Elucidation of the structure of the ribosome has stimulated numerous proposals for the roles of specific rRNA elements, including the universally conserved helix 69 (H69) of 23S rRNA, which forms intersubunit bridge B2a and contacts the D stems of A- and P-site tRNAs. H69 has been proposed to be involved not only in subunit association and tRNA binding but also in initiation, translocation, translational accuracy, the peptidyl transferase reaction, and ribosome recycling. Consistent with such proposals, deletion of H69 confers a dominant lethal phenotype. Remarkably, in vitro assays show that affinity-purified Deltah69 ribosomes have normal translational accuracy, synthesize a full-length protein from a natural mRNA template, and support EF-G-dependent translocation at wild-type rates. However, Deltah69 50S subunits are unable to associate with 30S subunits in the absence of tRNA, are defective in RF1-catalyzed peptide release, and can be recycled in the absence of RRF.

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.It is now generally accepted that picornavirus RNAs are translated by a mechanism of internal initiation, in which the ribosome enters directly at an internal site within the RNA rather than scanning from the physical 5Ј end (reviewed in reference 2). The 5Ј untranslated region (UTR) of the viral RNA has an IRES (internal ribosome entry site) about 450 nucleotides (nt) in length which is necessary and sufficient to promote internal ribosome entry and internal initiation. On the basis of primary and secondary structure conservation, the picornavirus IRESs can be divided into one minor and two major groups: (i) hepatitis A virus (HAV); (ii) entero-and rhinoviruses; and (iii) cardio-, aphtho-, and parechoviruses. Internal initiation of translation on the IRESs of the two major groups is thought to require all of the canonical initiation factors that are involved in the scanning mechanism except that eIF4E is completely redundant and the requirement for eIF4G can be fulfilled by just the C-terminal two-thirds fragment of this protein (26,27). Notably, the activity of these IRESs is not inhibited (and may even be actually stimulated in certain circumstances) when eIF4G is cleaved by entero-or rhinovirus 2A protease or foot-and-mouth disease virus (FMDV) L-protease (5, 6, 7, 28). These viral proteases cleave eIF4G to give (i) an N-terminal one-third fragment which has the site for interaction of eIF4G with eIF4E, the only translation initiation factor that binds directly to 5Ј caps, and also a site for binding poly(A)-binding protein; and (ii) a C-terminal two-thirds fragment which has two distinct sites for interaction with eIF4A, the RNA helicase initiation factor, and a site ...

Picornavirus proteases cleave translation initiation factor eIF4G into a C‐terminal two‐thirds fragment (hereafter named p100) and an N‐terminal one‐third fragment, which interacts with the cap‐binding factor eIF4E. As the timing of this cleavage correlates broadly with the shut‐off of host cell protein synthesis in infected cells, a very widespread presumption has been that p100 cannot support capped mRNA translation. Through the use of an eIF4G‐depleted reticulocyte lysate system, we show that this presumption is incorrect. Moreover, recombinant p100 can also reverse the inhibition of capped mRNA translation caused either by m7GpppG cap analogue, by 4E‐BP1, which sequesters eIF4E and thus blocks its association with eIF4G, or by cleavage of endogenous eIF4G by picornavirus proteases. The concentration of p100 required for maximum translation of capped mRNAs is ∼4‐fold higher than the endogenous eIF4G concentration in reticulocyte lysates. Our results imply that picornavirus‐induced shut‐off is not due to an intrinsic inability of p100 to support capped mRNA translation, but to the viral RNA outcompeting host cell mRNA for the limiting concentration of p100.

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