Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES

Abstract

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end‐independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2‐bound Met‐tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation‐competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met‐tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo‐EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2‐containing 48S initiation complexes, to eIF5B‐containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2‐ to eIF5B‐containing 48S complexes and prepare them for subunit joining. image Hepatitis C virus (HCV) RNA contains an internal ribosome entry site (IRES) that hijacks the cellular translation initiation machinery to produce viral proteins. Cryo‐EM studies now reveal the molecular role of specific HCV IRES domains in loading viral mRNA and how canonical initiation factors support this initiation process. Cryo‐EM allowed for the first time the visualization of HCV IRES initiation complexes from the binary complex to the eIF2‐containing and eIF5B‐contianing 48S initiation complexes. These structural studies reveal the role of IRES domain II in loading mRNA into the mRNA channel and the basis for the enhancement of this step by eIF1A. These structures revealed the dynamic nature of the contacts between the IRES and the 40S ribosomal subunit during translation initiation. tRNA is repositioned by eIF5B in preparation for joining of the large subunit.