L protease from foot and mouth disease virus confers eIF2-independent translation for mRNAs bearing picornavirus IRES

The leader protease (L^pro) from foot-and-mouth disease virus (FMDV) has the ability to cleave eIF4G, leading to a blockade of cellular protein synthesis. In contrast to previous reports, our present findings demonstrate that FMDV L^pro is able to increase translation driven by FMDV IRES. Additionally, inactivation of eIF2 subsequent to phosphorylation induced by arsenite or thapsigargin in BHK cells blocks protein synthesis directed by FMDV IRES, whereas in the presence of L^pro, significant translation is found under these conditions. This phenomenon was also observed in cell-free systems after induction of eIF2 phosphorylation by addition of poly(I:C).     

Translation Driven by Picornavirus IRES Is Hampered from Sindbis Virus Replicons: Rescue by Poliovirus 2A Protease

Alphavirus replicons are very useful for analyzing different aspects of viral molecular biology. They are also useful tools in the development of new vaccines and highly efficient expression of heterologous genes. We have investigated the translatability of Sindbis virus (SV) subgenomic mRNA bearing different 5′-untranslated regions, including several viral internal ribosome entry sites (IRESs) from picornaviruses, hepatitis C virus, and cricket paralysis virus. Our findings indicate that all these IRES-containing mRNAs are initially translated in culture cells transfected with the corresponding SV replicon but their translation is inhibited in the late phase of SV replication. Notably, co-expression of different poliovirus (PV) non-structural genes reveals that the protease 2A (2A^pro) is able to increase translation of subgenomic mRNAs containing the PV or encephalomyocarditis virus IRESs but not of those of hepatitis C virus or cricket paralysis virus. A PV 2A^pro variant deficient in eukaryotic initiation factor (eIF) 4GI cleavage or PV protease 3C, neither of which cleaves eIF4GI, does not increase picornavirus IRES-driven translation, whereas L protease from foot-and-mouth disease virus also rescues translation. These findings suggest that the replicative foci of SV-infected cells where translation takes place are deficient in components necessary to translate IRES-containing mRNAs. In the case of picornavirus IRESs, cleavage of eIF4GI accomplished by PV 2A^pro or foot-and-mouth disease virus protease L rescues this inhibition. eIF4GI co-localizes with ribosomes both in cells electroporated with SV replicons bearing the picornavirus IRES and in cells co-electroporated with replicons that express PV 2A^pro. These findings support the idea that eIF4GI cleavage is necessary to rescue the translation driven by picornavirus IRESs in baby hamster kidney cells that express SV replicons.     

Detailed analysis of the requirements of hepatitis A virus internal ribosome entry segment for the eukaryotic initiation factor complex eIF4F

The hepatitis A virus (HAV) internal ribosome entry segment (IRES) is unique among the picornavirus IRESs in that it is inactive in the presence of either the entero- and rhinovirus 2A or aphthovirus Lb proteinases. Since these proteinases both cleave eukaryotic initiation factor 4G (eIF4G) and HAV IRES activity could be rescued in vitro by addition of eIF4F to proteinase-treated extracts, it was concluded that the HAV IRES requires eIF4F containing intact eIF4G. Here, we show that the inability of the HAV IRES to function with cleaved eIF4G cannot be attributed to inefficient binding of the cleaved form of eIF4G by the HAV IRES. Indeed, the binding of both intact eIF4F and the C-terminal cleavage product of eIF4G to the HAV IRES was virtually indistinguishable from their binding to the encephalomyocarditis virus IRES, as assessed by UV cross-linking and filter retention assays. Rather, we show that HAV IRES activity requires, either directly or indirectly, components of the eIF4F complex which interact with the N-terminal fragment of eIF4G. Effectively, HAV IRES activity, but not that of the human rhinovirus IRES, was sensitive to the rotavirus nonstructural protein NSP3 [which displaces poly(A)-binding protein from the eIF4F complex], to recombinant eIF4E-binding protein (which prevents the association of the cap binding protein eIF4E with eIF4G), and to cap analogue.     

Activity of the hepatitis A virus IRES requires association between the cap-binding translation initiation factor (eIF4E) and eIF4G

The question of whether translation initiation factor eIF4E and the complete eIF4G polypeptide are required for initiation dependent on the IRES (internal ribosome entry site) of hepatitis A virus (HAV) has been examined using in vitro translation in standard and eIF4G-depleted rabbit reticulocyte lysates. In agreement with previous publications, the HAV IRES is unique among all picornavirus IRESs in that it was inhibited if translation initiation factor eIF4G was cleaved by foot-and-mouth disease L-proteases. In addition, the HAV IRES was inhibited by addition of eIF4E-binding protein 1, which binds tightly to eIF4E and sequesters it, thus preventing its association with eIF4G. The HAV IRES was also inhibited by addition of m(7)GpppG cap analogue, irrespective of whether the RNA tested was capped or not. Thus, initiation on the HAV IRES requires that eIF4E be associated with eIF4G and that the cap-binding pocket of eIF4E be empty and unoccupied. This suggests two alternative models: (i) initiation requires a direct interaction between an internal site in the IRES and eIF4E/4G, an interaction which involves the cap-binding pocket of eIF4E in addition to any direct eIF4G-RNA interactions; or (ii) it requires eIF4G in a particular conformation which can be attained only if eIF4E is bound to it, with the cap-binding pocket of the eIF4E unoccupied.     

IRES interaction with translation initiation factors: functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII

Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.     

The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule.     

Poly(A) binding protein, C-terminally truncated by the hepatitis A virus proteinase 3C, inhibits viral translation

Proteolytic cleavage of translation initiation factors is a means to interfere with mRNA circularization and to induce translation arrest during picornaviral replication or apoptosis. It was shown that the regulated cleavages of eukaryotic initiation factor (eIF) 4G and poly(A)-binding protein (PABP) by viral proteinases correlated with early and late arrest of host cap-dependent and viral internal ribosome entry site (IRES)-dependent translation, respectively. Here we show that in contrast to coxsackievirus, eIF4G is not a substrate of proteinase 3C of hepatitis A virus (HAV 3C^pro). However, PABP is cleaved by HAV 3C^pro in vitro and in vivo, separating the N-terminal RNA-binding domain (NTD) of PABP from the C-terminal protein-interaction domain. In vitro, NTD has a dominant negative effect on HAV IRES-dependent translation and an enhanced binding affinity to the RNA structural element pY1 in the 5′ nontranslated region of the HAV RNA that is essential for viral genome replication. The results point to a regulatory role of PABP cleavage in RNA template switching of viral translation to RNA synthesis.     

Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation

Translation of most eukaryotic mRNAs involves the synergistic action between the 5′ cap structure and the 3′ poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.     

Visualizing and quantifying the differential cleavages of the eukaryotic translation initiation factors eIF4GI and eIF4GII in the enterovirus‐infected cell

Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A^pro). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET‐based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP²) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real‐time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A^pro catalytic activity, irrespective of other viral‐encoded protease, the activated caspases or general inhibition of protein synthesis in the EV‐infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease–substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus‐induced host translation inhibition. Biotechnol. Bioeng. 2009; 104: 1142–1152. © 2009 Wiley Periodicals, Inc.     

A novel protein-RNA binding assay: functional interactions of the foot-and-mouth disease virus internal ribosome entry site with cellular proteins

Translation initiation on foot-and-mouth disease virus (FMDV) RNA occurs by a cap-independent mechanism directed by a highly structured element (approximately 435 nt) termed an internal ribosome entry site (IRES). A functional assay to identify proteins that bind to the FMDV IRES and are necessary for FMDV IRES-mediated translation initiation has been developed. In vitro-transcribed polyadenylated RNAs corresponding to the whole or part of the FMDV IRES were immobilized on oligo-dT Dynabeads and used to deplete rabbit reticulocyte lysate (RRL) of IRES-binding proteins. Translation initiation factors eIF4G, eIF4A, and eIF4B bound to the 3' domain of the FMDV IRES. Depletion of eIF4G from RRL by this region of the FMDV IRES correlated with the loss of translational capacity of the RRL for capped, uncapped, and FMDV IRES-dependent mRNAs. However, this depleted RRL still supported hepatitis C virus IRES-directed translation. Poly (rC) binding protein-2 bound to the central domain of the FMDV IRES, but depletion of RRL with this IRES domain had no effect on FMDV IRES-directed translation initiation.     

Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors

Type 2 internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and other picornaviruses comprise five major domains H-L. Initiation of translation on these IRESs begins with specific binding of the central domain of initiation factor, eIF4G to the J-K domains, which is stimulated by eIF4A. eIF4G/eIF4A then restructure the region of ribosomal attachment on the IRES and promote recruitment of ribosomal 43S pre-initiation complexes. In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45). To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G. The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.     

Proteomic elucidation of the targets and primary functions of the picornavirus 2A protease

Picornaviruses are small RNA viruses that hijack host cell machinery to promote their replication. During infection, these viruses express two proteases, 2A^pro and 3C^pro, which process viral proteins. They also subvert a number of host functions, including innate immune responses, host protein synthesis, and intracellular transport, by utilizing poorly understood mechanisms for rapidly and specifically targeting critical host proteins. Here, we used proteomic tools to characterize 2A^pro interacting partners, functions, and targeting mechanisms. Our data indicate that, initially, 2A^pro primarily targets just two cellular proteins: eukaryotic translation initiation factor eIF4G (a critical component of the protein synthesis machinery) and Nup98 (an essential component of the nuclear pore complex, responsible for nucleocytoplasmic transport). The protease appears to employ two different cleavage mechanisms; it likely interacts with eIF3L, utilizing the eIF3 complex to proteolytically access the eIF4G protein but also directly binds and degrades Nup98. This Nup98 cleavage results in only a marginal effect on nuclear import of proteins, while nuclear export of proteins and mRNAs were more strongly affected. Collectively, our data indicate that 2A^pro selectively inhibits protein translation, key nuclear export pathways, and cellular mRNA localization early in infection to benefit viral replication at the expense of particular cell functions.     

Foot-and-mouth disease virus 3C protease induces cleavage of translation initiation factors eIF4A and eIF4G within infected cells

Infection of cells by foot-and-mouth disease virus (FMDV) results in the rapid inhibition of host cell protein synthesis. This process is accompanied by the early cleavage of the translation initiation factor eIF4G, a component of the cap-binding complex eIF4F. This cleavage is mediated by the leader (L) protease. Subsequently, as the virus proteins accumulate, secondary cleavages of eIF4G occur. Furthermore, eIF4A (46 kDa), a second component of eIF4F, is also cleaved in these later stages of the infection cycle. The 33-kDa cleavage product of eIF4A has lost a fragment from its N terminus. Transient-expression assays demonstrated that eIF4A was not cleaved in the presence of FMDV L or with the poliovirus 2A protease (which also mediates eIF4G cleavage) but was cleaved when the FMDV 3C protease was expressed. The FMDV 3C protease was also shown in such assays to induce cleavage of eIF4G, resulting in the production of cleavage products different from those generated by the L protease. Consistent with these results, within cells infected with a mutant FMDV lacking the L protease or within cells containing an FMDV replicon lacking L-P1 coding sequences it was again shown that eIF4A and eIF4G were cleaved.     

Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo

The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein-RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1-2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3' region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.     

Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.     

Translation without eIF2 promoted by poliovirus 2A protease

Poliovirus RNA utilizes eIF2 for the initiation of translation in cell free systems. Remarkably, we now describe that poliovirus translation takes place at late times of infection when eIF2 is inactivated by phosphorylation. By contrast, translation directed by poliovirus RNA is blocked when eIF2 is inactivated at earlier times. Thus, poliovirus RNA translation exhibits a dual mechanism for the initiation of protein synthesis as regards to the requirement for eIF2. Analysis of individual poliovirus non-structural proteins indicates that the presence of 2A(pro) alone is sufficient to provide eIF2 independence for IRES-driven translation. This effect is not observed with a 2A(pro) variant unable to cleave eIF4G. The level of 2A(pro) synthesized in culture cells is crucial for obtaining eIF2 independence. Expression of the N-or C-terminus fragments of eIF4G did not stimulate IRES-driven translation, nor provide eIF2 independence, consistent with the idea that the presence of 2A(pro) at high concentrations is necessary. The finding that 2A(pro) provides eIF2-independent translation opens a new and unsuspected area of research in the field of picornavirus protein synthesis.     

Interaction of Eukaryotic Initiation Factor eIF4B with the Internal Ribosome Entry Site of Foot-and-Mouth Disease Virus Is Independent of the Polypyrimidine Tract-Binding Protein

Eukaryotic translation initiation factor 4B (eIF4B) binds directly to the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). Mutations in all three subdomains of the IRES stem-loop 4 reduce binding of eIF4B and translation efficiency in parallel, indicating that eIF4B is functionally involved in FMDV translation initiation. In reticulocyte lysate devoid of polypyrimidine tract-binding protein (PTB), eIF4B still bound well to the wild-type IRES, even after removal of the major PTB-binding site. In conclusion, the interaction of eIF4B with the FMDV IRES is essential for IRES function but independent of PTB.     

Differential processing of nuclear pore complex proteins by rhinovirus 2A proteases from different species and serotypes

Human rhinoviruses (HRVs) from the HRV-A, HRV-B, and HRV-C species use encoded proteases, 2A^pro and 3C^pro, to process their polyproteins and shut off host cell activities detrimental to virus replication. Reactions attributed to 2A^pro include cleavage of eIF4G-I and -II to inhibit cellular mRNA translation and cleavage of select nucleoporin proteins (Nups) within nuclear pore complexes (NPCs) to disrupt karyopherin-dependent nuclear-cytoplasmic transport and signaling. Sequence diversity among 2A^pro proteases from different HRV clades, even within species, suggested individual viruses might carry out these processes with unique mechanistic signatures. Six different recombinant 2A^pro proteases (A16, A89, B04, B14, Cw12, and Cw24) were compared for their relative substrate preferences and cleavage kinetics using eIF4G from cellular extracts and Nups presented in native (NPC) or recombinant formats. The enzyme panel attacked these substrates with different rates or processing profiles, mimicking the preferences observed during natural infection (A16 and B14). For eIF4G, all 2A^pro proteases cleaved at similar sites, but the comparative rates were species specific (HRV-A > HRV-C ≫ HRV-B). For Nup substrates, 5 of the 6 enzymes had unique product profiles (order of Nup selection) or reacted at different sites within Nup62, Nup98, and Nup153. Only A16 and A89 behaved similarly in most assays. Since each type of karyopherin receptor prefers particular Nups or uses a limited cohort of binding motifs within those Nups, the consequences of individual 2A^pro avidities could profoundly affect relative viral replication levels, intracellular signaling, or extracellular signaling, all of which are underlying triggers for different host immune responses.     

elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES-containing mRNAs.

Rhinovirus 2A and foot-and-mouth disease virus Lb proteinases stimulate the translation of uncapped messages and those carrying the rhinovirus and enterovirus Internal Ribosome Entry Segments (IRESes) by a mechanism involving the cleavage of host cell proteins. Here, we investigate this mechanism using an artificial dicistronic RNA containing the human rhinovirus IRES as intercistronic spacer. Because both proteinases cleave eukaryotic initiation factor 4G (eIF4G), we examined whether the cleavage products of eIF4G could stimulate uncapped or IRES-driven translation. Addition of intact eIF4F to translation extracts inhibited IRES-driven translation and reduced the translation stimulation observed in reactions pre-treated with Lb proteinase. Prolonged incubation of translation extracts with Lb proteinase removed all endogenous eIF4G and a substantial amount of the primary C- and N-terminal cleavage products. The translation of all mRNAs was reduced in such extracts. Capped mRNA translation was rescued by the addition of intact eIF4F. In contrast, addition of pre-cleaved eIF4F stimulated translation of uncapped or IRES-bearing messages to the levels seen upon proteinase addition. Furthermore, fractions containing the C-terminal, but not N-terminal, cleavage product of eIF4G stimulated translation moderately. These results demonstrate that the Lb and 2A proteinases stimulate translation of uncapped RNAs and those carrying IRESes by the production of cleavage products of eIF4G that enhance translation and by the removal of intact eIF4G that interferes with this stimulation.