The Israeli acute paralysis virus IRES captures host ribosomes by mimicking a ribosomal state with hybrid tRNAs
2019; Springer Nature; Volume: 38; Issue: 21 Linguagem: Inglês
10.15252/embj.2019102226
ISSN1460-2075
AutoresF.J. Acosta-Reyes, Ritam Neupane, Joachim Frank, I.S. Fernandez,
Tópico(s)Bacteriophages and microbial interactions
ResumoArticle14 October 2019Open Access Transparent process The Israeli acute paralysis virus IRES captures host ribosomes by mimicking a ribosomal state with hybrid tRNAs Francisco Acosta-Reyes Francisco Acosta-Reyes Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Ritam Neupane Ritam Neupane Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank [email protected] orcid.org/0000-0001-5449-6943 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Israel S Fernández Corresponding Author Israel S Fernández [email protected] orcid.org/0000-0001-7218-1603 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Francisco Acosta-Reyes Francisco Acosta-Reyes Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Ritam Neupane Ritam Neupane Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank [email protected] orcid.org/0000-0001-5449-6943 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Israel S Fernández Corresponding Author Israel S Fernández [email protected] orcid.org/0000-0001-7218-1603 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Author Information Francisco Acosta-Reyes1,‡, Ritam Neupane1,2,‡, Joachim Frank *,1,2 and Israel S Fernández *,1 1Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA 2Department of Biological Sciences, Columbia University, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 212 305 9512; E-mail: [email protected] *Corresponding author. Tel: +1 2 2 342 2385; E-mail: [email protected] The EMBO Journal (2019)38:e102226https://doi.org/10.15252/embj.2019102226 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Colony collapse disorder (CCD) is a multi-faceted syndrome decimating bee populations worldwide, and a group of viruses of the widely distributed Dicistroviridae family have been identified as a causing agent of CCD. This family of viruses employs non-coding RNA sequences, called internal ribosomal entry sites (IRESs), to precisely exploit the host machinery for viral protein production. Using single-particle cryo-electron microscopy (cryo-EM), we have characterized how the IRES of Israeli acute paralysis virus (IAPV) intergenic region captures and redirects translating ribosomes toward viral RNA messages. We reconstituted two in vitro reactions targeting a pre-translocation and a post-translocation state of the IAPV-IRES in the ribosome, allowing us to identify six structures using image processing classification methods. From these, we reconstructed the trajectory of IAPV-IRES from the early small subunit recruitment to the final post-translocated state in the ribosome. An early commitment of IRES/ribosome complexes for global pre-translocation mimicry explains the high efficiency observed for this IRES. Efforts directed toward fighting CCD by targeting the IAPV-IRES using RNA-interference technology are underway, and the structural framework presented here may assist in further refining these approaches. Synopsis Israeli acute paralysis virus (IAPV) affects honeybee hives worldwide and acts by hijacking the host ribosome for viral protein production through a non-coding RNA sequences termed IRESs. Cryo-EM studies now reveal how the IRES captures host ribosomes by mimicking tRNAs in hybrid configurations to bypass the initiation step, jumpstarting translation directly to the elongation phase. Cryo-EM reconstructions of the IAPV IRES in complex with eukaryotic ribosomes reveal molecular details of ribosome hijacking. Hybrid-tRNA mimicry allows the IAPV-IRES to divert ribosomes from canonical pathways thereby redirecting them towards the production of viral proteins. The IAPV IRES restricts the intrinsic dynamics of the 40S subunit's head to a configuration compatible with large subunit recruitment, and blocks binding sites for canonical initiation factors. Introduction Apis mellifera, the common western honey bee, is affected worldwide by an enigmatic syndrome characterized by a drastic disappearance of the workforce, causing the accelerated collapse of the hive (Ratnieks & Carreck, 2010). Given the essential role bees play in pollination of economically important crops, the impact of this syndrome, termed colony collapse disorder (CCD), has been estimated to cost the US economy $15 billion in direct loss of crops and $75 billion in indirect losses (Chopra et al, 2015). Though the exact etiology of CCD is unknown (Anderson & East, 2008), a group of viruses belonging to the Dicistroviridae family were found in metagenomic studies of CCD-affected hives (Cox-Foster et al, 2007; Chen et al, 2014). Among this group of viruses, the Israeli acute paralysis virus (IAPV) showed a strong correlation with CCD, revealing a prominent role in the development of the syndrome (Hou et al, 2014; Doublet et al, 2017). The Dicistroviridae family of viruses exhibits a wide environmental distribution, targeting invertebrates, mainly insects and other arthropods (Shi et al, 2016). The genetic architecture of these viruses is composed of a single positive-stranded RNA molecule which contains two open reading frames (ORF1 and ORF2; Wilson et al, 2000b; Pisarev et al, 2005). ORF1 encodes non-structural proteins: an RNA helicase, a cysteine protease, and an RNA-dependent RNA polymerase (RdRP). ORF2 encodes a single poly-protein that, upon proteolytic digestion, generates the structural proteins that will eventually compose the viral capsid (Kerr & Jan, 2016; Mullapudi et al, 2017). Both ORFs are preceded by non-coding RNA sequences responsible for the regulation of the expression of their downstream genes (Wilson et al, 2000a,2000b; Gross et al, 2017). A fine balance between the expression of ORF1 and ORF2 is required for the replication and expansion of the virus (Carrillo-Tripp et al, 2016; Khong et al, 2016). This is achieved through a precise exploitation of host resources, specially the machinery for protein synthesis (Kerr & Jan, 2016). The non-coding RNA regions preceding both ORFs harbor two different internal ribosomal entry sites (IRESs; Wilson et al, 2000b). IRESs are structured RNA sequences able to interfere with canonical translation, capturing host ribosomes in order to redirect them toward the production of viral proteins (Yamamoto et al, 2017). Eukaryotic ribosomes are operated by a complex collection of cellular factors that regulate the production of proteins in the cell (Jackson et al, 2010). Specially regulated in eukaryotes is the first step of translation, initiation (Aylett & Ban, 2017). During this initiation phase, the small ribosomal subunit (40S), in partnership with many initiation factors, is able to capture an mRNA, localize its AUG initiation codon, deliver the first aminoacyl-tRNA, and finally recruit the large subunit (60S) in order to assemble an elongation competent ribosome (80S) primed with an aminoacyl-tRNA in the P site and a vacant A site (Hinnebusch & Lorsch, 2012). The majority of IRES families leverage the complexity of initiation to hijack cellular ribosomes (Yamamoto et al, 2017; Jaafar & Kieft, 2019). The IAPV-IRES found in the intergenic region of the IAPV virus belongs to the well-characterized type IV family of viral IRESs. IRES sequences from this group dispense with all canonical initiation factors and are able to assemble by themselves an elongation competent ribosome, successfully redirecting the cellular machinery for viral protein production by an RNA-only mechanism (Hertz & Thompson, 2011). This is accomplished by an elaborate use of intrinsically dynamic elements of the ribosome, naturally involved in translocation (Noller et al, 2017a). These IRESs are able to induce an artificial state on the ribosome, mimicking a pre-translocation state with tRNAs. Elongation factors eEF2 and eEF1A can then be recruited to effectively by-pass the highly regulated initiation (Abeyrathne et al, 2016; Murray et al, 2016; Pisareva et al, 2018), jumpstarting directly in the elongation phase (Johnson et al, 2017). The type IV IRES family exhibits a remarkable structural diversity, which remains poorly characterized (Hertz & Thompson, 2011). Two genera, based on phylogenetic analysis of ORF2 as well as the intergenic region, have been defined: Aparaviruses and Cripaviruses. The cricket paralysis virus IRES (CrPV-IRES), the prototypical Cripavirus, has been extensively studied due to its early discovery and use as model mRNA of early studies in translation (Jan et al, 2001; Pestova et al, 2004). Recently, a divergent IRES sequence of a shrimp-infecting virus, the Taura virus syndrome IRES, has been visualized by cryo-EM in complex with yeast ribosomes (Koh et al, 2014; Abeyrathne et al, 2016). The IAPV-IRES presents the prototypical features of an Aparavirus, with an additional stem loop (SL-III) nested within the pseudoknot I (PKI) and an extended L1.1 region (Au et al, 2015). Importantly, this IRES can drive translation in two different ORFs, able to produce two different polypeptides from the same mRNA. A frameshift event at the first coding codon is responsible for this multi-coding capacity (Wang & Jan, 2014). Research efforts directed toward finding the cause of CCD and developing strategies to prevent it are underway (Hunter et al, 2010; Chen et al, 2014). RNA interference has proved effective in protecting against CCD. Directing double-stranded RNAs complementary to the IRES of the intergenic region of the IAPV virus decreases the probability of hive collapse, preventing effectively the massive death of the workforce, guaranteeing the protection of the queen and thus the survival of the colony (Maori et al, 2009). Using single-particle cryo-electron microscopy (cryo-EM), we have characterized how the IAPV-IRES redirects the host machinery for viral protein synthesis exploiting novel ribosomal sites. An early commitment of IRES/ribosome complexes toward global pre-translocation mimicry explains the high efficiency in ribosome hijacking observed for this IRES. These results may inspire structure-based rational designs for the fight against CCD by RNA-interference technology (Chen et al, 2014). Results Biochemical set-up and cryo-EM strategy Previous biochemical and genetic studies of IAPV-IRES established the secondary structure scheme displayed in Fig 1A (Au et al, 2015). The prototypical architecture of the type IV IRES family consisting of three nested pseudoknots is extended by a 5′ terminal stem loop (SL-VI) proposed to play functional roles in the early positioning of the IAPV-IRES in the ribosome (Schuler et al, 2006; Au et al, 2018). Additionally, the genus Aparavirus is characterized by an extended PKI which contains an insertion of a large stem loop (SL-III, Fig 1A, bottom). In the IAPV-IRES, SL-III consists of eight Watson–Crick canonical base pairs and a terminal loop of six nucleotides. Notably, two unpaired adenine residues are placed in a strategic position at the core of the three-way helical junction connecting SL-III, the anti-codon stem loop (ASL)-like element of the PKI (residues 6,546–6,574), and the double-helical segment connecting PKI and PKIII. A variable loop region (VLR) bridges the mRNA-like element of PKI (residues 6,613–6,617) with the helical region connecting PKI and PKIII. This single-stranded RNA loop is poorly conserved in sequence; however, even though its role in IRES functioning remains enigmatic, biochemical experiments have proved its integrity is mandatory for productive IRES-driven translation (Ruehle et al, 2015). Figure 1. IAPV-IRES secondary structure, experimental set-up, and cryo-EM image processing workflow IAPV-IRES diagram colored according to secondary structure motifs. Bottom, a closer view of the IAPV-IRES PKI highlighting its sequence, with base pairs indicated as well as the variable loop region (VLR) and stem loop III (SL-III). Sucrose gradient UV profile of 60S/40S/IAPV-IRES reaction mixture after an overnight run. The peaks corresponding to 80S and 40S were used for RNA extraction and UREA-PAGE shown in the inset. Representative cryo-EM image where roughly half of the particles correspond to 40S (blue) and the other half to 80S (orange). Two classes with robust density for the IAPV-IRES were found in the 40S group. After classification, three classes with clear IAPV-IRES density and small differences in the conformation of the 40S were found in the 80S group. Download figure Download PowerPoint In order to understand in structural terms how these constituent units of the IAPV-IRES are involved in ribosome hijacking, we produced a full, wild-type IAPV-IRES, including SL-VI and the first two coding codons. Binary complexes with mammalian ribosomes and IAPV-IRES were generated by incubating IRES with ribosomal subunits. We designed a reaction featuring an excess of 40S over 60S in an overall background of IRES excess, to test the ability of the IAPV-IRES to engage both 40S and full 80S ribosomes in a productive and stable binary interaction. The stability of these interactions was tested through a sucrose gradient run overnight (Fig 1B) where the different complexes could be resolved according to their size differences. Each peak was subjected to RNA extraction and UREA-PAGE analysis where the presence of IAPV-IRES bound in the 80S peak as well as in the 40S peak could be confirmed (Fig 1B). Leveraging latest cryo-EM maximum-likelihood classification methods implemented in RELION 3.0 (Scheres, 2012; von Loeffelholz et al, 2017; Zivanov et al, 2018), we decided to directly image the above reaction without the sucrose gradient step. A large dataset ensuing from this experiment was subjected to an optimized classification scheme combining different in silico classification approaches (Appendix Fig S1). This allowed us to identify and refine to high-resolution five distinctive classes from a single dataset (Fig 1C–E). The nominal resolution of the maps was calculated to be around 3 Å (Appendix Figs S2, S3, and S6). In the best areas, such as the 60S subunit and the body of the 40S subunit, the maps exhibit characteristics in accordance with this resolution, with very well-resolved side chains in proteins and clear base separation in the ribosomal RNA components (Appendix Fig S4A and B). However, the resolution of the IRES density, due to the intrinsic flexibility of this component, deviates from the nominal resolution. Areas of the IRES stabilized by ribosomal components are well-resolved, with local resolution better than 4 Å (Fig 3D and E), while areas not stabilized by the ribosome or in contact with intrinsically dynamic elements of the ribosome like the L1 stalk exhibit lower local resolution (Appendix Figs S2, S3, and S6). In order to properly visualize the continuity of the maps for the full IRES, we show the unsharpened maps in the figures, especially where large areas of the maps are depicted. In those regions of the maps exhibiting resolution better than 4 Å for the IRES, maps sharpened with B factors reported in Appendix Table S1are shown. The IAPV-IRES restricts the conformational freedom of the 40S blocking functional sites The 40S subunit can be roughly divided into two parts: the body, which forms the bulk of the subunit accounting for two-thirds of its mass, and a more mobile part roughly comprising the remaining third, designated as the head (Fig 2A). The interface between these two components forms the tRNA binding sites of the small subunit. The head of the 40S subunit is a dynamic component, modifying its relative orientation with respect to the body. This dynamics is of critical importance in two aspects of translation: the positioning of the initiator aminoacyl-tRNA and in the concerted movement of mRNA and tRNAs during elongation (Ramrath et al, 2013; Llacer et al, 2015). We identified two classes of particles showing robust density for IAPV-IRES in the context of a binary interaction with the 40S (Fig 2B and C). All elements of the IAPV-IRES included in the produced construct were identified in the maps except SL-VI, which proved disordered—no density could be assigned to it even in low-pass filtered maps. The L1.1 region in the context of a binary interaction with the 40S shows a high degree of mobility and can only be modeled in maps filtered to 4 Å. Figure 2. Structure of the IAPV-IRES in complex with the 40S ribosomal subunit Overview of the mammalian 40S in complex with IAPV-IRES. Left, cryo-EM final post-processed map of class 1 with 40S colored yellow and IAPV-IRES maroon. Right, corresponding final refined model with IAPV-IRES colored according to Fig 1A. Ribbon diagram of the 40S colored by pairwise root-mean-square deviation displacements observed between the two IAPV-IRES/40S classes. The different position of the 40S head between both classes is a composition of swiveling and tilt movements (indicated by arrows in orthogonal views). Close-up view of the ribosomal sites of the 40S for IAPV-IRES/40S class 1 showing cryo-EM unsharpened cryo-EM density. Sequence of the PKI three-way helical junction. Unsharpened cryo-EM density for the PKI region of the IAPV-IRES in class 1 with the SL-III and the tRNA/mRNA mimicking domain indicated. Download figure Download PowerPoint The IAPV-IRES inserts two elements of its structure between the head and the body of the 40S subunit, effectively restricting the dynamics of the 40S head to specific ranges of conformations. The ASL/mRNA mimicking part of the PKI is inserted in the decoding site (A site) of the small subunit stabilized by the decoding bases of the 18S rRNA A1824-A1825 and G626 [A1492, A1494, and G530 in Escherichia coli (Ogle & Ramakrishnan, 2005)], inducing a decoding event. SL-IV is deeply inserted in the interface of head and body, in the surroundings of the E site, clamped by stacking interactions established with residue A6498 of the IRES and tyrosine 72 from uS7 and arginine 135 from uS11 (Fig 3E). In this conformation, the IAPV-IRES fully blocks all three tRNA binding sites of the 40S subunit, interfering with early steps of canonical initiation (Fig 2C; Aylett & Ban, 2017). Figure 3. IAPV-IRES conformation in the context of a 40S interaction Superposition of IAPV-IRES models corresponding to IAPV-IRES/40S class 1 and class 2 after alignments excluding the IRES and the 40S head. A similar conformation can be observed with distinctive relative orientation with respect to the 40S body. The movement characteristic of the 40S head is indicated by arrows in orthogonal views. Ribbon diagram of the IAPV-IRES colored by pairwise root-mean-square deviation displacements observed between the two IAPV-IRES/40S classes. The ASL/mRNA-like regions of the PKI and well as the SL-IV show the lowest degree of displacement (blue), whereas the apical part of SL-III and the L1.1 the highest (red). Superposition of the IAPV-IRES with tRNAs in different configurations indicated at the bottom. IAPV-IRES is depicted as ribbons colored according to the secondary structure elements, and tRNAs are represented as gray ribbons. Alignments of the models were computed with the 40S body, excluding from the computation the ligands (IRES/tRNAs) and the 40S head. Detailed view of the refined model for IAPV-IRES/40S class 1 inserted in the post-processed cryo-EM density focused on the decoding center of the 40S. PKI of IAPV-IRES is depicted green and 18S rRNA yellow. Close-up view of the refined model for IAPV-IRES/40S class 1 inserted in the post-processed cryo-EM density focused on the SL-IV of the IAPV-IRES (depicted blue). Download figure Download PowerPoint Density for the full PKI, including SL-III, was clearly visible in the maps (Fig 2D and E, and Appendix S3) which allowed accurate modeling of the three-way helical junction characteristic of Aparavirus IRESs. The VLR was also visible in the maps, partially occupying the P site, stabilized by a stacking interaction between A6609 of the IRES and A1085 of the 18S rRNA (Fig 2E). The two classes present a conformation of the IAPV-IRES nearly identical (r.m.s.d. = 1.12 Å between the two IRES conformations) but in displaced position with respect to the 40S body (Fig 3A and B). The IAPV-IRES seems to follow the movement of the 40S head, pivoting around the anchored PKI and SL-IV which are exceptionally stabilized by ribosomal elements from both head and body, effectively "clamping" the IRES to the 40S subunit (Fig 3D and E). The three-way helical junction modeled in the PKI of the IAPV-IRES resembles a "hammer" shape, with SL-III coaxially stacked on top of the ASL-like stem (Fig 2D and E). Perpendicular to both and situated in between them, a helical segment connects PKI and PKIII. The coaxially aligned SL-III and ASL-like domain forms a straight unit of shape and dimensions similar to a tRNA, excluding the acceptor stem (Fig 3C). Alignments of structures containing tRNAs in several configurations [canonical tRNA PDBID:4V5D (Voorhees et al, 2009), with A/T-tRNA PDBID:5LZS (Shao et al, 2016) and hybrid tRNA PDBID:3J7R (Voorhees et al, 2014)] with the structure of the IAPV-IRES in complex with the 40S, reveal an interesting positioning of the coaxial unit formed by SL-III and the ASL-like part of PKI (Fig 3C). Notably, the SL-III/ASL-like unit of IAPV-IRES populates a space more similar to a hybrid A/P-tRNA than a canonical, A/A-tRNA or A/T-tRNA [following nomenclature of hybrid states previously proposed (Ratje et al, 2010)]. Similarly, PKIII overlaps with the position occupied by a hybrid P/E-tRNA, mimicking its helical components of the elbow region of a tRNA in this intermediate configuration. Overall, the IAPV-IRES is able to manipulate the 40S subunit in isolation, blocking the functional sites where canonical initiation factors eIF1, eIF1A, and eIF5B bind and, at the same time, steering the intrinsic dynamics of the 40S head toward a configuration reminiscent of an early elongation, pre-translocated state. SL-III interacts with ribosomal protein uL16 stabilizing the 80S in a pre-translocation configuration mimicking hybrid tRNAs The binary IAPV-IRES/80S complex populates three major conformations, with limited differences between them (Fig 1E). The majority of particles populated a class where the 40S subunit exhibits a small degree of intersubunit rotation (approx. 1°) compared with the unrotated, canonical configuration (Fig 5A). No major swiveling or tilt of the 40S head is visible in this conformation. The IAPV-IRES maintains a similar global conformation as in the binary complex with 40S, but in the 80S map, both the L1.1 region and the tip of the SL-III show good density as their dynamics are restricted by specific contacts with elements of the 60S: The L1 stalk stabilizes the L1.1 region and the A site finger and the ribosomal protein uL16 the SL-III. The A site finger (28S rRNA helix 38) is a flexible component of the 28S rRNA which plays an important role in translocation of tRNAs from the A to the P site (Nguyen et al, 2017). In many structures, it is not visible due to its intrinsic flexibility, required to perform its role escorting in-transit tRNAs (Brown et al, 2016; Nguyen et al, 2017). The SL-III of IAPV-IRES contacts the A site finger, stabilizing it in a fixed conformation, which allows the apical loop of SL-III to reach deep into the 60S, establishing a novel interaction with the ribosomal protein uL16 (Fig 4A and B). The IAPV-IRES positions the apical loop of SL-III (nucleotides 6,585–6,590) in direct contact with basic residues of uL16, which are in electrostatic interacting distance with negatively charged phosphates of the RNA backbone of the IRES (Fig 4C). The additional anchoring points to the ribosome contributed by SL-III, allows the IAPV-IRES, in the context of an 80S interaction, to be stabilized in a conformation that overlaps with the space occupied by a hybrid A/P-tRNA. The coaxial unit SL-III/ASL-like domain of the IAPV-IRES functionally mimics an A/P-tRNA priming the 80S for eEF2 recruitment, effectively bypassing the initiation stage (Fig 4D). Additionally, the anchoring points provided by the IAPV-IRES along the intersubunit space probably contribute to an effective recruitment of the 60S in the absence of the dedicated factor responsible for such function in canonical translation, eIF5B (Pestova et al, 2000). Figure 4. SL-III of IAPV-IRES engages novel sites of the 60S ribosomal subunit Overall view of the IAPV-IRES/80S complex class 1 with 60S represented as cyan ribbons, the 40S as yellow ribbons, and the IAPV-IRES represented as solid Van der Waals surface colored by secondary structure motifs. Close-up view of the intersubunit space with the IAPV-IRES depicted as cartoons colored as in (A) inserted in the unsharpened cryo-EM density. Zoomed view of the A site finger in interacting distance with the SL-III (green). The apical loop of SL-III reaches deep into the 60S contacting the ribosomal protein uL16. Superposition of the IAPV-IRES in complex with 80S (class 1) with tRNAs in different configurations indicated at the bottom. Alignments of the models were computed with the 40S body, excluding from the computation the ligands (IRES/tRNAs) and the 40S head. IAPV-IRES PKI component SL-III/ASL-like domain populates a space of the intersubunit space similar to a A/P-tRNA. IAPV-IRES PKIII (red) mimics the elbow region of a hybrid P/E-tRNA. Download figure Download PowerPoint Aparavirus IRESs restrict the small subunit rotation dynamics in the pre-translocation state No populations with wide rotations of the small subunit were identified in our large 80S/IAPV-IRES dataset. This suggests that, in contrast with Cripavirus IRESs (Fernandez et al, 2014; Koh et al, 2014), the IAPV-IRES is able to restrict the dynamics of the small subunit, channeling it toward a canonical, non-rotated configuration (Fig 5A). This is accomplished by a solid anchoring of the PKI in the A site, which not only mimics the ASL of a tRNA interacting with a cognate codon in the A site, but also, by placing the SL-III in a similar position as a hybrid A/P-tRNA (Moazed & Noller, 1989; Frank, 2012), mimics the T and D arms of a tRNA (Figs 4D and 5B, and Appendix Fig S7). In such position, the PKI of the IAPV-IRES establishes a network of interactions with both the large and the small subunits, effectively restricting the rotation of the 40S. Apart from the interactions established by the apical loop of the SL-III with ribosomal protein uL16 (Fig 4C), the decoding event elicited by the placement of the PKI in the decoding center allows the establishment of an interaction with the 28S rRNA base A3760 (A1913 in E. coli), normally involved in decoding (Fig 5C; Demeshkina et al, 2010). This interaction is maintained along the small fluctuations of the 40S, which, locally, are restricted to displacements of a few Angströms (Fig 5D). The IAPV-IRES seems to bind very tightly in a binary, pre-translocation complex with the 80S. The recruitment of elongation factors to commit the ribosome to the production of viral proteins seems to be achieved not through a dynamic manipulation of the 40S rotation, but by directly adopting a configuration reminiscent of a ribosome with tRNAs in hybrid configurations. Figure 5. The IAPV-IRES restricts the small subunit rotational dynamics in a pre-translocation complex with 80S Ribbon diagram of IAPV-IRES/80S complex viewed from the 40S colored by pairwise root-mean-square deviation displacements observed between the classes indicated on the left. Class 1 is unrotated, while classes 2 and 3 exhibit a small rotational movement of the 40S. Top, general overview of the non-rotated IAPV-IRES/80S class 1 structure. IAPV-IRES is depicted as solid Van Der Waals surface colored according to the secondary structure motifs. The PKI (green) is solidly anchored to the A site. Bottom, close-up view of the IAPV-IRES PKI inserted in unsharpened map. Zoomed view of A3760, a nucleotide belonging to the helix 69 (H69) of the 28S rRNA interacting with PKI. Final refined model inserted in the post-processed map is shown. This interaction is not disrupted along the small fluctuations of the 40S. An apical view along the axis of the PKI of a superposition of class 1 versus class 2 shows the IRES displacements are minimal and are followed by the H69 which constantly interacts with the IRES. Download figure Download PowerPoint Remodeling of specific components of the IAPV-IRES allows its translocation through the ribosome Due to their intrinsic flexibility, IRESs are able to populate multiple
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