Laboratory for Translation Structural Biology

Our laboratory aims to elucidate the molecular mechanisms underlying complex biological systems through three-dimensional structural analyses using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), with a particular focus on eukaryotic translation.

Hepatitis C virus (HCV) initiates protein synthesis through an internal ribosome entry site (IRES), allowing viral translation independently of the 5′ cap structure used by host mRNAs. A key host factor involved in this process is eukaryotic initiation factor 3 (eIF3), although its precise role has remained unclear. We combined in vitro reconstituted translation, cryo-electron microscopy (cryo-EM), cross-linking mass spectrometry, and biochemical analyses to elucidate how eIF3 functions during HCV IRES-mediated translation.

We determined multiple cryo-EM structures representing successive stages of HCV IRES-mediated translation, from initiation to elongation. These structures revealed that the HCV IRES binds simultaneously to the ribosome and eIF3 throughout translation. Unlike canonical cap-dependent initiation, in which the eIF3 core binds directly to the 40S ribosomal subunit, the HCV IRES sequesters the eIF3 core by interacting with subdomain IIIb of the IRES. This rearrangement allows viral RNA to occupy the ribosomal binding site normally used by host mRNAs while retaining the functional contribution of eIF3.

A particularly notable finding is that the N-terminal domain of the eIF3c subunit binds directly to the 60S large ribosomal subunit during elongation. Biochemical experiments confirmed this interaction and suggested that it stabilizes the 80S ribosome, facilitating large subunit joining during initiation and potentially promoting efficient translation reinitiation after termination. Furthermore, cross-linking mass spectrometry indicated that the noncore eIF3 subunits remain positioned similarly to those in canonical translation, despite the displacement of the eIF3 core by the HCV IRES.

Functional translation assays demonstrated that eIF3 markedly enhances HCV IRES-dependent translation, particularly under eIF2-limited conditions that mimic the integrated stress response induced during HCV infection. This mechanism enables efficient viral protein synthesis even when host cap-dependent translation is suppressed.

Overall, we provided the first structural view of eIF3 throughout HCV IRES-mediated translation and revealed previously unrecognized roles of eIF3 during elongation and reinitiation. These findings substantially advance our understanding of viral translation mechanisms and suggest that eIF3 dynamics may represent a potential target for antiviral intervention and broader translational regulation. (Iwasaki et al., Proc Natl Acad Sci U S A, 2025).