Differential stimulation of phosphorylation of initiation factors eIF-4F, eIF-4B, eIF-3, and ribosomal protein S6 by insulin and phorbol esters

Exposure of quiescent, serum-starved 3T3-L1 cells to insulin promotes phosphorylation of initiation factors eIF-4F, eIF-4B, and eIF-3 p120, as well as ribosomal protein S6. Phosphorylation of both the p25 and p220 subunits of eIF-4F is stimulated typically by 2.5-5-fold, with a 2-4-fold increase in phosphorylation of eIF-4B and eIF-3 p120. Optimal stimulation is observed by 10(-9) M insulin. A similar pattern of stimulation is seen upon treatment of 3T3-L1 cells with 1 x 10(-6) M phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide mapping of p25, isolated from quiescent, insulin- or PMA-stimulated cells, results in a single tryptic phosphopeptide, indicating a single phosphorylation site identical to that obtained with protein kinase C. A more complex phosphopeptide map is observed with the p220 subunit. Following PMA-stimulation of 3T3-L1 cells, phosphopeptide mapping of p220 results in a pattern similar to that observed in vitro with Ca2+/phospholipid-dependent protein kinase (protein kinase C). Following insulin stimulation, mapping of p220 results in the appearance of novel peptides. Upon prolonged exposure to PMA, the cells are no longer responsive to this mitogen and no stimulation of phosphorylation of eIF-4F, eIF-4b, eIF-3 p120, or S6 via a protein kinase C-dependent mechanism is observed. Addition of insulin to these down-regulated cells leads to stimulation of phosphorylation of eIF-4F p220, ribosomal protein S6, and to a lesser extent, eIF-4B; little or no stimulation of phosphorylation of eIF-4F p25 and eIF-3 p120 is observed. Thus, eIF-4F p220, eIF-4B and ribosomal protein S6 are phosphorylated via PMA-dependent and insulin-dependent pathways, whereas phosphorylation of eIF-4F p25 and eIF-3 p120 is stimulated only upon activation of protein kinase C. Phosphopeptide maps of eIF-4F p220 and ribosomal protein S6 suggest that protease-activated kinase II is one of the protein kinases involved in the insulin-stimulated response in protein kinase C-depleted cells.     

Epidermal growth factor or okadaic acid stimulates phosphorylation of eukaryotic initiation factor 4F

Eukaryotic initiation factor 4F, a multi-protein mRNA cap binding complex, was isolated by m7GTP-Sepharose affinity chromatography from human mammary epithelial cells (184A1N4) incubated with [32P] orthophosphate. Treatment of cells with epidermal growth factor resulted in enhanced phosphorylation of both p28 (eIF-4E) and p220 subunits. The identities of the p28 and p220 subunits were confirmed by immunoprecipitation. The phosphorylation was both rapid and sustained in duration; p28 attained maximal levels (2-3-fold) within 30 min of treatment and remained elevated for at least 2 h, while p220 reached one-half maximal levels by 30 min, and maximal levels (3-4-fold) by 2 h of treatment. Two phosphorylated isoforms of p28 and multiple phosphorylated forms of p220 were detected by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of 6 N HCl hydrolyzates of p28 and p220 isolated from epidermal growth factor-treated and control cells indicated that serine is the predominant phosphorylated amino acid in both instances. In no case was phosphotyrosine observed. Pretreatment of cells with 1 microM okadaic acid resulted in the hyperphosphorylation of both p28 and p220 subunits. These results suggest that mitogenic growth factors and cellular serine/threonine phosphatases (pp1 and/or pp2A) serve essential roles in regulating phosphorylation levels of eukaryotic initiation factor 4F and support the concept that translational control is a component of the signal transduction mechanisms involved in growth regulation.     

Plant cap-binding complexes eukaryotic initiation factors eIF4F and eIFISO4F: molecular specificity of subunit binding

The initiation of translation in eukaryotes requires a suite of eIFs that include the cap-binding complex, eIF4F. eIF4F is comprised of the subunits eIF4G and eIF4E and often the helicase, eIF4A. The eIF4G subunit serves as an assembly point for other initiation factors, whereas eIF4E binds to the 7-methyl guanosine cap of mRNA. Plants have an isozyme form of eIF4F (eIFiso4F) with comparable subunits, eIFiso4E and eIFiso4G. Plant eIF4A is very loosely associated with the plant cap-binding complexes. The specificity of interaction of the individual subunits of the two complexes was previously unknown. To address this issue, mixed complexes (eIF4E-eIFiso4G or eIFiso4E-eIF4G) were expressed and purified from Escherichia coli for biochemical analysis. The activity of the mixed complexes in in vitro translation assays correlated with the large subunit of the respective correct complex. These results suggest that the eIF4G or eIFiso4G subunits influence translational efficiency more than the cap-binding subunits. The translation assays also showed varying responses of the mRNA templates to eIF4F or eIFiso4F, suggesting that some level of mRNA discrimination is possible. The dissociation constants for the correct complexes have K(D) values in the subnanomolar range, whereas the mixed complexes were found to have K(D) values in the ～10 nm range. Displacement assays showed that the correct binding partner readily displaces the incorrect binding partner in a manner consistent with the difference in K(D) values. These results show molecular specificity for the formation of plant eIF4F and eIFiso4F complexes and suggest a role in mRNA discrimination during initiation of translation.     

RNA unwinding in translation: assembly of helicase complex intermediates comprising eukaryotic initiation factors eIF-4F and eIF-4B.

Ribosome binding to mRNA requires the concerted action of three initiation factors, eIF-4A, eIF-4B, and eIF-4F, and the hydrolysis of ATP in a mechanism that is not well understood. Several lines of evidence support a model by which these factors bind to the 5' end of mRNA and unwind proximal secondary structure, thus allowing 40S ribosomal subunits to bind. We have previously used an unwinding assay to demonstrate that eIF-4A or eIF-4F in combination with eIF-4B functions as an RNA helicase. To elucidate the molecular mechanism of RNA unwinding, we used a mobility shift electrophoresis assay which allows the simultaneous analysis of unwinding and complex formation between these factors and RNA. eIF-4F forms a stable complex (complex A) with duplex RNA in the absence of ATP. Addition of eIF-4B results in the formation of a second complex (complex B) of slower mobility in the gel. In the presence of ATP, both complexes dissociate, concomitant with the unwinding of the duplex RNA. We present evidence to suggest that unwinding occurs in a processive as opposed to distributive manner. Thus, we conclude that helicase complexes that are formed in the absence of ATP on duplex RNA translocate processively along the RNA in an ATP-dependent reaction and melt secondary structure. These helicase complexes therefore represent intermediates in the unwinding process of mRNA that could precede ribosome binding.     

Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI

The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C-terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide-3-kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12-rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N-terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C-terminus to kinases is modulated by this pathway(s).     

Binding of inosine-substituted mRNA to reticulocyte ribosomes and eukaryotic initiation factors 4A and 4B requires ATP

We examined the hypothesis that initiation of eukaryotic protein synthesis involves ATP-dependent melting of 5'-cap-proximal secondary structure in mRNA by eukaryotic initiation factors 4A and 4B. In reticulocyte lysate depleted of ribonucleoside triphosphates by pretreatment with hexokinase/glucose, initiation complex formation by native reovirus mRNA showed a strict requirement for ATP. The corresponding mRNA synthesized with ITP in place of GTP to minimize secondary structure also required ATP for binding to 40 S ribosomal subunits in complexes characteristic of initiation. In a partial reaction without ribosomes, purified eukaryotic initiation factors 4A and 4B bound and cross-linked to the capped 5'-end of oxidized mRNA. This interaction was ATP-dependent with inosine-substituted or bromouridine-containing reovirus RNAs as observed previously with native mRNA. The results indicate that if initiation involves ATP-dependent denaturation of mRNA, the effect must occur after initiation factor-mediated attachment of mRNA to the 40 S ribosomal subunit.     

mRNP proteins,initiation factors and phosphorylation

Information has been collected to stimulate interest regarding the nature and the possible role of mRNP protein phosphorylation in a cytoplasmic control mechanism for protein synthesis. It does not imply a direct relationship between mRNP protein and initiation factors. These proteins have some properties in common (e.g. molecular weight, phosphorylation, protein kinase, mRNA binding activity). We emphasize that some free mRNP may be translatable after modification of their protein by interference factors belonging to other cellular compartments. Thus, some mRNP proteins might possess initiation factor or protein synthetic activity if we accept Spirin's theory, i.e., Eukaryotic messenger RNA and informosomes omnia mea mecum porto     

Phorbol esters stimulate cyclic adenosine 3', 5'-monophosphate accumulation in hamster spermatozoa during in vitro capacitation

Phorbol ester, phorbol 12-myristate 13-acetate (PMA), induced a 20- to 50-fold increase (ED50: 2 microM) in cyclic adenosine 3', 5'-monophosphate (cAMP) levels in spermatozoa incubated in capacitation medium for short periods of time (30 min). Similar results were obtained with 1-oleoyl 2-acetylglycerol (OAG), whereas 1, 2 diolein, 1-oleoyl glycerol, or 4 alpha-phorbol 12, 13-didecanoate had no effect. When extracellular Ca2+ was complexed by [ethylenebis(oxyethyleneitrilo)] tetraacetic acid (EGTA), a 50% reduction of maximal stimulation was observed, and 90% inhibition was seen after chelation of both extra- and intracellular Ca2+ with EGTA and 2-[[2-[bis [(carbonyl) methyl] amino]-5-methylphenoxy] methyl]-6-methoxy-8-[bis[(carbonyl) methyl] amino] quinoline acetoxy methyl (Quin 2). The acrosome reaction was not affected by similar concentration of PMA or OAG at different periods of incubation. These results suggest the involvement of protein kinase C activity in the regulation of cAMP levels in sperm during capacitation. This stimulation is dependent on intracellular Ca2+ and probably is not linked to the process of the acrosome reaction.     

The phosphorylation state of poly(A)-binding protein specifies its binding to poly(A) RNA and its interaction with eukaryotic initiation factor (eIF) 4F, eIFiso4F, and eIF4B

The poly(A)-binding protein (PABP) interacts with the eukaryotic initiation factor (eIF) 4G (or eIFiso4G), the large subunit of eIF4F (or eIFiso4F) to promote translation initiation. In plants, PABP also interacts with eIF4B, a factor that assists eIF4F function. PABP is a phosphoprotein, although the function of its phosphorylation has not been previously investigated. In this study, we have purified the phosphorylated and hypophosphorylated isoforms of PABP from wheat to examine whether its phosphorylation state affects its binding to poly(A) RNA and its interaction with eIF4G, eIFiso4G, or eIF4B. Phosphorylated PABP exhibited cooperative binding to poly(A) RNA even under non-stoichiometric binding conditions, whereas multiple molecules of hypophosphorylated PABP bound to poly(A) RNA only after free poly(A) RNA was no longer available. Together, phosphorylated and hypophosphorylated PABP exhibited synergistic binding. eIF4B interacted with PABP in a phosphorylation state-specific manner; native eIF4B increased the RNA binding activity specifically of phosphorylated PABP and was greater than 14-fold more effective than was recombinant eIF4B, whereas eIF4F promoted the cooperative binding of hypophosphorylated PABP. These data suggest that the phosphorylation state of PABP specifies the type of binding to poly(A) RNA and its interaction with its partner proteins.     

Protein synthesis eukaryotic initiation factors 4A and 4B are not altered by poliovirus infection of HeLa cells

Infection of HeLa cells by poliovirus results in the inhibition of translation of capped cellular mRNA. A plausible mechanism for this inhibition is that the structure of one or more initiation factors involved in the recognition of capped mRNA is altered. Eukaryotic initiation factor (eIF) 4A and eIF-4B are implicated in mRNA binding to 40 S ribosomal subunits and can be cross-linked to oxidized capped mRNA. We examined these factors in HeLa cell lysates by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. No alterations in the number of molecules/cell, in the molecular size, or in extents of covalent modification were detected when lysates from infected and mock-infected cells were compared. The integrity of eIF-2 and several eIF-3 polypeptides was also examined and likewise no alterations were detected. The failure of the translational machinery to recognize capped mRNA therefore is not due to a change in the structure of these initiation factors.     

Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase.

Insulin rapidly stimulates protein synthesis in a wide variety of tissues. This stimulation is associated with phosphorylation of several translational initiation and elongation factors, but little is known about the signaling pathways to these events. To study these pathways, we have used a myeloid progenitor cell line (32D) which is dependent on interleukin 3 but insensitive to insulin because of the very low levels of insulin receptor (IR) and the complete lack of insulin receptor substrate (IRS)-signaling proteins (IRS-1 and IRS-2). Expression of more IR permits partial stimulation of mitogen-activated protein kinase by insulin, and expression of IRS-1 alone mediates insulin stimulation of the 70-kDa S6 kinase (pp70S6K) by the endogenous IR. However, expression of both IR and IRS-1 is required for stimulation of protein synthesis. Moreover, this effect requires activation of phosphatidylinositol 3-kinase (PI3K), as determined by wortmannin inhibition and the use of an IRS-1 variant lacking all Tyr residues except those which activate PI3K. Stimulation of general protein synthesis does not involve activation by IRS-1 of GRB-2-SOS-p21ras or SH-PTP2, since IRS-1 variants lacking the SH2-binding Tyr residues for these proteins are fully active. Nor does it involve pp70S6K, since rapamycin, while strongly inhibiting the synthesis of a small subset of growth-regulated proteins, only slightly inhibits total protein synthesis. Recruitment of mRNAs to the ribosome is enhanced by phosphorylation of eIF4E, the cap-binding protein, and PHAS-I, a protein that specifically binds eIF4E. The behavior of cell lines containing IRS-1 variants and inhibition by wortmannin and rapamycin indicate that the phosphorylation of both proteins requires IRS-1-mediated stimulation of PI3K and pp70S6K but not mitogen-activated protein kinase or SH-PTP2.     

Inhibition of mammalian target of rapamycin induces phosphatidylinositol 3-kinase-dependent and Mnk-mediated eukaryotic translation initiation factor 4E phosphorylation

The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its entry into initiation complexes. Mammalian target of rapamycin (mTOR) inhibitors suppress cap-dependent translation through inhibition of the phosphorylation of eIF4E-binding protein 1. Paradoxically, we have shown that inhibition of mTOR signaling increases eIF4E phosphorylation in human cancer cells. In this study, we focused on revealing the mechanism by which mTOR inhibition increases eIF4E phosphorylation. Silencing of either mTOR or raptor could mimic mTOR inhibitors' effects to increase eIF4E phosphorylation. Moreover, knockdown of mTOR, but not rictor or p70S6K, abrogated rapamycin's ability to increase eIF4E phosphorylation. These results indicate that mTOR inhibitor-induced eIF4E phosphorylation is secondary to mTOR/raptor inhibition and independent of p70S6K. Importantly, mTOR inhibitors lost their ability to increase eIF4E phosphorylation only in cells where both Mnk1 and Mnk2 were knocked out, indicating that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. Given that mTOR inhibitors failed to increase Mnk and eIF4E phosphorylation in phosphatidylinositol 3-kinase (PI3K)-deficient cells, we conclude that mTOR inhibition increases eIF4E phosphorylation through a PI3K-dependent and Mnk-mediated mechanism. In addition, we also suggest an effective therapeutic strategy for enhancing mTOR-targeted cancer therapy by cotargeting mTOR signaling and Mnk/eIF4E phosphorylation.     

Phorbol esters stimulate the transport of anionic amino acids in cultured human fibroblasts

The effect of phorbol esters on the transport of amino acids has been evaluated in cultured human fibroblasts. The activity of the Na(+)-dependent system XAG- for anionic amino acids is selectively and markedly stimulated by phorbol esters. The effect is maximal within 15 min; it is attributable to an increase in transport maximum (Vmax) and not prevented by inhibitors of protein synthesis. The half-maximal stimulation is observed at concentrations of phorbol 12,13-dibutyrate lower than 100 nM. Prolonged incubations in the presence of 1 microM phorbol 12,13-dibutyrate lower the binding of the ligand to its receptor with a loss of the stimulatory effect on transport. The results presented indicate that the stimulation of amino acid transport through system XAG- by phorbol esters requires the activation of protein kinase C.     

Selective modification of eukaryotic initiation factor 4F (eIF4F) at the onset of cell differentiation: recruitment of eIF4GII and long-lasting phosphorylation of eIF4E

mRNA translation is mainly regulated at the level of initiation, a process that involves the synergistic action of the 5' cap structure and the 3' poly(A) tail at the ends of eukaryotic mRNA. The eukaryote initiation factor 4G(eIF4G) is a pivotal scaffold protein that forms a critical link between mRNA cap structure, poly(A) tail, and the small ribosomal subunit. There are two functional homologs of eIF4G in mammals, the original eIF4G, renamed eIF4GI, and eIF4GII that functionally complements eIF4GI. To date, biochemical and functional analysis have not identified differential activities for eIF4GI and eIF4GII. In this report, we demonstrate that eIF4GII, but not eIF4GI, is selectively recruited to capped mRNA at the onset of cell differentiation. This recruitment is coincident with a strong and long-lasting phosphorylation of eIF4E and the release of 4E-BP1, a suppressor of eIF4E function, from the cap structure, without a concomitant change in 4E-BP1's phosphorylation. Our data further indicate that cytokines such as thrombopoietin can differentially regulate eIF4GI/II activities. These results provide the first evidence that eIF4GI/II does fulfill selective roles in mammalian cells.     

Discriminatory interaction of purified eukaryotic initiation factors 4F plus 4A with the 5' ends of reovirus messenger RNAs

The interaction of several reovirus mRNAs with cap-binding initiation factors has been investigated. Two quantitative experimental techniques have been applied to this question: (a) the rates of reaction of different mRNAs with tobacco acid pyrophosphatase and (b) the extent of cross-linking of different mRNAs to initiation factors in the presence and absence of ATP. The effects of ionic strength on these reactions have also been investigated. Our results demonstrate for the first time that the purified initiation factors interact differentially with purified reovirus mRNAs under competitive conditions and thus confirm earlier interpretations based on kinetic data. Comparison of the data from these studies with the translational behavior of the reovirus mRNAs, both in vitro and in vivo, has also led to specific predictions about features of these mRNAs that determine their competitive efficiencies. 1) Under ordinary ionic conditions, the steric accessibility of the m7G cap moiety of a reovirus mRNA appears to be a major determinant of its translation rate. 2) When the ionic strength is increased to supranormal levels, an additional feature, which may simply be the amount of secondary structure formed by sequences proximal to the cap, can become rate-limiting for several, but not all, of these mRNAs.     

Hypoxia enhances phosphorylation of eukaryotic initiation factor 4A in maize root tips.

We have identified two isoforms of initiation factor 4A (eIF-4A) in maize root tips, with distinct isoelectric points and similar molecular mass (approximately 50 kDa). Both isoforms of maize eIF-4A cross-react with antibodies raised against wheat germ eIF-4A, and one of the maize proteins (higher pI isoform) comigrates with purified wheat germ eIF-4A on two-dimensional gels. The two maize eIF-4As were indistinguishable by comparative peptide fingerprint analysis, which also showed a very strong similarity between eIF-4A in maize roots and wheat germ. Maize eIF-4As copurify with eIF-4F and eIF-(iso)4F on a 7-methyl-GTP-Sepharose affinity column, indicating that they are part of the 5'-cap-binding complex. Two-dimensional gel electrophoresis and immunoblotting of proteins from 32P-labeled maize root tips revealed that the lower pI isoform of eIF-4A is phosphorylated. Two-dimensional phosphopeptide maps of trypsin-digested eIF-4A contained one principal phosphorylated fragment; phosphoamino acid analysis indicated phosphorylation of threonine. In oxygenated maize root tips, the ratio of phosphorylated to nonphosphorylated eIF-4A is approximately 0.2. This ratio increases to approximately 1 within 20 min following the onset of hypoxia, due to interconversion between the two maize eIF-4A isoforms. The hypoxia-induced phosphorylation of eIF-4A is discussed with respect to metabolic responses, and the translational control of gene expression, in hypoxic plant tissues.     

Interaction of wheat germ protein synthesis initiation factors eIF-3, eIF-(iso)4F, and eIF-4F with mRNA analogues

The interaction of wheat germ eIF-3 with the wheat germ cap-binding proteins eIF-(iso)4F and eIF-4F as a function of pH and ionic strength is described. Direct fluorescence titration experiments are used to measure the equilibrium association constants (Keq) for the binary protein/protein complexes as well as for the interaction of eIF-3 with methylated cap analogues and rabbit alpha-globin mRNA oligonucleotide analogues. The Keq values for ternary eIF-3/eIF-(iso)4F/analogue and eIF-3/eIF-4F/analogue interactions were also measured. The equilibrium binding constants were used to calculate coupling free energies, which provide an estimate of the cooperativity for the interaction of the mRNA analogues, eIF-3, and either eIF-4F or eIF-(iso)4F. These data suggest a mechanism in which the binding of eIF-(iso)4F or eIF-4F to mRNA enhances the subsequent binding of eIF-3 to the message. This may lead to favorable positioning of the complex on the ribosome and thereby enhance translation.     

Modification of eukaryotic initiation factor 4F during infection by influenza virus.

Influenza virus infection of cells is accompanied by a striking shutoff of cellular protein synthesis, resulting in the exclusive translation of viral mRNAs. The mechanism for control of cellular protein synthesis by influenza virus is poorly understood, but several translation properties of influenza virus mRNAs which are potentially involved have been described. Influenza virus mRNAs possess the surprising ability to translate in the presence of inhibitory levels of inactive (phosphorylated) eukaryotic initiation factor 2 (eIF-2). In addition, influenza virus mRNAs were shown to be capable of translating in cells during the late phase of adenovirus infection but not in cells infected by poliovirus. Since both adenovirus and poliovirus facilitate virus-specific translation by impairing the activity of initiation factor eIF-4F (cap-binding protein complex) but through different mechanisms, we investigated the translation properties of influenza virus mRNAs in more detail. We show that influenza virus infection is associated with the significant dephosphorylation and inactivation of eIF-4E (cap-binding protein), a component of eIF-4F, and accordingly that influenza virus mRNAs possess a moderate ability to translate by using low levels of eIF-4F. We also confirm the ability of influenza virus mRNAs to translate in the presence of high levels of inactive (phosphorylated) eIF-2 but to a more limited extent than reported previously. We suggest a potential mechanism for the regulation of protein synthesis by influenza virus involving a decreased requirement for large pools of active eIF-4F and eIF-2.     

Granzyme B inhibits vaccinia virus production through proteolytic cleavage of eukaryotic initiation factor 4 gamma 3

Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD(1408)S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.