Sindbis virus translation is inhibited by a PKR/RNase L-independent effector induced by alpha/beta interferon priming of dendritic cells.

The tropism of Sindbis virus (SB) for cells of the dendritic cell (DC) lineage and the virulence of SB in vivo are largely determined by the efficacy of alpha/beta interferon (IFN-alpha/beta)-mediated antiviral responses. These responses are essentially intact in the absence of PKR and/or RNase L (K. D. Ryman, L. J. White, R. E. Johnston, and W. B. Klimstra, Viral Immunol. 15:53-76, 2002). In the present studies, we investigated the nature of antiviral effects and identity of antiviral effectors primed by IFN-alpha/beta treatment of bone marrow-derived DCs (BMDCs) generated from mice deficient in PKR and RNase L (TD). IFN-alpha/beta priming exerted significant antiviral activity at very early stages of SB replication and most likely inhibited the initial translation of infecting genomes. The early effect targeted cap-dependent translation as protein synthesis from an SB-like and a simple RNA were inhibited by interferon treatment, but an encephalomyocarditis virus internal ribosome entry site-driven element exhibited no inhibition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 was defective after virus infection of TD cells, suggesting other mechanisms of translation inhibition. To identify components of these alternative antiviral pathway(s), we have compared global gene regulation in BMDCs derived from normal 129 Sv/Ev, IFNAR1-/-, and TD mice following infection with SB or treatment with IFN-alpha/beta. Candidate effectors of alternative antiviral pathways were those genes induced by virus infection or IFN-alpha/beta treatment in 129 Sv/Ev and TD-derived BMDC but not in virus-infected or IFN-alpha/beta-treated IFNAR1-/- cells. Statistical analyses of gene array data identified 44 genes that met these criteria which are discussed.

Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG.

The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications. However, Pasquinelli et al. [RNA (1995) 1:957-967] found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3'-5' phosphodiester bond to the first nucleotide residue of the RNA chain. Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs. We therefore designed two novel cap analogs, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-3'-O,7-dimethylguanosine-5' P3-guanosine-5' triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation. The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures were proven by 1H NMR. Transcripts produced with SP6 polymerase using "anti-reverse" cap analogs (ARCAs) were of the predicted length and indistinguishable in size and homogeneity from those produced with m7GpppG or GpppG. Analysis of the transcripts with RNase T2 and tobacco acid pyrophosphatase indicated that reverse caps were formed with m7GpppG but not with ARCAs. Both of the ARCAs inhibited cell-free translation with a K(I) similar to that of m7GpppG. Finally, the translational efficiency of ARCA-capped transcripts in a rabbit reticulocyte lysate was 2.3- to 2.6-fold higher than that of m7GpppG-capped transcripts. This suggests the presence of reverse caps in conventional in vitro-synthesized mRNAs reduces their translational efficiency.

An isoform of eIF4E is a component of germ granules and is required for spermatogenesis in C. elegans.

Control of gene expression at the translational level is crucial for many developmental processes. The mRNA cap-binding protein, eIF4E, is a key player in regulation of translation initiation; appropriate levels of eIF4E are essential for normal cell-cycle regulation and tissue differentiation. The observation that eIF4E levels are elevated during gametogenesis in several organisms suggests that eIF4E might have a specific role in gamete formation as well. We show that one of the five isoforms of C. elegans eIF4E, IFE-1, is enriched in the germline and is a component of germ granules (P granules). The association of IFE-1 with P granules requires the P-granule protein PGL-1. In vitro PGL-1 interacts directly with IFE-1, but not with the other four isoforms of eIF4E. Analysis of animals depleted of IFE-1 by RNAi shows that IFE-1 is required for spermatogenesis, specifically for efficient progression through the meiotic divisions and for the production of functional sperm, in both hermaphrodites and males. The requirement for IFE-1 is highly sensitive to temperature. IFE-1 is not required for oogenesis, as ife-1(RNAi) hermaphrodites produce viable progeny when normal sperm are supplied. Consistent with a primary role in spermatogenesis, ife-1 mRNA levels are highest in regions of the gonad undergoing spermatogenesis. Our results suggest that C. elegans spermatogenesis requires either this specific isoform of eIF4E or an elevated level of eIF4E.

Characterization of the two eIF4A-binding sites on human eIF4G-1.

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1.2 x 10(5) m(-1) s(-1), 2.1 x 10(-3) s(-1), and 17 nm for the central site and 5.1 x 10(3) m(-1) s(-1), 1.7 x 10(-3) s(-1), and 330 nm for the COOH-terminal site.

Nitric oxide inhibits dystrophin proteolysis by coxsackieviral protease 2A through S-nitrosylation: A protective mechanism against enteroviral cardiomyopathy.

BACKGROUND: Infection with enteroviruses like coxsackievirus B3 (CVB3) as well as genetic dystrophin deficiency can cause dilated cardiomyopathy. We recently identified cleavage and functional impairment of dystrophin by the viral protease 2A during CVB3-infection as a molecular mechanism that may contribute to the pathogenesis of enterovirus-induced cardiomyopathy. Nitric oxide (NO) is elevated in human dilated cardiomyopathy, but the relevance of this finding is unknown. In mice, NO inhibits CVB3 myocarditis. Therefore, we investigated the effects of NO on the coxsackieviral protease 2A. METHODS AND RESULTS: In vitro, NO donors like PAPA-NONOate inhibited the cleavage of human and mouse dystrophin by recombinant coxsackievirus B protease 2A in a dose-dependent manner (IC(50), 51 micromol/L). In CVB3-infected HeLa cells, addition of the NO donor SNAP inhibited protease 2A catalytic activity on dystrophin. Because this inhibitory effect was reversed by the thiol-protecting agent DTT, we investigated whether NO S:-nitrosylates the protease 2A. In vitro, NO nitrosylated the active-site cysteine (C110) of the coxsackieviral protease 2A, as demonstrated by site-directed mutagenesis. Within living COS-7 cells, SNAP-induced S:-nitrosylation of this site was confirmed with electron spin resonance spectroscopy. CONCLUSIONS: These data demonstrate inactivation of a coxsackieviral protease 2A by NO through active-cysteine S:-nitrosylation in vitro and intracellularly. Given that the enteroviral protease 2A cleaves mouse and human dystrophin, NO may be protective in human heart failure with an underlying enteroviral pathogenesis through inhibition of dystrophin proteolysis.

Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1.

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. The central region of eIF4G binds the ATP-dependent RNA helicase eIF4A, the 40 S binding factor eIF3, and RNA. In the present work, we have further characterized the binding properties of the central region of human eIF4G. Both titration and competition experiments were consistent with a 1:1 stoichiometry for eIF3 binding. Surface plasmon resonance studies showed that three recombinant eIF4G fragments corresponding to amino acids 642-1560, 613-1078, and 975-1078 bound eIF3 with similar kinetics. A dissociation equilibrium constant of approximately 42 nm was derived from an association rate constant of 3.9 x 10(4) m(-1) s(-1) and dissociation rate constant of 1.5 x 10(-3) s(-1). Thus, the eIF3-binding region is included within amino acid residues 975-1078. This region does not overlap with the RNA-binding site, which suggests that eIF3 binds eIF4G directly and not through an RNA bridge, or the central eIF4A-binding site. Surprisingly, the binding of eIF3 and eIF4A to the central region was mutually cooperative; eIF3 binding to eIF4G increased 4-fold in the presence of eIF4A, and conversely, eIF4A binding to the central (but not COOH-terminal) region of eIF4G increased 2.4-fold in the presence of eIF3.

Functional characterization of five eIF4E isoforms in Caenorhabditis elegans.

Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.

Enteroviral protease 2A directly cleaves dystrophin and is inhibited by a dystrophin-based substrate analogue.

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy through unknown pathological mechanism(s). Dystrophin is a large extrasarcomeric cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy. In addition, we have recently shown that dystrophin is proteolytically cleaved by the Coxsackievirus protease 2A leading to functional impairment and morphological disruption. However, the mechanism of dystrophin cleavage and the exact cleavage site remained to be identified. Antibody epitope mapping of endogenous dystrophin indicated protease 2A-mediated cleavage at the site in the hinge 3 region predicted by a neural network algorithm (human, amino acid 2434; mouse, amino acid 2427). Using site-directed mutagenesis, peptide sequencing, and fluorescence resonance energy transfer assays with recombinant dystrophin, we demonstrate that this putative site in mouse and human dystrophin is a direct substrate for the Coxsackieviral protease 2A both in vitro and in vivo. The substrate analogue protease inhibitor z-LSTT-fmk was designed based on the dystrophin sequence that interacts with the protease 2A and was found to have an IC(50) of 550 nM in vitro. Dystrophin is the first cellular substrate of the enteroviral protease 2A that was identified using by a bioinformatic approach and for which the cleavage site was molecularly mapped within living cells.

Cloning and characterization of the prostate-specific membrane antigen promoter.

Prostate-specific membrane antigen (PSMA) is a protein that is expressed predominantly in normal prostate epithelial cells and in most adenocarcinomas of the prostate (Cap) and in virtually all Cap metastases. In this article we describe the cloning of a 2-kb human genomic DNA fragment containing the 5' upstream untranslated region of the PSMA gene and present evidence that it provides promoter activity. When the DNA fragment was cloned into transient expression vectors to examine promoter activity, the vectors were functional in promoting expression in several prostate and nonprostate cell lines in transient transfection assays. A 614-bp fragment derived from the 3' end of the 2-kb fragment may represent the minimal PSMA promoter as determined by deletion mutagenesis. The 2-kb fragment compared with the 614-bp fragment provided higher expression levels when using prostate-derived cell lines (DU 145 and LNCaP). The increased transcription using the 2-kb fragment was not as great in non-prostate cell lines. Little or no transcription over basal levels was seen with a 232-bp promoter fragment. When the concentration of dihydrotestosterone was depleted or supplemented in the growth medium, no significant effect was seen on PSMA-promoted transient expression in LNCaP cells, a prostate cell line. J. Cell. Biochem. 74:395-405, 1999. Published 1999 Wiley-Liss, Inc.

Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation.

Fifty-eight analogues of the 5'-terminal 7-methylguanosine-containing cap of eukaryotic messenger RNA were synthesized and tested for their ability to inhibit in vitro protein synthesis. A new algorithm was developed for extracting KI, the dissociation constant for the cap analogue.eIF4E complex, from protein synthesis data. The results indicated that addition of a methyl group to the N2 of guanine produced more inhibitory compounds, but addition of a second methyl group to N2 decreased the level of inhibition dramatically. Aryl substitution at N7 improved the efficacy of guanine nucleoside monophosphate analogues. Substitution of the aromatic ring at the para position with methyl or NO2 groups abolished this effect, but substitution with Cl or F enhanced it. By contrast, aryl substitution at N7 in nucleoside di- or triphosphate analogues produced only minor effects, both positive and negative. By far the strongest determinants of inhibitory activity for cap analogues were phosphate residues. The beneficial effect of more phosphate residues was related more to anionic charge than to the number of phosphate groups per se. The second nucleotide residue in analogues of the form m7GpppN affected inhibitory activity in the order G > C > U > A, but there was no effect of 2'-O-modification. Opening the first ribose ring of m7GpppG analogues dramatically decreased activity, but alterations at the 2'-position of this ribose had no effect. Non-nucleotide-based cap analogues containing benzimidazole derivatives were inhibitory, though less so than those containing 7-methylguanine.

Protein synthesis initiation factor 4G.

eIF4G is a member of the class of translational initiation factors involved in mRNA recruitment to the 43S initiation complex. The proteins from yeast to mammals are present in multiple isoforms of 82-176 kDa. Mammalian eIF4G-1 is synthesized by internal initiation of translation and is specifically degraded by viral and host proteases activated by stress conditions. The role of eIF4G in protein synthesis is inferred from the presence of binding sites for other initiation factors that serve to co-localize the 5'- and 3'-termini of mRNA with RNA-helicase activity and the 40S ribosomal subunit. Growth-regulated mRNAs are preferentially translated under conditions of accentuated eIF4E-eIF4G interaction. Proteolysis of eIF4G or expression of competitor proteins interferes with its binding to either the 5'- or 3'-termini, changing the spectrum of mRNAs translated. Elevated eIF4G levels correlate with malignant cell transformation and diminished eIF4G levels, with nutritional deprivation and anoxia.

Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy.

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy, but the mechanism of this pathology is unknown. Mutations in cytoskeletal proteins such as dystrophin cause hereditary dilated cardiomyopathy, but it is unclear if similar mechanisms underlie acquired forms of heart failure. We demonstrate here that purified Coxsackievirus protease 2A cleaves dystrophin in vitro as predicted by computer analysis. Dystrophin is also cleaved during Coxsackievirus infection of cultured myocytes and in infected mouse hearts, leading to impaired dystrophin function. In vivo, dystrophin and the dystrophin-associated glycoproteins alpha-sarcoglycan and beta-dystroglycan are morphologically disrupted in infected myocytes. We suggest a molecular mechanism through which enteroviral infection contributes to the pathogenesis of acquired forms of dilated cardiomyopathy.

Translational recruitment of Xenopus maternal mRNAs in response to poly(A) elongation requires initiation factor eIF4G-1.

Xenopus oocytes accumulate maternal mRNAs which are then recruited to ribosomes during meiotic cell cycle progression in response to progesterone and coincident with poly(A) elongation. Prior to stimulation, most protein synthesis ( approximately 70%) does not require intact translation factor eIF4G (B. D. Keiper and R. E. Rhoads, 1997, Nucleic Acids Res. 25, 395-402). In the present study we have addressed the requirement of eIF4G in the recruitment of mRNAs during meiosis. Cleavage of eIF4G by coxsackievirus protease 2A inhibited progesterone-induced meiotic progression in 88% of the oocytes; prevented the recruitment of maternal mRNAs encoding cyclin B1, c-Mos, D7, and B9; and disrupted the association of eIF4G with poly(A)-binding protein. Poly(A) elongation, however, was not inhibited by eIF4G cleavage. Injection of MPF restored meiotic cell cycle progression to >60% of the oocytes but not the recruitment of cyclin B1 or B9 mRNA. Previously recruited maternal mRNAs were removed from polyribosomes following subsequent cleavage of eIF4G, indicating that eIF4G is required both to recruit and also to maintain maternal mRNAs on polyribosomes. The expression of a cleavage-resistant variant of human eIF4G-1 (G486E) significantly restored the ability to synthesize c-Mos in response to progesterone and to translate exogenous beta-globin mRNA, indicating that the inhibition by protease 2A is due to cleavage of eIF4G alone. These results indicate that intact eIF4G is required for the poly(A)-dependent recruitment of several maternal mRNAs (cyclin B1, c-Mos, D7, and B9) during meiotic cell cycle progression but not for the synthesis of most proteins.

Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff?

Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at 482VANTSTQTM downward arrowGPRPAAAAAA500, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.

Direct cleavage of elF4G by poliovirus 2A protease is inefficient in vitro.

Previously, the purified recombinant 2A proteases (2Apro) of coxsackievirus B4 (CVB4) and human rhinovirus type 2 (HRV2) were shown to cleave synthetic peptides derived from human or rabbit elF4G as well as elF4G protein purified from rabbit reticulocytes. These results were in contrast to previous evidence which supported the view that elF4G cleavage activity in poliovirus-infected HeLa cells required a cellular factor(s) activated by poliovirus (PV) 2Apro. In the present study, recombinant PV 2Apro was shown to cleave either rabbit or human elF4G or their derived peptides in direct cleavage reactions, but cleaved the 4G-derived peptides with 100-fold lower efficiency than with a peptide derived from the poliovirus polyprotein. In these experiments, up to 25-fold molar excess of 2Apro over elF4G protein was required to cause greater than 50% cleavage. CVB4 2Apro was also tested in peptide cleavage assays under the same conditions as PV 2Apro and was found to cleave all elF4G substrates with efficiencies similar to PV 2Apro. Finally, cleavage reactions utilizing recombinant elF4G containing a G486E substitution at the cleavage site for CVB4 and HRV2 proteases resulted in drastically reduced cleavage by PV 2Apro, similar to the reduction previously seen with HRV2 and CVB4 2Apro, confirming that all three viral 2A proteases recognize the same cleavage site on elF4G. These data show that PV 2Apro can directly cleave elF4G in vitro with efficiencies similar to those of CVB 2Apro, but cleavage efficiency of elF4G is approximately 1000-fold lower than cleavage of a peptide derived from the authentic 2A cleavage site on the poliovirus polyprotein.

Multiple isoforms of eukaryotic protein synthesis initiation factor 4E in Caenorhabditis elegans can distinguish between mono- and trimethylated mRNA cap structures.

The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5'-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m32,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegans may possess a specialized form of elF4E that can recognize m32,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, and ife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m32,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m32,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m32,2, 7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.

Cloning and characterization of human eIF4E genes.

Two human eukaryotic initiation factor 4E (eIF4E) genes were isolated and characterized from placental and chromosome 4-specific genomic libraries. One of the genes (EIF4E1) contained six introns, but the other gene (EIF4E2) was intronless, flanked by Alu sequences and 14-base pair (bp) direct repeats, and terminated by a short poly(A) stretch, all characteristics of retrotransposons. Numerous additional intronless eIF4E pseudogenes were found, but unlike EIF4E2, all contained premature in-frame stop codons. The entire EIF4E1 gene spanned >50 kilobase pairs. The coding regions of these two genes differed in four nucleotide residues, resulting in two amino acid differences in the predicted proteins. The promoter of EIF4E1 has been characterized previously. The putative promoter of EIF4E2 contained no TATA box but did contain a transcription initiator region (Inr) and numerous other sequence motifs characteristic of regulated promoters. EIF4E2 contained only two of the three polyadenylation signals present in EIF4E1. Evidence for transcription of both genes was obtained from primer extension, S1 mapping, ribonuclease protection, and reverse transcriptase-polymerase chain reaction experiments. Transcription was found to initiate 19 bp upstream of the translational initiation codon in the case of EIF4E1 and 80 bp in the case of EIF4E2. The two genes were differentially expressed in four human cell lines, Wish, Chang, K562, and HeLa.

Functional characterization of the internal ribosome entry site of eIF4G mRNA.

The eIF4 group initiation factors are required for cap-dependent translation initiation. Infection of mammalian cells by picornaviruses results in proteolytic cleavage of one of these factors, eIF4G, which severely restricts cap-dependent initiation but permits cap-independent initiation to proceed from an internal ribosome entry site (IRES) in picornaviral RNAs. The first 357 nucleotides (nt) of the 5'-untranslated region of eIF4G mRNA also contains an IRES. Using bicistronic constructs for expression in K562 cells, we have now shown that progressive deletions of the 5'-untranslated region can have either stimulatory or inhibitory effects. Furthermore, a 101-nt segment exhibits full IRES activity, and an 81-nt segment exhibits detectable IRES activity. A polypyrimidine tract (PPT) at the 3' terminus is essential for internal initiation, a property which is characteristic of picornaviral IRESs but not the other host cellular IRESs studied to date. IRES activity does not require sequences beyond 357 nt. Out-of-frame AUGs have no effect on IRES-driven luciferase expression when introduced upstream of the PPT but markedly decrease expression when introduced at sites between the PPT and the authentic initiation codon at nt 369. These results suggest that the ribosomal subunit enters at or near the PPT and then scans downstream for the initiation codon.