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

The EMBO Journal (2022) e110581https://doi.org/10.15252/embj.2022110581

Here is an annotated abstract:

“Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring [only] a subset of eukaryotic initiation factors (eIFs).”

An internal ribosome entry site, abbreviated IRES, is an RNA element that allows for translation initiation in a cap-independent manner, as part of the greater process of protein synthesis.

These are the eukaryotic initiation factors: eIF1, eIF1A, eIF2, eIF3, eIF4, eIF5, eIF5A, eIF5B, eIF6 (to the tune of “Old McDonald had a farm”).

All of these are normally required for translation initiation.  But the IRES-mediated initiation pathway, which is employed by many viruses uses shortcuts, allowing the virus to hijack the ribosome for the synthesis of its own proteins.  Depending on the class of IRES, only a subset of the host’s initiation factors are used by the virus.

Hepatitis C virus IRES was first visualized in our lab by Christian Spahn as we teamed up with Jennifer Doudna’s group, then still at Yale. Jennifer’s postdoc Jeff Kieft prepared the IRES.

Spahn, C.M., Kieft, J.S., Grassucci, R.A., Penczek, P.A., Zhou, K., Doudna, J.A., and Frank, J. (2001). Hepatitis C virus IRES RNA-induced changes in the conformation of the 40S ribosomal subunit. Science 291, 1959-1962.

“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-tRNA_i^Met forming a 48S initiation complex.”

Note that the placement of the initiation codon (AUG) is normally done through an elaborate scanning process.  But here the IRES, upon binding to the 40S subunit, places the AUG codon directly into the P site and thereby jump-starts initiation with the foreign mRNA.

“Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome.”

So what this means is that the virus uses only a minimum set of host initiation factors: eIF2, eIF5, and eIF5b.

“Initiation can also proceed without eIF2, in which case Met-tRNA_i^Met is recruited directly by eIF5B.”

Here is a different additional pathway in which eIF2 is not even required!

“However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown.”

This was the state of knowledge before our study was done.

“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.”

Essentially, this has been a gigantic structural inventory, which has been made possible by single-particle cryo-EM with the tools of 3D classification.  While the biochemical samples were prepared in the labs of Chris Hellen and Tatyana Pestova, the bulk of the work on visualization and modeling was done by Zuben in our lab.  In particular, it was necessary to employ focused classification to obtain the details of interactions between the key players.  Dozens of 3D maps had to be modeled and ordered on timelines that comport with existing knowledge.

Without exaggeration, this work ˙as been the largest project we have ever done in our lab. It required close to a month of continuous data collection, yielding 78,622 micrographs and 6 million picked particles. To decipher the complicated conformation and compositional landscape of the multiple HCV IRES complexes, over 500 refinement jobs and more than 150 classification jobs were required, and the result was 18 full maps and models of different 48S initiation complexes with the HCV IRES and an additional 30 maps showing various conformational or classification stages, all submitted to the PDB and EMDB.

“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.”