Siridechadilok B, Fraser CS, Hall RJ, Doudna JA, Nogales E. Structural roles for human translation factor eIF3 in initiation of protein synthesis. Science 2005;310:1513–1515. (Reprinted with permission from AAAS.)
Protein synthesis in mammalian cells requires initiation factor eIF3, a ∼750-kilodalton complex that controls assembly of 40S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5′-cap or an internal ribosome entry site (IRES). Cryoelectron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5′-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly.
Translation initiation in eukaryotes is a complex process that requires interaction of the messenger RNA (mRNA) with various cellular factors, the initiator transfer RNA (Met-tRNAi) as well as the small (40S) and large (60S) subunits of the ribosome.1–3 First, the 5′ end of the mRNA bearing a methylated guanosine (5′-m7G) cap is recognized by eukaryotic initiation factor 4F (eIF4F). Subsequently, the mRNA · eIF4F complex is recruited by the 43S preinitiation complex composed of eIF1, eIF1A, eIF3, eIF5, the 40S small ribosomal subunit and the eIF2 · Met-tRNAi · GTP ternary complex to form a 48S initiation complex. This complex scans the mRNA for the start codon, where the initiation factors dissociate, the 60S ribosomal subunit joins the 40S subunit to form the 80S ribosome, and protein synthesis begins.
Different from this canonical mechanism, translation of HCV is initiated in a cap-independent manner on a highly structured RNA element located in the 5′ noncoding region (5′ NCR) of the viral positive-strand RNA genome, termed internal ribosome entry site (IRES). The HCV 5′ NCR contains 4 highly ordered domains numbered I to IV. Domains II, III, and IV, together with the first 24-40 nucleotides of the core coding region, constitute the IRES (reviewed in Moradpour and Rice.4). The key element is domain III that permits direct binding of the 40S ribosomal subunit and of eIF3.5 Domain III includes a large stem-loop, with subdomains a, b, and c forming a critical four-way junction. Previous studies have shown that this part of domain III interacts with eIF3. However, the exact binding site remains controversial.5–7 Domain III also includes subdomains d, e, and f of which the latter forms a so-called pseudoknot with domain IV that contains the translation initiation codon.
IRES-mediated translation has been discovered in several viral RNAs and, more recently, in a number of cellular mRNAs.8–10 In the case of HCV, translation initiation occurs through formation of a binary complex between the IRES and the 40S ribosomal subunit, followed by assembly of a 48S-like complex at the AUG initiation codon upon association of eIF3 and ternary complex (eIF2 · Met-tRNAi · GTP) and, in a rate limiting step, GTP-dependent association of the 60S subunit to form the 80S complex.11
The 3-dimensional structure of the HCV IRES bound to the 40S ribosomal subunit was resolved at 20 Å resolution by cryoelectron microscopy.12 Strikingly, it was found that IRES binding induces significant conformational changes in the 40S subunit, indicating that the HCV IRES dynamically modulates the host translational machinery.
Although HCV translation can be initiated without the presence of canonical initiation factors like eIF4F, eIF3 is indispensable for the formation of the active 80S complex.13 Thus, eIF3, a large complex comprising at least 12 proteins, plays a central role in both cap-dependent as well as IRES-mediated translation initiation. Moreover, eIF3 prevents premature association of 40S and 60S ribosomal subunits,14 interacts with other initiation factors, and helps assemble active ribosomes.
In the fascinating paper under discussion, Siridechadilok et al. provide a structural basis for the activities of eIF3 and its interaction with the translation machinery. Their work reveals unexpected structural similarities between the HCV IRES and the canonical initiation factor eIF4F.15
The 3-dimensional structure of eIF3 was determined at ∼30 Å resolution by cryoelectron microscopy and image reconstruction. The authors show that eIF3 forms a five-lobed particle with anthropomorphic features that have been used to name the 5 domains according to body parts, including a head, arms, and legs (Fig. 1). Further analyses allowed the authors to resolve the structure of the eIF3 · HCV IRES complex and to identify the IRES binding sites on eIF3. The IRES was found to extend across eIF3, from the left arm to the right leg (Fig. 2). Based on previous mapping,12 domain IIId/e/f appears to be located near the central region of eIF3, domain IIIa/b/c is near the right leg, and domain II corresponds to a flexible region emanating from the left arm of eIF3.
By using the structure of the HCV IRES · 40S binary complex described by Spahn et al.,12 the authors produced a model of the eIF3 · HCV IRES · 40S complex (Fig. 3). In this ternary complex, the front of the eIF3 left leg was found to be located below the so-called platform domain of the 40S ribosomal subunit, near the binding site of the 60S subunit, whereas the left arm was found to point toward the tRNA exit (E) site. The position of eIF3 in this model provides a structural explanation for its role in preventing premature joining of 40S and 60S subunits in that the left toe of eIF3 covers a critical contact site between the 2 subunits.
Finally, the authors compared the roles of the HCV IRES and the canonical initiation factor eIF4G, a component of the eIF4F cap-binding complex, during translation initiation. This factor has been shown to directly interact with eIF3 in cap-mediated translation initiation.2, 3 Interestingly, the authors showed that eIF3 interacts with part of the HCV IRES and the cap-binding complex via the same domain at the tip of its left arm. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit revealed that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the cellular mRNA or positive-strand viral RNA, respectively, near the E site of 40S, promoting initiation complex assembly. This structurally analogous positioning implies mechanistic overlap, possibly explaining why HCV does not need eIF4F for polyprotein translation.
Taken together, the interaction of eIF3 with eIF4F or the HCV IRES is an important step in the recruitment of active ribosomes, by correctly positioning the mRNA or the viral RNA, respectively, on the 40S subunit. In cap-mediated translation, the AUG translation initiation codon is recognized by a scanning mechanism in which eIF4F plays a crucial role. By contrast, in HCV IRES-mediated translation, the initiation codon is directly positioned in the ribosomal peptidyl (P) site, i.e., in the site where amino acids are added to the nascent polypeptide chain. Conformational changes in both the HCV IRES and the 40S ribosome may be required for this positioning. Indeed, as discussed above, domain II induces conformational changes within the 40S subunit that appear essential to position the viral RNA in the mRNA channel.12 However, it is unknown if these domain II-dependent conformational changes are sufficient to correctly position the start codon in the P site. Based on the results of the study by Siridechadilok et al., eIF3 may play a crucial role in this process by interacting with domain III. In this context, a recent study comparing HCV IRES · 40S and HCV IRES · 80S structures has demonstrated conformational changes in the IIIabc four-way junction as well as the pseudoknot that could have been induced by eIF3.16 Higher resolution structures should yield further insight into the interactions of the HCV IRES with eIF3 and 40S and should result in an improved understanding of the molecular mechanisms involved in formation of active 80S ribosomes.
In conclusion, the work by Siridechadilok et al. provides exciting structural insights into a fundamental biological process and an essential step of the HCV life cycle that represents a potential target for antiviral intervention. Understanding of the complex interactions between the HCV IRES and the host cell translation machinery is crucial for the design of compounds intervening at the step of polyprotein translation. Rational design and optimization of small molecules that block the interaction of the HCV IRES with eIF3 or the 40S ribosome may provide a new class of antivirals against hepatitis C.