliovirus-infected HeLa cells. Cap-binding protein was found in the postribosomal supernatant (S-200) and in the ribosomal salt wash. The cap-binding protein in the S-200 had a sedimentation coefficient of 5 to 7S and lacked the ability to restore translation in extracts of poliovirus-infected cells.     

The cap-binding protein complex in uninfected and poliovirus-infected HeLa cells

In poliovirus-infected HeLa cells, the mechanism of protein synthesis initiation factor recognition of m7G cap groups on mRNA is impaired. Translation of capped host cell mRNAs is inhibited, whereas translation of uncapped poliovirus mRNA proceeds exclusively. The site of this defect has been localized to the cap-binding protein complex (CBPC). To elucidate the specific structural and functional defects of the CBPC following poliovirus infection, the CBPC and/or its polypeptide components were purified from uninfected and poliovirus-infected HeLa cells. The CBPC from uninfected cells consisted of tightly associated 24- and 220-kDa polypeptides; minor amounts of polypeptides of 40, 44, and 80 kDa also consistently co-purified with the p24/p220 cores. No evidence of a 50-kDa, eIF-4A-related polypeptide subunit of the CBPC was obtained. The CBPC from poliovirus-infected cells had undergone major structural alterations. The 220-kDa component was absent; antigenically related (100-130 kDa) degradation products were present instead. The 24-kDa component co-purified with the p220 degradation products, but other components were missing. The association of the infected cell CBPC components was quite labile compared with that demonstrated by the components of CBPC from uninfected cells. Differential stimulation of capped, but not uncapped mRNAs in a cell-free translation assay was demonstrated by unmodified CBPC. Conversely, modified CBPC from poliovirus-infected cells differentially stimulated in vitro translation of uncapped poliovirus mRNA but not capped mRNAs. The implications of these results for the mechanism of cap-independent translation are briefly discussed.     

Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex

Following poliovirus infection of HeLa cells, the synthesis of cellular proteins is inhibited but translation of poliovirus mRNA proceeds. The defect in the recognition of host cell mRNA may be due to a change in a cap recognition complex which, when added to an infected cell lysate, restores the ability to translate capped mRNAs. We employed immunoblotting techniques to examine initiation factors in crude lysates from uninfected and poliovirus-infected HeLa cells. Using an antiserum against eucaryotic initiation factor 3, we detected an antigen of approximate molecular weight 220,000 in uninfected cell lysates but not in infected cell lysates. Antigenically related polypeptides of 100,000 to 130,000 daltons, presumably degradation products, were detected in the infected cell lysate. The time course for degradation of the 220,000-dalton polypeptide correlates with that for inhibition of cellular protein synthesis in vivo. A portion of the population of 220,000-dalton polypeptides apparently associates with initiation factor eIF3 but is readily dissociated in buffers containing high salt. Affinity-purified antibodies against the polypeptide recognize a protein of the same size in a purified preparation of a cap binding protein complex obtained by cap-affinity chromatography. We postulate that the 220,000-dalton polypeptide is an essential component of the cap recognition complex and that its degradation in poliovirus-infected cells results in the inhibition of host cell translation. These results are in the first demonstration of a specific structural defect in an initiation factor resulting from poliovirus infection.     

Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

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.     

Functional characterization of eukaryotic mRNA cap binding protein complex: effects on translation of capped and naturally uncapped RNAs

We examined the effects of a eukaryotic mRNA cap binding protein (CBP) complex purified by cap analogue affinity chromatography [Edery, I., Humebelin, M., Darveau, A., Lee, K.A. W., Milburn, S., Hershey, J.W.B., Trachsel, H., & Sonenberg, N. (1983) J. Biol. Chem. 258, 11398 11403], on translation of several capped and naturally uncapped mRNAs in extracts prepared from poliovirus-infected or mock-infected HeLa cells. The CBP complex has activity that restores capped mRNA (globin, tobacco mosaic virus, and others) function in extracts from poliovirus-infected HeLa cells. Translation of two naturally uncapped RNAs (poliovirus and mengovirus RNAs), the translation of which is not restricted in extracts from poliovirus-infected cells, is also not stimulated by the CBP complex. Translation of several capped eukaryotic mRNAs (vesicular stomatitis virus, reovirus, and tobacco mosaic virus) in extracts from mock-infected cells is inhibited when the potassium ion concentration is increased. However, translation of capped AMV-4 RNA, which has negligible secondary structure at its 5' end, is resistant to this inhibition. Furthermore, the CBP complex reverses the high salt induced inhibition of translation of the former mRNAs. Since mRNA secondary structure is more stable at elevated salt concentrations, these data are consistent with a model in which the CBP complex has a role in melting mRNA secondary structure involving 5'-proximal sequences, to facilitate ribosome binding.     

Selective inactivation of rabbit reticulocyte initiation factor eIF-3 by helenalin

Helenalin, a sesquiterpene lactone which reacts primarily with exposed sulfhydryl groups, was shown to be an effective inhibitor of protein synthesis in rabbit reticulocyte lysates. Optimal inhibition required a 30 min preincubation in the absence of any added thiol compound. β-Mercaptoethanol was more effective than reduced glutathione in protecting enzyme sulfhydryl groups from inactivation by helenalin. Using partially fractionated systems, it was possible to show that helenalin had no effect on the elongation reactions or on the formation of the ternary initiation complex. However, the conversion of the ternary complex to the 48 S initiation complex was strongly inhibited. In this assay, only the initiation factor(s) were sensitive to helenalin. Using an assay system which requires all the initiation factors for optimal activity it was possible to show that the 0–40% ammonium sulfate cut of intiation factors (containing eIF-3 and eIF-4B) was sensitive to helenalin, while the 40–50% ammonium sulfate cut (containing eIF-2 and eIF-5) was not. Both ammonium sulfate cuts were equally sensitive to inhibition by the sulfhydryl reagent N-ethylmaleimide. Three purified rabbit reticulocyte initiation factors were then tested in the same assay system. Only eIF-3 showed appreciable sensitivity to helenalin, while eIF-2, eIF-3 and eIF-4B were all sensitive to inactivation by N-ethylmaleimide. These data suggest that helenalin may possess a relatively high degree of specificity as a sulfhydryl reagent.     

Eukaryotic protein synthesis initiation factor eIF-3: determination of concentration and association with ribosomes in rabbit reticulocyte and HeLa cell lysates

Monoclonal and polyclonal antibodies against eukaryotic protein synthesis initiation factor eIF-3 were produced and used to determine the factor concentration and its association with ribosomes in rabbit reticulocyte and HeLa cell lysates. In rabbit reticulocyte lysate we found 3-5 micrograms eIF-3 per mg total protein and in HeLa cell lysate 8-15 micrograms eIF-3 per mg total protein. The initiation factor eIF-3 was found both associated with 40 S ribosomal subunits and free in the post-ribosomal supernatant. However, no eIF-3 could be detected on mono- or polyribosomes.     

Variations in cap-binding complexes from uninfected and poliovirus-infected HeLa cells

Poliovirus infection of HeLa cells results in cleavage of the p220 subunit of eukaryotic initiation factor eIF-4F and inhibits cap-dependent initiation of protein synthesis. To examine the effect of virus-induced inhibition on the structure of initiation factor complexes involved in cap binding, the polypeptide compositions of cap affinity-purified complexes from uninfected and poliovirus-infected HeLa cells were analyzed. Monoclonal antibodies directed against p220 and an eIF-3 subunit, p170, were utilized to locate eIF-3 and eIF-4F on sucrose gradients and in fractions eluting from cap analog columns. This approach resulted in the purification of several different cap-binding complexes from different cellular subfractions and revealed significant differences in their composition after infection. The results indicate that eIF-3 and eIF-4F bind to the cap structure, possibly in the form of a complex, and that a modified form of eIF-3 alone has some cap-binding activity in the complete absence of p220, eIF-4A, and eIF-4E. Ribosome-derived complexes containing cleaved p220 are no longer associated with eIF-3 or eIF-4A, and a significant amount of cleaved p220 is associated with a unique cytoplasmic cap-binding complex. The cytoplasmic complex also contains Mr = 170,000 and 80,000 polypeptides, neither of which are major components of eIF-4F. These results demonstrate significant variation in the composition of cap-binding complexes from both infected and uninfected cells. They indicate that eIF-3 might play a direct role in cap binding and suggest that poliovirus-induced cleavage of p220 results in the release of the eIF-4A subunit from eIF-4F and abolishes an association between eIF-4F and eIF-3 which may function during the multifactor steps involved in initiation of cap-mediated translation.     

Catalytic utilization of eIF-2 and mRNA binding proteins are limiting in lysates from vesicular stomatitis virus infected L cells

Infection of mouse L cells by vesicular stomatitis virus results in the inhibition of cellular protein synthesis. Lysates prepared from these infected cells are impaired in their ability to translate endogenous or exogenous cellular and viral mRNAs. The ability of initiation factors from rabbit reticulocytes to stimulate protein synthesis in these lysates was examined. Preparations of eukaryotic initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) stimulated protein synthesis strongly in L cell lysates from infected cells but only slightly in lysates from mock-infected cells. Maximal stimulation was obtained when a fraction containing eukaryotic initiation factors 4B (eIF-4B) and 4F (eIF-4F) was also present. In lysates from infected cells, these initiation factors increased endogenous cellular mRNA translation on the average 2-fold. In contrast, endogenous viral mRNA translation was increased to a much greater extent: the M protein was stimulated 8-fold, NS 5-fold, N 2.5-fold, and G 12-fold. When fractions containing eIF-4B, eIF-4F, or eIF-4A were added to these lysates in the presence of eIF-2, all three stimulated translation. Fractions containing rabbit reticulocyte initiation factors eIF-3 and eIF-6 had no effect on translation in either lysate. The results suggest that lysates from infected L cells are defective in the catalytic utilization of eIF-2 and deficient in mRNA binding protein activity.     

Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II

Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.     

Cloning of eukaryotic protein synthesis initiation factor genes: isolation and characterization of cDNA clones encoding factor eIF-4A.

Monoclonal antibodies directed against rabbit reticulocyte protein synthesis initiation factor 4A (eIF-4A) were used to isolate mouse cDNA clones expressing eIF-4A protein sequences in E. coli. The identity of cDNA clones encoding eIF-4A sequences was confirmed by hybrid-selected translation and peptide mapping of the translation product. Analysis of the mRNA coding for eIF-4A from mouse liver and HeLa cells by Northern hybridization revealed two discrete mRNA species of approximately 2000 and 1600 nucleotides in length. The existence of two mRNAs in mouse and HeLa cells encoding eIF-4A was confirmed by cDNA sequencing.     

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.