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Structural and mechanistic insights into hepatitis C viral translation initiation

Key Points

  • The RNA genome of the flavivirus hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) that allows protein synthesis to occur in a cap-independent manner. The HCV IRES binds specifically to the 40S ribosomal subunit in the absence of canonical initiation factors and places the 40S subunit directly at the initiation codon.

  • Biochemical studies have revealed specific roles for the HCV IRES domains in recruiting the 40S ribosomal subunit and the initiation factors required for efficient protein synthesis.

  • HCV-type IRESs are distinct from picornavirus IRESs by virtue of their requirement for only two initiation factors, eIF2 and eIF3.

  • Structures of individual IRES domains, determined by X-ray crystallography and nuclear magnetic resonance, have provided information about discrete domain folding. The structure of the intact IRES in complex with eIF3, the 40S ribosomal subunit and the 80S ribosome has been studied using cryo-electron microscopy.

  • Conformational changes in the 40S subunit on association with the IRES and with the 60S ribosomal subunit, particularly in the head domain and the mRNA binding cleft, indicate a mechanism for IRES positioning of mRNA during translation initiation.

Abstract

Hepatitis C virus uses an internal ribosome entry site (IRES) to control viral protein synthesis by directly recruiting ribosomes to the translation-start site in the viral mRNA. Structural insights coupled with biochemical studies have revealed that the IRES substitutes for the activities of translation-initiation factors by binding and inducing conformational changes in the 40S ribosomal subunit. Direct interactions of the IRES with initiation factor eIF3 are also crucial for efficient translation initiation, providing clues to the role of eIF3 in protein synthesis.

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Figure 1: Secondary structure of the 5′ UTR of the HCV genome and model for the formation of the 80S initiation complex.
Figure 2: Cryo-electron microscopy (EM) reconstruction of the HCV IRES associated with the 40S ribosomal subunit.
Figure 3: Cryo-electron microscopy (EM) reconstruction of the initiation factor eIF3, and a structural model of the IRES–40S–eIF3 complex.
Figure 4: Cryo-electron microscopy (EM) reconstruction of the 80S–HCV IRES complex.
Figure 5: Capped mRNA and viral mRNA ribosome recruitment strategies.
Figure 6: Model of cap-dependent mRNA loading onto the 40S ribosomal subunit.

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Acknowledgements

We would like to thank H. Stark and B. Siridechadilok for providing help with the figures. We gratefully acknowledge support from the Howard Hughes Medical Institute and the National Institutes of Health.

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Correspondence to Jennifer A. Doudna.

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classical swine fever virus

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encephalomyocarditis virus

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Glossary

Shine–Dalgarno sequence

(AGGAGG) This sequence is located 5′ of the AUG codon on bacterial mRNAs and functions as the signal for the initiation of protein synthesis.

Peptidyl (P) site

The site on the small ribosomal subunit that holds the tRNA molecule that is linked to the growing end of the polypeptide chain.

Sarcin–ricin loop

A highly conserved RNA loop in the rRNA from the large ribosomal subunit that forms a site for the binding of protein synthesis elongation factors. This association is inhibited by the ribotoxins α-sarcin and ricin.

Platform region

A large domain in the small ribosomal subunit above which is the mRNA and tRNA binding cleft.

Exit (E) site

The site from which the deacylated tRNA molecule is ejected.

Difference mapping

This is used to determine conformational changes between closely related structures. The electron density map of one structure is subtracted from another structure revealing the difference in conformation between the structures.

Modification interference

An RNA sequence is chemically modified so that a proportion of molecules cannot function correctly in a given assay. The RNA that cannot function is recovered and the site of modification is determined.

Footprinting technique

A technique that determines the site of a nucleic acid–protein interaction using the fact that a protein bound to a nucleic-acid region will protect it from enzymatic cleavage.

Translocation

The movement of an mRNA across the ribosome by one codon together with the movement of tRNAs between the aminoacyl, peptidyl and exit sites, catalysed by elongation factors and GTP hydrolysis.

Aminoacyl (A) site

The site on the small ribosomal subunit that holds the incoming tRNA molecule that is charged with an amino acid.

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Fraser, C., Doudna, J. Structural and mechanistic insights into hepatitis C viral translation initiation. Nat Rev Microbiol 5, 29–38 (2007). https://doi.org/10.1038/nrmicro1558

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