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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Rosenberg, S. Recent advances in the molecular biology of hepatitis C virus. J. Mol. Biol. 313, 451–464 (2001).
Friebe, P., Lohmann, V., Krieger, N. & Bartenschlager, R. Sequences in the 5′ nontranslated region of hepatitis C virus required for RNA replication. J. Virol. 75, 12047–12057 (2001).
Jopling, C. L., Yi, M., Lancaster, A. M., Lemon, S. M. & Sarnow, P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 309, 1577–1581 (2005).
Sarnow, P., Jopling, C. L., Norman, K. L., Schutz, S. & Wehner, K. A. MicroRNAs: expression, avoidance and subversion by vertebrate viruses. Nature Rev. Microbiol. 4, 651–659 (2006). Shows that a liver-specific miRNA binds to the 5′UTR of HCV and increases viral RNA production, most likely at the stage of replication.
Tsukiyama-Kohara, K., Iizuka, N., Kohara, M. & Nomoto, A. Internal ribosome entry site within hepatitis C virus RNA. J. Virol. 66, 1476–1483 (1992).
Wang, C., Sarnow, P. & Siddiqui, A. Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J. Virol. 67, 3338–3344 (1993).
Brown, E. A., Zhang, H., Ping, L. H. & Lemon, S. M. Secondary structure of the 5′ non-translated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acids Res. 20, 5041–5045 (1992).
Honda, M., Brown, E. A. & Lemon, S. M. Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA. RNA 2, 955–968 (1996).
Hellen, C. U. & Pestova, T. V. Translation of hepatitis C virus RNA. J. Viral Hepat. 6, 79–87 (1999).
Rijnbrand, R. C. & Lemon, S. M. Internal ribosome entry site-mediated translation in hepatitis C virus replication. Curr. Top. Microbiol. Immunol. 242, 85–116 (2000).
Kieft, J. S. et al. The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold. J. Mol. Biol. 292, 513–529 (1999).
Melcher, S. E., Wilson, T. J. & Lilley, D. M. The dynamic nature of the four-way junction of the hepatitis C virus IRES. RNA 9, 809–820 (2003).
Fukushi, S. et al. Complete 5′ noncoding region is necessary for the efficient internal initiation of hepatitis C virus RNA. Biochem. Biophys. Res. Commun. 199, 425–432 (1994).
Rijnbrand, R. et al. Almost the entire 5′ non-translated region of hepatitis C virus is required for cap-independent translation. FEBS Lett. 365, 115–119 (1995).
Honda, M. et al. Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA. Virology 222, 31–42 (1996).
Kamoshita, N., Tsukiyama-Kohara, K., Kohara, M. & Nomoto, A. Genetic analysis of internal ribosomal entry site on hepatitis C virus RNA: implication for involvement of the highly ordered structure and cell type-specific transacting factors. Virology 233, 9–18 (1997).
Rijnbrand, R., Abell, G. & Lemon, S. M. Mutational analysis of the GB virus B internal ribosome entry site. J. Virol. 74, 773–783 (2000).
Psaridi, L., Georgopoulou, U., Varaklioti, A. & Mavromara, P. Mutational analysis of a conserved tetraloop in the 5′ untranslated region of hepatitis C virus identifies a novel RNA element essential for the internal ribosome entry site function. FEBS lett. 453, 49–53 (1999).
Staehelin, T., Erni, B. & Schreier, M. H. Purification and characterization of seven initiation factors for mammalian protein synthesis. Methods Enzymol. 60, 136–165 (1979).
Benne, R., Brown-Luedi, M. L. & Hershey, J. W. Protein synthesis initiation factors from rabbit reticulocytes: purification, characterization, and radiochemical labeling. Methods Enzymol. 60, 15–35 (1979).
Pestova, T. V., Shatsky, I. N., Fletcher, S. P., Jackson, R. J. & Hellen, C. U. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev. 12, 67–83 (1998). This paper provided the first evidence that a eukaryotic ribosome could recognize and bind directly to an mRNA in the absence of initiation factors. This was significant as at the time it was believed that only the prokaryotic ribosome could bind directly to an mRNA.
Shine, J. & Dalgarno, L. Determinant of cistron specificity in bacterial ribosomes. Nature 254, 34–38 (1975).
Reynolds, J. E. et al. Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J. 14, 6010–6020 (1995).
Reynolds, J. E. et al. Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon. RNA 2, 867–878 (1996).
Rijnbrand, R. C., Abbink, T. E., Haasnoot, P. C., Spaan, W. J. & Bredenbeek, P. J. The influence of AUG codons in the hepatitis C virus 5′ nontranslated region on translation and mapping of the translation initiation window. Virology 226, 47–56 (1996).
Kolupaeva, V. G., Pestova, T. V. & Hellen, C. U. An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. J. Virol. 74, 6242–6250 (2000).
Kieft, J. S., Zhou, K., Jubin, R. & Doudna, J. A. Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 7, 194–206 (2001).
Lytle, J. R., Wu, L. & Robertson, H. D. The ribosome binding site of hepatitis C virus mRNA. J. Virol. 75, 7629–7636 (2001).
Lytle, J. R., Wu, L. & Robertson, H. D. Domains on the hepatitis C virus internal ribosome entry site for 40s subunit binding. RNA 8, 1045–4055 (2002).
Rijnbrand, R., van der Straaten, T., van Rijn, P. A., Spaan, W. J. & Bredenbeek, P. J. Internal entry of ribosomes is directed by the 5′ noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon. J. Virol. 71, 451–457 (1997).
Trachsel, H., Erni, B., Schreier, M. H. & Staehelin, T. Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors. J. Mol. Biol. 116, 755–767 (1977).
Benne, R. & Hershey, J. W. The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes. J. Biol. Chem. 253, 3078–3087 (1978).
Ji, H., Fraser, C. S., Yu, Y., Leary, J. & Doudna, J. A. Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA. Proc. Natl Acad. Sci. USA 101, 16990–16995 (2004).
Sizova, D. V., Kolupaeva, V. G., Pestova, T. V., Shatsky, I. N. & Hellen, C. U. Specific interaction of eukaryotic translation initiation factor 3 with the 5′ nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J. Virol. 72, 4775–4782 (1998).
Buratti, E., Tisminetzky, S., Zotti, M. & Baralle, F. E. Functional analysis of the interaction between HCV 5′UTR and putative subunits of eukaryotic translation initiation factor eIF3. Nucleic Acids Res. 26, 3179–3187 (1998).
Otto, G. A. & Puglisi, J. D. The pathway of HCV IRES-mediated translation initiation. Cell 119, 369–380 (2004).
Collier, A. J. et al. A conserved RNA structure within the HCV IRES eIF3-binding site. Nature Struct. Biol. 9, 375–380 (2002).
Kieft, J. S., Zhou, K., Grech, A., Jubin, R. & Doudna, J. A. Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation. Nature Struct. Biol. 9, 370–374 (2002).
Rijnbrand, R., Thiviyanathan, V., Kaluarachchi, K., Lemon, S. M. & Gorenstein, D. G. Mutational and structural analysis of stem-loop IIIC of the hepatitis C virus and GB virus B internal ribosome entry sites. J. Mol. Biol. 343, 805–817 (2004).
Lukavsky, P. J., Otto, G. A., Lancaster, A. M., Sarnow, P. & Puglisi, J. D. Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function. Nature Struct. Biol. 7, 1105–1110 (2000).
Lukavsky, P. J., Kim, I., Otto, G. A. & Puglisi, J. D. Structure of HCV IRES domain II determined by NMR. Nature Struct. Biol. 10, 1033–1038 (2003). The NMR structure of domain II shows that this domain can fold in the absence of the 40S ribosomal subunit.
Klinck, R. et al. A potential RNA drug target in the hepatitis C virus internal ribosomal entry site. RNA 6, 1423–1431 (2000).
Spahn, C. M. et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit. Science 291, 1959–1962 (2001). The first report of a cryo-EM reconstruction of an IRES bound to a eukaryotic ribosome. It identifies how the HCV IRES can position itself on the surface of the 40S ribosomal subunit and induce conformational changes in the 40S ribosomal subunit.
Laletina, E. et al. Proteins surrounding hairpin IIIe of the hepatitis C virus internal ribosome entry site on the human 40S ribosomal subunit. Nucleic Acids Res. 34, 2027–2036 (2006).
Otto, G. A., Lukavsky, P. J., Lancaster, A. M., Sarnow, P. & Puglisi, J. D. Ribosomal proteins mediate the hepatitis C virus IRES-HeLa 40S interaction. RNA 8, 913–923 (2002).
Lata, K. R. et al. Three-dimensional reconstruction of the Escherichia coli 30 S ribosomal subunit in ice. J. Mol. Biol. 262, 43–52 (1996).
Yusupova, G. Z., Yusupov, M. M., Cate, J. H. & Noller, H. F. The path of messenger RNA through the ribosome. Cell 106, 233–241 (2001).
Siridechadilok, B., Fraser, C. S., Hall, R. J., Doudna, J. A. & Nogales, E. Structural roles for human translation factor eIF3 in initiation of protein synthesis. Science 310, 1513–1515 (2005). This cryo-EM reconstruction revealed how the HCV IRES can associate with eIF3.
Srivastava, S., Verschoor, A. & Frank, J. Eukaryotic initiation factor 3 does not prevent association through physical blockage of the ribosomal subunit–subunit interface. J. Mol. Biol. 226, 301–304 (1992).
Hershey, J. W. B. & Merrick, W. C. in Translational Control of Gene Expression ( eds Sonenberg, N. et al.) 33–188 (Cold Spring Harbour Laboratory Press, New York, 2000).
Culver, G. M., Cate, J. H., Yusupova, G. Z., Yusupov, M. M. & Noller, H. F. Identification of an RNA-protein bridge spanning the ribosomal subunit interface. Science 285, 2133–2136 (1999).
Maivali, U. & Remme, J. Definition of bases in 23S rRNA essential for ribosomal subunit association. RNA 10, 600–604 (2004).
Spahn, C. M. et al. Structure of the 80S ribosome from Saccharomyces cerevisiae — tRNA-ribosome and subunit–subunit interactions. Cell 107, 373–386 (2001).
Kolupaeva, V. G., Unbehaun, A., Lomakin, I. B., Hellen, C. U. & Pestova, T. V. Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association. RNA 11, 470–486 (2005).
Boehringer, D., Thermann, R., Ostareck-Lederer, A., Lewis, J. D. & Stark, H. Structure of the hepatitis C Virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES. Structure (Camb) 13, 1695–1706 (2005). This cryo-EM reconstruction of the HCV IRES bound to the 80S ribosome shows details of the changes in conformation of the 40S ribosomal subunit on 60S joining.
Frank, J. & Agrawal, R. K. A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406, 318–322 (2000).
Schuwirth, B. S. et al. Structures of the bacterial ribosome at 3.5 Å resolution. Science 310, 827–834 (2005).
Valle, M. et al. Locking and unlocking of ribosomal motions. Cell 114, 123–134 (2003).
Pisarev, A. V., Shirokikh, N. E. & Hellen, C. U. Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites. C. R. Biol. 328, 589–605 (2005).
Bordeleau, M. E. et al. Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A. Nature Chem. Biol. 2, 213–220 (2006). A study that used an inhibitor of the eIF4A to distinguish between protein synthesis directed by HCV-type IRES elements and picornavirus IRES elements.
Wilson, J. E., Pestova, T. V., Hellen, C. U. & Sarnow, P. Initiation of protein synthesis from the A site of the ribosome. Cell 102, 511–520 (2000).
Cevallos, R. C. & Sarnow, P. Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp. J. Virol. 79, 677–683 (2005).
Spahn, C. M. et al. Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor. Cell 118, 465–475 (2004).
Jang, S. K. et al. A segment of the 5′ non-translated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J. Virol. 62, 2636–2643 (1988).
Pelletier, J. & Sonenberg, N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320–325 (1988). References 64 and 65 describe the identification of the first mRNAs to initiate protein synthesis by a cap-independent mechanism.
Chen, C. Y. & Sarnow, P. Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science 268, 415–417 (1995).
Jackson, R. in Translational Control of Gene Expression ( eds Sonenberg, N. et al.) 127–184 (Cold Spring Harbour Laboratory Press, New York, 2000).
Hellen, C. U. & Sarnow, P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15, 1593–1612 (2001).
Jackson, R. J. Alternative mechanisms of initiating translation of mammalian mRNAs. Biochem. Soc. Trans. 33, 1231–1241 (2005).
Pestova, T. V., Hellen, C. U. & Shatsky, I. N. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol. Cell Biol. 16, 6859–6869 (1996).
Pestova, T. V., Shatsky, I. N. & Hellen, C. U. Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Mol. Cell. Biol. 16, 6870–6878 (1996).
Kolupaeva, V. G., Lomakin, I. B., Pestova, T. V. & Hellen, C. U. Eukaryotic initiation factors 4G and 4A mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal entry site. Mol. Cell. Biol. 23, 687–698 (2003).
Sengupta, J. et al. Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM. Nature Struct. Mol. Biol. 11, 957–962 (2004).
Ceci, M. et al. Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly. Nature 426, 579–584 (2003).
Morley, S. J. & Traugh, J. A. Differential stimulation of phosphorylation of initiation factors eIF-4F, eIF-4B, eIF-3, and ribosomal protein S6 by insulin and phorbol esters. J. Biol. Chem. 265, 10611–10616 (1990).
Lachance, P. E., Miron, M., Raught, B., Sonenberg, N. & Lasko, P. Phosphorylation of eukaryotic translation initiation factor 4E is critical for growth. Mol. Cell. Biol. 22, 1656–1663 (2002).
Raught, B. et al. Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI. EMBO J. 19, 434–444 (2000).
Raught, B., Gingras, A. C. & Sonenberg, N. in Translational Control of Gene Expression ( eds Sonenberg, N. et al.) 245–294 (Cold Spring Harbour Laboratory Press, New York, 2000).
Ruggero, D. & Sonenberg, N. The Akt of translational control. Oncogene 24, 7426–7434 (2005).
Nilsson, J., Sengupta, J., Frank, J. & Nissen, P. Regulation of eukaryotic translation by the RACK1 protein: a platform for signalling molecules on the ribosome. EMBO Rep. 5, 1137–1141 (2004).
Holz, M. K., Ballif, B. A., Gygi, S. P. & Blenis, J. mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events. Cell 123, 569–580 (2005).
Harris, T. E. et al. mTOR-dependent stimulation of the association of eIF4G and eIF3 by insulin. EMBO J. 25, 1659–1668 (2006).
Lindenbach, B. D. et al. Complete replication of hepatitis C virus in cell culture. Science 309, 623–626 (2005).
Wakita, T. et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nature Med. 11, 791–796 (2005).
Zhong, J. et al. Robust hepatitis C virus infection in vitro. Proc. Natl Acad. Sci. USA 102, 9294–9299 (2005). References 83, 84 and 85 describe the recent development of a cell-culture system to study the replication of HCV infectious particles.
Lindenbach, B. D. et al. Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc. Natl Acad. Sci. USA 103, 3805–3809 (2006).
Kozak, M. Pushing the limits of the scanning mechanism for initiation of translation. Gene 299, 1–34 (2002).
Kapp, L. D. & Lorsch, J. R. The molecular mechanics of eukaryotic translation. Annu. Rev. Biochem. 73, 657–704 (2004).
Merrick, W. C. Cap-dependent and cap-independent translation in eukaryotic systems. Gene 332, 1–11 (2004).
Hinnebusch, A. G. in Translational Control of Gene Expression ( eds Sonenberg, N. et al.) 185–244 (Cold Spring Harbour Laboratory Press, New York, 2000).
Hinnebusch, A. G. eIF3: a versatile scaffold for translation initiation complexes. Trends Biochem. Sci. 31, 553–562 (2006).
Kozak, M. Inability of circular mRNA to attach to eukaryotic ribosomes. Nature 280, 82–85 (1979).
Hinnebusch, A. G. Translational regulation of GCN4 and the general amino acid control of yeast. Annu. Rev. Microbiol. 59, 407–450 (2005).
Holcik, M. & Sonenberg, N. Translational control in stress and apoptosis. Nature Rev. Mol. Cell Biol. 6, 318–327 (2005).
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Related links
Related links
DATABASES
Entrez Genome Project
FURTHER INFORMATION
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.
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nrmicro1558
This article is cited by
-
A conserved RNA structural motif for organizing topology within picornaviral internal ribosome entry sites
Nature Communications (2019)
-
Heterogeneity and specialized functions of translation machinery: from genes to organisms
Nature Reviews Genetics (2018)
-
Direct and indirect parieto-medial temporal pathways for spatial navigation in humans: evidence from resting-state functional connectivity
Brain Structure and Function (2017)
-
Hepatitis C Virus-Genotype 3: Update on Current and Emergent Therapeutic Interventions
Current Infectious Disease Reports (2017)
-
RNA regulons in Hox 5′ UTRs confer ribosome specificity to gene regulation
Nature (2015)