The initiation of eukaryotic protein synthesis is one of the most highly regulated steps in gene expression. Cap-dependent translation requires ≥12 initiation factors (eIFs) to associate with the 7-methyl guanosine cap at the 5′ end of mRNA and the 40S ribosomal subunit, leading to the formation of a 43S preinitiation complex that scans to the correct initiation codon and positions the initiating methionyl-tRNA (Met-tRNAi). The resulting 48S preinitiation complex then associates with the 60S ribosomal subunit to form an active 80S ribosome (1–3
). In viruses such as hepatitis C virus (HCV), structured RNA sequences called internal ribosome entry sites (IRESs) located in the genomic 5′-untranslated region trigger an alternative cap-independent mechanism of assembling the eukaryotic protein synthesis machinery at the correct start codon (3
). The HCV IRES drives translation of its genomic RNA, helping the virus to evade the host immune response that suppresses canonical cap-dependent translation (4
). However, the specific molecular events that accompany preinitiation complex formation in an mRNA cap- or IRES-dependent manner remain poorly understood.
Translation initiation factor eIF3 is central to the assembly of competent translation preinitiation complexes (3
). Evidence now indicates that eIF3 stimulates most of the reactions in cap-dependent translation initiation, including initiation factor binding to the 40S subunit in the 43S preinitiation complex, mRNA recruitment to the 43S preinitiation complex, mRNA scanning for the start codon (2
) and release of eIF2–guanosine diphosphate (GDP) after start codon recognition (5
). Human eIF3 also extends the length of the mRNA-binding channel of the 40S subunit in initiation complexes (6
). In humans, eIF3 is important for cell homeostasis, and misregulation of eIF3 subunit expression correlates with cancer progression (7
). In addition, human eIF3 plays an important role in translation initiation mediated by the HCV IRES. Defined secondary and tertiary structural elements of the HCV IRES (8
) bind specifically to eIF3 and the 40S ribosomal subunit (4
). These structural interactions position the genomic RNA at the correct start codon without the need for the 5′-cap binding translation initiation machinery and mRNA scanning (12
). HCV IRES–eIF3 association has been proposed to substitute for translation initiation factor eIF4G, which is required for cap-dependent translation (14
). However, the molecular consequences of HCV IRES interactions with eIF3 are unknown.
Despite the biological significance of eIF3, its molecular contributions to translation initiation remain unclear owing to the scarcity of structural information. Human eIF3 is an 800 kDa complex containing 13 proteins (eIF3a–eIF3m) (15
). The cryo-electron microscopic reconstruction of endogenously purified human eIF3 revealed a five-lobed structure with anthropomorphic features that binds to the platform of the 40S ribosomal subunit (14
). Remarkably, our recent 3D reconstruction of a bacterially reconstituted octameric core complex of eIF3 showed it to be structurally similar to the larger native eIF3, likely due to the inherent flexibility of large regions within the complex beyond the central core (16
In the case of HCV IRES-mediated translation, cryo-EM reconstructions of the binary HCV IRES–eIF3 complex revealed extensive interactions between the IRES RNA and eIF3. However, the specific regions of contact could not be identified definitively (14
). In cross-linking experiments, eIF3 subunits a, b, d and f were shown to be located near to or contacting the HCV IRES (10
). The functional importance of these interactions remains obscure owing to the paucity of structural information about them.
Here we used EM, biochemical and bioinformatics approaches to determine the critical domains responsible for eIF3 binding to the HCV IRES and 40S ribosomal subunit, and for the formation of translation initiation complexes. Together, these results provide fundamental mechanistic insights into eIF3 function during HCV IRES-mediated translation, identify possible new targets for HCV therapeutic intervention and reveal motifs in eIF3 that may be important for the control of translation initiation on cellular mRNAs.