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A new twist to translation initiation of the hepatitis C virus
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HEPATOLOGY ELSEWHERE
HEPATOLOGY, March 2002
Comments
A New Twist to Translation Initiation of the Hepatitis C Virus Spahn CMT, Kieft JS, Grassucci RA, Penczek PA, Zhou KH, Doudna JA, Frank J. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40S ribosomal subunit. Reprinted with permission from Science 2001;291:1959-1962. Copyright 2001 American Association for the Advancement of Science.
Abstract Abstract available in print only.
Translation initiation in eukaryotes is a complex process that requires interaction of the messenger RNA (mRNA) with various cellular factors, the initiator tRNA (Met-tRNAi) as well as the large (60S) and small (40S) subunits of the ribosome.1 First, the 5⬘ end of the mRNA bearing a m7GpppN cap structure (where N can be any nucleotide) is recognized by eukaryotic initiation factor 4F (eIF4F). Subsequently, the 43S particle (comprising the 40S small ribosomal subunit, eIF2/MettRNAi, and eIF3) binds to the mRNA/eIF4F complex to form a functional 48S preinitiation complex. This complex scans the mRNA for the first AUG initiation codon, where the 60S ribosomal subunit joins the paused 40S subunit to form the 80S ribosome, and protein synthesis begins. In contrast, hepatitis C virus (HCV) uses an alternative mechanism to initiate translation of its genome.2 Here, translation is initiated in a cap-independent manner on an RNA element located in the 5⬘ noncoding region, termed internal ribosome entry site (IRES). HCV IRES– driven translation initiation occurs through direct recognition of the 40S subunit and eIF3 by the IRES RNA tertiary structure, eliminating the need for the cap structure and other cap-binding factors. This mechanism of translation initiation, which was first described for picornavirus genomes,3,4 is utilized only by a few cellular mRNAs.5 Although secondary structure predictions based on chemical and enzymatic probing have been reported for the HCV IRES, allowing the division into domains II-IV, and cofactors such as eIF3 have been defined,6 the molecular mechanisms underlying recruitment of eIF3 and precise placement of the start codon in the decoding center of the ribosome are not understood. In their very elegant work, Spahn et al. combined cryo-electron microscopy (cryo-EM) and 3-dimensional modeling to reveal the first 3-dimensional view of the interaction between the HCV IRES and the 40S subunit of the ribosome (Fig. 1). Three-dimensional reconstruction of a vacant 40S ribosomal subunit at 20 Å resolution clearly showed its main structural features, such as the body, head, and platform domains (Fig. 1 A-C). When bound to the 40S ribosomal subunit, the HCV IRES RNA adopts a single conformation (Fig. 1D-F). This is in contrast to previous observations with the unbound HCV IRES RNA,7 but would be consistent with the notion that the IRES RNA may hold the downstream coding RNA in position until the translation machinery is assembled and the ribosome is in transition to form the first peptide bond.8 The investigators took advantage of a domain II deletion construct with nearly wild-type binding affinity to assign the structural domains of the IRES RNA (Fig. 1G-I). Domain II was found to be in contact with the 40S ribosome head and platform. This is the exclusive interaction in close vicinity to the active site of the ribosome. Therefore, domain II may facilitate the placement of the coding RNA in the ribosomal decoding center. Domain III is making various contacts with the 40S ribosomal subunit.9 How-
HEPATOLOGY, Vol. 35, No. 3, 2002
ever, domain IIIb is not involved in 40S subunit binding, but points away from the surface of the 40S subunit and is responsible for the interaction with eIF3 (Fig. 2). Most remarkably, interaction with the HCV IRES was found to induce a conformational change of the 40S ribosomal subunit (compare Fig. 1A-C with D-F), which is absent when domain II is missing (Fig. 1G-I). Thus, this structured viral RNA element actively manipulates the cellular translation machinery. The conformational alterations are rather complex, but the net outcome is an alignment of the ribosome head toward the body. Rotation of the head domain results in an extensive fusion of the head with the shoulder part of the body. In addition, the different parts of the 40S ribosomal subunit, such as the platform and the head, change their shape (marked by asterisks in Fig. 1). Currently, the purpose of the ribosomal bending during the initiation step of cap-independent translation is unknown. However, it may structurally mimic the function of canonical initiation factors involved in capdependent translation.
HEPATOLOGY ELSEWHERE
725
Image available in print only
Fig. 2. Assignment of the structural domains identified previously by chemical and enzymatic probing to the cryo-EM structure. Arrows link the tertiary structure domains to the corresponding elements within the IRES RNA cryo-EM structure. Nucleotides that were previously reported to be inaccessible to solvent upon 40S subunit binding are circled in red.14 The location of the eIF3 binding site on domain IIIa/b is boxed in blue. The magenta arrow delineates the probable location of the coding RNA in the mRNA binding groove. (Reprinted with permission from Spahn CMT, et al. Science 2001;291:1959-1962. Copyright 2001 American Association for the Advancement of Science.)
Image available in print only
Fig. 1. Surface representation of the vacant 40S ribosomal subunit (A-C), and the 40S subunit in complex with the HCV IRES RNA (D-F), or the domain II-deleted HCV IRES RNA (G-I). Cryo-EM maps are shown in yellow. Difference maps corresponding to the HCV RNA are superposed and shown in purple. The views are from the 60S side of the 40S subunit (A, D, and G), the platform side (B, E, and H), and the solvent side (C, F, and I). b, body; bk, beak; h, head; pt, platform; sh, shoulder. Entry and exit denote the proposed entry and exit path, respectively, of the mRNA. Conformational changes in the HCV IRES RNA-40S subunit complex are indicated by asterisks (D and F). (Reprinted with permission from Spahn CMT, et al. Science 2001;291:1959-1962. Copyright 2001 American Association for the Advancement of Science.)
At the fundamental level, the data raise the fascinating question whether all IRES elements actively manipulate the cellular translation machinery to initiate translation. Furthermore, it is of considerable interest whether eukaryotic mRNAs translated by a capdependent mechanism induce conformational changes of the ribosome as well, which would suggest a phylogenically highly conserved mechanism. Finally, the findings reported in this study provide a first glimpse into an essential step of the HCV life cycle that represents an attractive target for antiviral intervention.10 Understanding of the complex interactions between the HCV IRES and the host cell translation machinery is crucial for the design of drugs intervening at the step of HCV IRES–mediated translation. In this context, antisense oligonucleotides,11 ribozymes,12 and a small yeast inhibitory RNA13 have been found, among others, to inhibit translation from the HCV IRES. Rational design and optimization of small molecules with similar properties may provide a new class of antivirals against hepatitis C. RAINER GOSERT, PH.D. DARIUS MORADPOUR, M.D. Department of Medicine II University of Freiburg Freiburg, Germany
References 1. Kozak M. The scanning model for translation: an update. J Cell Biol 1989;108:229-241.
726
HEPATOLOGY ELSEWHERE
2. Tsukiyama-Kohara K, Iizuka N, Kohara M, Nomoto A. Internal ribosome entry site within hepatitis C virus RNA. J Virol 1992;66: 1476-1483. 3. Pelletier J, Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 1988;334:320-325. 4. Jang SK, Kra¨usslich H-G, Nicklin MJH, Duke GM, Palmenberg AC, Wimmer E. A segment of the 5⬘ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 1988;62:2636-2643. 5. Hellen CUT, Sarnow P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev 2001;15:1593-1612. 6. Rijnbrand RC, Lemon SM. Internal ribosome entry site-mediated translation in hepatitis C virus replication. Curr Top Microbiol Immunol 2000;242:85-116. 7. Kieft JS, Zhou K, Jubin R, Murray MG, Lau JY, Doudna JA. The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold. J Mol Biol 1999;292:513-529. 8. Kolupaeva VG, Pestova TV, Hellen CUT. An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. J Virol 2000;74: 6242-6250.
HEPATOLOGY, March 2002
9. Sizova DV, Kolupaeva VG, Pestova TV, Shatsky IN, Hellen CU. 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 1998;72:4775-4782. 10. Jubin R. Hepatitis C IRES: translating translation into a therapeutic target. Curr Opin Mol Ther 2001;3:278-287. 11. Wakita T, Wands JR. Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides. J Biol Chem 1994;269: 14205-14210. 12. Macejak DG, Jensen KL, Jamison SF, Domenico K, Roberts EC, Chaudhary N, von Carlowitz I, et al. Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes. HEPATOLOGY 2000;31:769-776. 13. Das S, Ott M, Yamane A, Tsai W, Gromeier M, Lahser F, Gupta S, et al. A small yeast RNA blocks hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation and inhibits replication of a chimeric poliovirus under translational control of the HCV IRES element. J Virol 1998;72:5638-5647. 14. Kieft JS, Zhou K, Jubin R, Doudna JA. Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 2001;7:194-206.
HEPATOLOGY ELSEWHERE
HEPATOLOGY, March 2002
Comments
A New Twist to Translation Initiation of the Hepatitis C Virus Spahn CMT, Kieft JS, Grassucci RA, Penczek PA, Zhou KH, Doudna JA, Frank J. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40S ribosomal subunit. Reprinted with permission from Science 2001;291:1959-1962. Copyright 2001 American Association for the Advancement of Science.
Abstract Abstract available in print only.
Translation initiation in eukaryotes is a complex process that requires interaction of the messenger RNA (mRNA) with various cellular factors, the initiator tRNA (Met-tRNAi) as well as the large (60S) and small (40S) subunits of the ribosome.1 First, the 5⬘ end of the mRNA bearing a m7GpppN cap structure (where N can be any nucleotide) is recognized by eukaryotic initiation factor 4F (eIF4F). Subsequently, the 43S particle (comprising the 40S small ribosomal subunit, eIF2/MettRNAi, and eIF3) binds to the mRNA/eIF4F complex to form a functional 48S preinitiation complex. This complex scans the mRNA for the first AUG initiation codon, where the 60S ribosomal subunit joins the paused 40S subunit to form the 80S ribosome, and protein synthesis begins. In contrast, hepatitis C virus (HCV) uses an alternative mechanism to initiate translation of its genome.2 Here, translation is initiated in a cap-independent manner on an RNA element located in the 5⬘ noncoding region, termed internal ribosome entry site (IRES). HCV IRES– driven translation initiation occurs through direct recognition of the 40S subunit and eIF3 by the IRES RNA tertiary structure, eliminating the need for the cap structure and other cap-binding factors. This mechanism of translation initiation, which was first described for picornavirus genomes,3,4 is utilized only by a few cellular mRNAs.5 Although secondary structure predictions based on chemical and enzymatic probing have been reported for the HCV IRES, allowing the division into domains II-IV, and cofactors such as eIF3 have been defined,6 the molecular mechanisms underlying recruitment of eIF3 and precise placement of the start codon in the decoding center of the ribosome are not understood. In their very elegant work, Spahn et al. combined cryo-electron microscopy (cryo-EM) and 3-dimensional modeling to reveal the first 3-dimensional view of the interaction between the HCV IRES and the 40S subunit of the ribosome (Fig. 1). Three-dimensional reconstruction of a vacant 40S ribosomal subunit at 20 Å resolution clearly showed its main structural features, such as the body, head, and platform domains (Fig. 1 A-C). When bound to the 40S ribosomal subunit, the HCV IRES RNA adopts a single conformation (Fig. 1D-F). This is in contrast to previous observations with the unbound HCV IRES RNA,7 but would be consistent with the notion that the IRES RNA may hold the downstream coding RNA in position until the translation machinery is assembled and the ribosome is in transition to form the first peptide bond.8 The investigators took advantage of a domain II deletion construct with nearly wild-type binding affinity to assign the structural domains of the IRES RNA (Fig. 1G-I). Domain II was found to be in contact with the 40S ribosome head and platform. This is the exclusive interaction in close vicinity to the active site of the ribosome. Therefore, domain II may facilitate the placement of the coding RNA in the ribosomal decoding center. Domain III is making various contacts with the 40S ribosomal subunit.9 How-
HEPATOLOGY, Vol. 35, No. 3, 2002
ever, domain IIIb is not involved in 40S subunit binding, but points away from the surface of the 40S subunit and is responsible for the interaction with eIF3 (Fig. 2). Most remarkably, interaction with the HCV IRES was found to induce a conformational change of the 40S ribosomal subunit (compare Fig. 1A-C with D-F), which is absent when domain II is missing (Fig. 1G-I). Thus, this structured viral RNA element actively manipulates the cellular translation machinery. The conformational alterations are rather complex, but the net outcome is an alignment of the ribosome head toward the body. Rotation of the head domain results in an extensive fusion of the head with the shoulder part of the body. In addition, the different parts of the 40S ribosomal subunit, such as the platform and the head, change their shape (marked by asterisks in Fig. 1). Currently, the purpose of the ribosomal bending during the initiation step of cap-independent translation is unknown. However, it may structurally mimic the function of canonical initiation factors involved in capdependent translation.
HEPATOLOGY ELSEWHERE
725
Image available in print only
Fig. 2. Assignment of the structural domains identified previously by chemical and enzymatic probing to the cryo-EM structure. Arrows link the tertiary structure domains to the corresponding elements within the IRES RNA cryo-EM structure. Nucleotides that were previously reported to be inaccessible to solvent upon 40S subunit binding are circled in red.14 The location of the eIF3 binding site on domain IIIa/b is boxed in blue. The magenta arrow delineates the probable location of the coding RNA in the mRNA binding groove. (Reprinted with permission from Spahn CMT, et al. Science 2001;291:1959-1962. Copyright 2001 American Association for the Advancement of Science.)
Image available in print only
Fig. 1. Surface representation of the vacant 40S ribosomal subunit (A-C), and the 40S subunit in complex with the HCV IRES RNA (D-F), or the domain II-deleted HCV IRES RNA (G-I). Cryo-EM maps are shown in yellow. Difference maps corresponding to the HCV RNA are superposed and shown in purple. The views are from the 60S side of the 40S subunit (A, D, and G), the platform side (B, E, and H), and the solvent side (C, F, and I). b, body; bk, beak; h, head; pt, platform; sh, shoulder. Entry and exit denote the proposed entry and exit path, respectively, of the mRNA. Conformational changes in the HCV IRES RNA-40S subunit complex are indicated by asterisks (D and F). (Reprinted with permission from Spahn CMT, et al. Science 2001;291:1959-1962. Copyright 2001 American Association for the Advancement of Science.)
At the fundamental level, the data raise the fascinating question whether all IRES elements actively manipulate the cellular translation machinery to initiate translation. Furthermore, it is of considerable interest whether eukaryotic mRNAs translated by a capdependent mechanism induce conformational changes of the ribosome as well, which would suggest a phylogenically highly conserved mechanism. Finally, the findings reported in this study provide a first glimpse into an essential step of the HCV life cycle that represents an attractive target for antiviral intervention.10 Understanding of the complex interactions between the HCV IRES and the host cell translation machinery is crucial for the design of drugs intervening at the step of HCV IRES–mediated translation. In this context, antisense oligonucleotides,11 ribozymes,12 and a small yeast inhibitory RNA13 have been found, among others, to inhibit translation from the HCV IRES. Rational design and optimization of small molecules with similar properties may provide a new class of antivirals against hepatitis C. RAINER GOSERT, PH.D. DARIUS MORADPOUR, M.D. Department of Medicine II University of Freiburg Freiburg, Germany
References 1. Kozak M. The scanning model for translation: an update. J Cell Biol 1989;108:229-241.
726
HEPATOLOGY ELSEWHERE
2. Tsukiyama-Kohara K, Iizuka N, Kohara M, Nomoto A. Internal ribosome entry site within hepatitis C virus RNA. J Virol 1992;66: 1476-1483. 3. Pelletier J, Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 1988;334:320-325. 4. Jang SK, Kra¨usslich H-G, Nicklin MJH, Duke GM, Palmenberg AC, Wimmer E. A segment of the 5⬘ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 1988;62:2636-2643. 5. Hellen CUT, Sarnow P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev 2001;15:1593-1612. 6. Rijnbrand RC, Lemon SM. Internal ribosome entry site-mediated translation in hepatitis C virus replication. Curr Top Microbiol Immunol 2000;242:85-116. 7. Kieft JS, Zhou K, Jubin R, Murray MG, Lau JY, Doudna JA. The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold. J Mol Biol 1999;292:513-529. 8. Kolupaeva VG, Pestova TV, Hellen CUT. An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. J Virol 2000;74: 6242-6250.
HEPATOLOGY, March 2002
9. Sizova DV, Kolupaeva VG, Pestova TV, Shatsky IN, Hellen CU. 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 1998;72:4775-4782. 10. Jubin R. Hepatitis C IRES: translating translation into a therapeutic target. Curr Opin Mol Ther 2001;3:278-287. 11. Wakita T, Wands JR. Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides. J Biol Chem 1994;269: 14205-14210. 12. Macejak DG, Jensen KL, Jamison SF, Domenico K, Roberts EC, Chaudhary N, von Carlowitz I, et al. Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes. HEPATOLOGY 2000;31:769-776. 13. Das S, Ott M, Yamane A, Tsai W, Gromeier M, Lahser F, Gupta S, et al. A small yeast RNA blocks hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation and inhibits replication of a chimeric poliovirus under translational control of the HCV IRES element. J Virol 1998;72:5638-5647. 14. Kieft JS, Zhou K, Jubin R, Doudna JA. Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 2001;7:194-206.