Eukaryotic Initiation Factors 4G and 4A Mediate Conformational Changes Downstream of the Initiation Codon of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

Abstract

Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG 834 ). Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds. Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains. Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function. Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex. eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES. This complex also induced more extensive conformational rearrangements at the 3 border of the ribosome binding site that required ATP and active eIF4A. We propose that these conformational changes prepare the region flanking AUG 834 for productive binding of the ribosome.Translation of a subset of cellular and viral mRNAs is initiated by end-independent binding of ribosomes to an internal ribosome entry site (IRES) in the 5Ј untranslated region. IRESs were first identified in different picornavirus RNA genomes, and these IRESs are now classified into two major groups, one of which includes those of the encephalomyocarditis virus (EMCV), Theiler's murine encephalitis virus (TMEV), and foot-and-mouth disease virus (FMDV) (17). The EMCV IRES is ϳ450 nucleotides (nt) long and comprises H, I, J-K, and L structural domains upstream of the initiation codon AUG at nucleotide 834 (AUG 834 ). Ribosomal initiation complexes attach directly to AUG 834 , and initiation does not involve scanning (19,35). Recent analysis has begun to resolve molecular details of the mechanism of initiation on EMCVlike IRESs. Viral and cellular IRES-containing mRNAs can be translated under conditions when the cap-mediated mode of end-dependent initiation that is used by most mRNAs is inhibited (12), and studies of initiation on viral IRESs are therefore yielding insights into mechanisms that enable selective translation of mRNAs in the cell.The stage that differentiates IRES-mediated initiation from the conventional cap-mediated mode of initiation is the mechanism by which the 43S ribosomal preinitiation complex engages mRNA to form a 48S complex at the initiation codon (16,36). The 43S complex consists of a 40S ribosomal subunit, initiator tRNA, and eukaryotic initiation factors (eIFs), including eIF2 and eIF3. In the process of cap-mediated initiation, eIF4F is thought to create a binding site for this complex adjacent to the 5Ј-terminal m 7 G cap by ...

Direct functional interaction of initiation factor eIF4G with type 1 internal ribosomal entry sites

“…The finding that recruitment of eIF4G/eIF4A to domain V led to these changes in domain VI is consistent with observations suggesting that domains V and VI are functionally linked and might interact structurally, such as their synergy in promoting UV crosslinking to the IRES of a 36-kDa protein in cellular extracts (22) and determining PV neurovirulence (23). Importantly, binding of eIF4G/eIF4A to the type 2 EMCV IRES induced analogous toe prints at its 3Ј border (7). These conformational changes induced at the 3Ј borders of type 1 and 2 IRESs could be essential for subsequent attachment of 43S complexes to these regions.…”

“…2 I and J), and in both instances its C terminus is oriented toward the apex and the N terminus is oriented toward the base of these domains (this paper and ref. 7). As with type 2 IRESs (5), eIF4G recruits eIF4A to type 1 IRESs, and eIF4A may in turn enhance the eIF4G-type 1 IRES interaction (e.g., Fig.…”

“…eIF4F was purified from RRL (Green Hectares), and recombinant eIF4A, eIF4B, eIF4G 737-1116, and eIF4G737-1600 were expressed and purified from Escherichia coli DE3 (4,7,24).…”

“…mutants with unique surface-exposed cysteines (Fig. 1B) were fully active in 48S complex formation on the EMCV IRES in an in vitro-reconstituted system and induced specific cleavage in its J-K domain in eIF4G/IRES complexes (7).…”

Bunyavirus N: eIF4F surrogate and cap-guardian

“…In many of the picornaviruses, such as encephalomyocarditis virus, localization of the 43S pre-initiation complex requires the eIF4G and eIF4A components of eIF4F. [14][15][16] However, in the case of the flavivirus, hepatitis C virus (HCV), as well as some of the avian and porcine picornaviruses, the pre-initiation complex is recruited directly to the IRES by way of just the 40S ribosomal subunit and eIF3, and without eIF4F components. [17][18][19] Further, the dicistoviruses, such as cricket paralysis virus, recruit the 40S subunit its IRES without either eIF4F or eIF3.…”