Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry.

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.

Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes.

Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G.

Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation.

Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not beta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.

Translation of encephalomyocarditis virus RNA by internal ribosomal entry.

Picornavirus 5' NCRs contain IRES elements that have been divided into two groups, exemplified by PV (type 1) and EMCV (type 2). These elements are functionally related and have an intriguing level of structural and sequence similarity. Some conserved RNA sequences and/or structures may correspond to cis-acting elements involved in IRES function, so that there may also be similarities in the mechanism by which the two types or IRES promote initiation. The function of both types of IRES element appears to depend on a cellular 57 kDa polypeptide, which has been identified as the predominantly nuclear hnRNP protein PTB. However, a specific function for p57/PTB in translation has not yet been established. These two groups can be differentiated on the basis of their requirements for trans-acting factors. The EMCV IRES functions efficiently in a broader range of eukaryotic cell types than type 1 IRES elements, probably because the latter require additional factor(s). A second distinction between these IRES element is that initiation occurs directly at the 3' border of type 2 IRES elements, whereas a nonessential spacer of between 30 nt and 154 nt separates type 1 IRES elements from the downstream initiation codon.

Viral proteases as targets for chemotherapeutic intervention.

Many viruses encode proteinases that are essential for infectivity, and are consequently attractive chemotherapeutic targets. The biochemistry and structure of the human immunodeficiency virus proteinase have been characterized extensively, and potent peptide-mimetic inhibitors have been developed. Techniques and strategies used to improve the efficiency of these compounds are likely to be applicable to other viral proteinases.

Assay methods for retroviral proteases.

A variety of assay methods for retroviral proteases have been developed in response to different experimental requirements, such as initial identification of a protease, subsequent enzymatic characterization, and high-capacity automated screening of potential inhibitors. This chapter has reviewed a number of these methods above; most have been closely tailored to match specific experimental requirements, and some of them are described in greater detail in other chapters in this volume. They include analysis of polyprotein cleavage using SDS-PAGE, analysis of the determinants of substrate cleavage using either chromogenic peptides or reversed-phase HPLC for product separation after cleavage of unmodified peptides, and the design and utilization of quenched fluoregenic peptides for use in continuous assay.

The structure and function of initiation factors in eukaryotic protein synthesis.

Protein synthesis is one of the most complex cellular processes, involving numerous translation components that interact in multiple sequential steps. The most complex stage in protein synthesis is the initiation process. It involves initiation factor-mediated assembly of a 40S ribosomal subunit and initiator tRNA into a 48S initiation complex at the initiation codon of an mRNA and subsequent joining of a 60S ribosomal subunit to form a translationally active 80S ribosome. The basal set of factors required for translation initiation has been determined, and biochemical, genetic, and structural studies are now beginning to reveal details of their individual functions in this process. The mechanism of translation initiation has also been found to be influenced significantly by structural properties of the 5' and 3' termini of individual mRNAs. This review describes some of the major developments in elucidating molecular details of the mechanism of initiation that have occurred over the last decade.

The role of proteolytic processing in the morphogenesis of virus particles.

Proteinases are encoded by many RNA viruses, all retroviruses and several DNA viruses. They play essential roles at various stages in viral replication, including the coordinated assembly and maturation of virions. Most of these enzymes belong to one of three (Ser, Cys or Asp) of the four major classes of proteinases, and have highly substrate-selective and cleavage specific activities. They can be thought of as playing one of two general roles in viral morphogenesis. Structural proteins are encoded by retroviruses and many RNA viruses as part of large polyproteins. Their proteolytic release is a prerequisite to particle assembly; consequent structural rearrangement of the capsid domains serves to regulate and direct association and assembly of capsid subunits. The second general role of proteolysis is in assembly-dependent maturation of virus particles, which is accompanied by the acquisition of infectivity.

Internal initiation of translation of bovine viral diarrhea virus RNA.

Initiation of translation on the bovine viral diarrhea virus (BVDV) internal ribosomal entry site (IRES) was reconstituted in vitro from purified translation components to the stage of 48S ribosomal initiation complex formation. Ribosomal binding and positioning on this mRNA to form a 48S complex did not require the initiation factors eIF4A, eIF4B, or eIF4F, and translation of this mRNA was resistant to inhibition by a trans-dominant eIF4A mutant that inhibited cap-mediated initiation of translation. The BVDV IRES contains elements that are bound independently by ribosomal 40S subunits and by eukaryotic initiation factor (eIF) 3, as well as determinants that mediate direct attachment of 43S ribosomal complexes to the initiation codon.

Preparation and activity of synthetic unmodified mammalian tRNAi(Met) in initiation of translation in vitro.

Translation of eukaryotic mRNA is initiated by a unique amino-acyl tRNA, Met-tRNAi(Met), which passes through a complex series of highly specific interactions with components of the translation apparatus during the initiation process. To facilitate in vitro biochemical and molecular biological analysis of these interactions in fully reconstituted translation initiation reactions, we generated mammalian tRNAi(Met) by in vitro transcription that lacked all eight base modifications present in native tRNAi(Met). Here we report a method for in vitro transcription and aminoacylation of synthetic unmodified initiator tRNAi(Met) that is active in every stage of the initiation process, including aminoacylation by methionyl-tRNA synthetase, binding of Met-tRNAi(Met) to eIF2-GTP to form a ternary complex, binding of the ternary complexes to 40S ribosomal subunits to form 43S complexes, binding of the 43S complex to a native capped eukaryotic mRNA, and scanning on its 5' untranslated region to the correct initiation codon to form a 48S complex, and finally joining with a 60S subunit to assemble an 80S ribosome that is competent to catalyze formation of the first peptide bond using the [35S]methionine residue attached to the acceptor terminus of the tRNAi(Met) transcript.

Translation of hepatitis C virus RNA.

The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.

Ribosomal binding to the internal ribosomal entry site of classical swine fever virus.

Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.

Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs.

Initiation of translation of a subset of eukaryotic mRNAs results from internal ribosomal entry. This process is exemplified by encephalomyocarditis virus (EMCV), which contains an internal ribosomal entry site (IRES) within its 5' nontranslated region that is approximately 450-nt long and consists of a series of stem-loops designated H-L. We have previously identified a cellular 58-kDa polypeptide that binds specifically to this IRES and that is implicated in its function as the pyrimidine tract-binding protein PTB. We have now mapped PTB binding sites directly on the IRES elements of EMCV and the related foot-and-mouth disease virus (FMDV) using structure-specific enzymatic probes and base-specific chemical probes. PTB bound to six sites on the EMCV IRES: site 1 (UCUU401) is upstream of domain H, site 2 is the basal helix of domain H (nt 407-410 and 440-443), site 3 (UCUUU423) is the apical loop of domain H, site 4 is the apical helix and adjacent internal bulged loop of domain K, site 5 (CUUUA750) is the apical loop of domain K, and site 6 (CCUUU815) is downstream of domain L. PTB bound to sites on the FMDV IRES that correspond precisely to EMCV sites 3, 5, and 6. These sites have the consensus sequence CUUU and form two groups that are located near to the 5' and 3' borders of these IRES elements. Their position, and the effects of mutation of them on IRES function are consistent with PTB's role in IRES-mediated initiation being to bind to multiple sites in the IRES, thereby stabilizing a specific active conformation.

Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis.

Initiation of poliovirus translation is mediated by a large, structured segment of the 5' nontranslated region known as the internal ribosome entry site (IRES) and normally occurs 155 nucleotides (nt) downstream of the IRES at AUG743 (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG743 by 10 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG743 was reduced by only 50 to 80%, depending on the number of upstream initiation codons. Initiation at AUG743 was also reduced following insertion of a stable hairpin at nt 630, but the reduction was modest in an ascites carcinoma cell extract. Initiation was more frequent at AUG743 than at AUG683 if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all initiation events at AUG743 can be accounted for by a scanning-dependent mechanism. Translation of bicistronic mRNAs in which the intercistronic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry point for ribosomes independently of the IRES. Insertion of increasingly longer sequences immediately downstream of the stable hairpin progressively reduced initiation at AUG743 without affecting initiation at AUG683. These results are discussed in terms of a model for initiation of poliovirus translation in which a complex RNA superstructure upstream of nt 586 promotes ribosome binding at an entry point determined by specific downstream cis-acting elements.

Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons.

The scanning model of translation initiation is a coherent description of how eukaryotic ribosomes reach the initiation codon after being recruited to the capped 5' end of messenger RNA. Five eukaryotic initiation factors (eIF 2, 3, 4A, 4B and 4F) with established functions have been assumed to be sufficient to mediate this process. Here we report that eIF1 and eIF1A are also both essential for translation initiation. In their absence, 43S ribosomal preinitiation complexes incubated with ATP, eIF4A, eIF4B and eIF4F bind exclusively to the cap-proximal region but are unable to reach the initiation codon. Individually, eIF1A enhances formation of this cap-proximal complex, and eIF1 weakly promotes formation of a 48S ribosomal complex at the initiation codon. These proteins act synergistically to mediate assembly of ribosomal initiation complexes at the initiation codon and dissociate aberrant complexes from the mRNA.

Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.

Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.

Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5' nontranslated region and involvement of a cellular 57-kilodalton protein.

Translation of poliovirus RNA is initiated by cap-independent internal entry of ribosomes into the 5' nontranslated region. This process is dependent on elements within the 5' nontranslated region (the internal ribosomal entry site) and may involve novel translation factors. Systematic mutation of a conserved oligopyrimidine tract has revealed a cis-acting element that is essential for translation in vitro. The function of this element is related to its position relative to other cis-acting domains. This element is part of a more complex structure that interacts with several cellular factors, but changes in protein binding after mutation of this element were not detected in a UV cross-linking assay. A 57-kDa protein from the ribosomal salt wash fraction of HeLa cells was identified that binds upstream of the oligopyrimidine tract. Translation of poliovirus mRNA in vitro was strongly and specifically inhibited by competition with the p57-binding domain (nucleotides 260 to 488) of the 5' nontranslated region of encephalomyocarditis virus, indicating a probable role for p57 in poliovirus translation. p57 is likely to be identical to the ribosome-associated factor that binds to and is necessary for the function of the internal ribosomal entry site of encephalomyocarditis virus RNA.

Synthesis and proteolytic processing of arabis mosaic nepovirus, cherry leaf roll nepovirus, and strawberry latent ringspot nepovirus proteins in reticulocyte lysate.

The genomic RNA components of three nepoviruses, arabis mosaic (ArMV), cherry leaf roll (CLRV), and strawberry latent ringspot (SLRV), were translated in rabbit reticulocyte lysate. Each component (except the RNA-2 of CLRV) directed the synthesis of proteins that corresponded in size to their theoretical coding capacity. The RNA-1 components of all three viruses were translated to yield polyproteins of Mr 250k, which were autocatalytically processed to yield up to five cleavage products. The primary products of translation of the RNA-2 components of ArMV (Mr 115k and 105k), CLRV (Mr 165k) and SLRV (Mr 99k and 96k) were polyproteins that were stable on incubation, but which underwent proteolytic processing in the presence of the corresponding RNA-1 and its translation products. These polyproteins were immunoprecipitated using antisera to appropriate virions indicating that the RNA-2 sequences encode the coat protein cistrons.