Final reaction concentrations are as follows: 38 mM HEPES-KOH, pH 7.4, 135 mM KOAc, 3.25 mM MgOAc 2, 2.7 mM dithiothreitol, 25 mM sucrose, 2.5% glycerol, 1 mM GMP-PNP, 200 nM eIF2, 1 μM eIF1, 400 nM eIF1A, 200 nM 40S ribosomal subunits, and 10 or 100 μM test compound (NCS119889). TC was then added to 40S ribosomal subunits, eIF1A, eIF1, a minimal message (5′-GGAA 7UAUG 10C-3′), and NCS119889 (or other compounds). Essentially, purified eIF2 was incubated for 10 min with saturating 5′-guanylylimidodiphosphate (GMP-PNP) (1 mM final) to facilitate the exchange of eIF2-bound GDP for GMP-PNP, followed by the addition of Met-tRNA i Met and a 5-min incubation to form TC. Our results indicate that the HCV IRES has evolved a mechanism to facilitate recruitment of TC under conditions when these are limiting for initiation, as occurs during the cellular antiviral interferon response.Ĥ3S preinitiation complexes were formed essentially as described by Lorsch and Herschlag (1999). In addition, we demonstrate that the HCV IRES is still capable of recruiting eIF2α and Met-tRNA i Met at concentrations of NSC119889 that block cap-dependent translation. We find that translation from the HCV IRES proceeds to near wild-type levels in the presence of this class of TC inhibitors in vitro and in vivo. Herein, we characterize the mode of action of NSC119889 and find that it prevents the association of Met-tRNA i Met to eIF2. Recently, an initiation factor-independent mode of ribosome recruitment has also been described for the HCV IRES under high Mg ++ conditions in vitro ( Lancaster et al., 2006).Ī previous forward chemical genetic screen identified a new inhibitor of translation initiation, named NSC119889, that suppressed cap-dependent translation but did not significantly affect translation driven by the HCV IRES ( Novac et al., 2004). Whether the TC is recruited to the 40S ribosome before binding the HCV IRES or assembles once the 40S ribosome has loaded onto the HCV IRES is not known. Recent mutational analysis suggests that the HCV IRES coordinates eIF3 and eIF2 interaction with the ribosome, leading to correct positioning of the Met-tRNA i Met in the P site ( Ji et al., 2004). Although the order in which the 40S ribosomal subunit, the TC, and eIF3 are recruited to the HCV IRES remains to be defined, addition of TC to an HCV-bound 40S docks the AUG into the ribosomal P site and the presence of eIF3 allows for formation of the 80S initiation complex ( Pestova et al., 1998). After binding of TC, a 60S subunit is recruited to generate an 80S ribosomal complex competent for elongation ( Pestova et al., 1998). In hepatitis C virus (HCV), the IRES can recruit the 40S ribosomal subunit independently of translation factors, followed by formation of a 48S complex that contains eIF3 ( Buratti et al., 1998 Sizova et al., 1998 Kolupaeva et al., 2000 Kieft et al., 2001 Otto and Puglisi, 2004).

The latter case is exemplified by the cricket paralysis virus (CrPV) IRES, which recruits 40S ribosomal subunits and initiates translation from the A site, in the absence of a Met-tRNA i Met positioned in the P site ( Wilson et al., 2000 Jan and Sarnow, 2002 Pestova and Hellen, 2003). This mode of ribosome recruitment is driven by the ability of internal ribosome entry sites (IRESs) to either interact with initiation factors ( Pestova et al., 1996, 1998) and recruit 43S preinitiation complexes ( Pestova et al., 1996) or to directly engage the 40S ribosomal subunit ( Wilson et al., 2000 Jan and Sarnow, 2002 Pestova and Hellen, 2003).

Some cellular and viral mRNAs initiate in a cap-independent manner, involving direct binding of components of the translation machinery at or upstream of the initiation codon.

Met-tRNA i Met] (hereafter referred to as TC), eIF1, eIF1A, eIF5, and the multisubunit complex eIF3.

In this process, the 40S ribosomal subunit is converted to a 43S preinitiation complex by recruitment of the ternary complex [eIF2 For the majority of eukaryotic mRNAs, ribosome recruitment is mediated by the 5′ cap structure (m 7GpppN, where N is any nucleotide) and involves reorganization of the mRNA template by the eukaryotic initiation factor (eIF) 4 class of translation factors. Translation initiation in eukaryotes occurs by at least two distinct pathways.

Ribosome initiater t rna