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The mechanism of translation initiation on Aichivirus RNA mediated by a novel type of picornavirus IRES

2011; Springer Nature; Volume: 30; Issue: 21 Linguagem: Inglês

10.1038/emboj.2011.306

1460-2075

Yingpu Yu, Trevor R. Sweeney, Panagiota Kafasla, Richard J. Jackson, Tatyana V. Pestova, Christopher U.T. Hellen,

Plant Virus Research Studies

Article26 August 2011free access The mechanism of translation initiation on Aichivirus RNA mediated by a novel type of picornavirus IRES Yingpu Yu Yingpu Yu Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Trevor R Sweeney Trevor R Sweeney Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Panagiota Kafasla Panagiota Kafasla Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Richard J Jackson Richard J Jackson Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Tatyana V Pestova Tatyana V Pestova Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Christopher UT Hellen Corresponding Author Christopher UT Hellen Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Yingpu Yu Yingpu Yu Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Trevor R Sweeney Trevor R Sweeney Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Panagiota Kafasla Panagiota Kafasla Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Richard J Jackson Richard J Jackson Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Tatyana V Pestova Tatyana V Pestova Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Christopher UT Hellen Corresponding Author Christopher UT Hellen Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Author Information Yingpu Yu1, Trevor R Sweeney1, Panagiota Kafasla2, Richard J Jackson2, Tatyana V Pestova1 and Christopher UT Hellen 1 1Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA 2Department of Biochemistry, University of Cambridge, Cambridge, UK *Corresponding author. Department of Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Box 44, Brooklyn, NY 11203-2098, USA. Tel.: +1 718 221 1034; Fax: +1 718 270 2656; E-mail: [email protected] The EMBO Journal (2011)30:4423-4436https://doi.org/10.1038/emboj.2011.306 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 Picornavirus mRNAs contain IRESs that sustain their translation during infection, when host protein synthesis is shut off. The major classes of picornavirus IRESs (Types 1 and 2) have distinct structures and sequences, but initiation on both is determined by their specific interaction with eIF4G. We report here that Aichivirus (AV), a member of the Kobuvirus genus of Picornaviridae, contains an IRES that differs structurally from Type 1 and Type 2 IRESs. Its function similarly involves interaction with eIF4G, but its eIF4G-interacting domain is structurally distinct, although it contains an apical eIF4G-interacting motif similar to that in Type 2 IRESs. Like Type 1 and Type 2 IRESs, AV IRES function is enhanced by pyrimidine tract-binding protein (PTB), but the pattern of PTB's interaction with each of these IRESs is distinct. Unlike all known IRESs, the AV IRES is absolutely dependent on DHX29, a requirement imposed by sequestration of its initiation codon in a stable hairpin. Introduction The first stage in eukaryotic translation initiation is assembly of the 48S initiation complex on the initiation codon of mRNA. On most mRNAs, it occurs by the scanning mechanism (Jackson et al, 2010). The first step is formation of a 43S preinitiation complex comprising a 40S subunit, an eIF2/GTP/Met-tRNAMeti ternary complex, eIF3, eIF1, eIF1A and eIF5. 43S complexes attach to the 5′-proximal region of mRNA and scan along the 5′-untranslated region (5′-UTR) to the initiation codon where they stop and form 48S complexes. Attachment of 43S complexes is mediated by eIFs 4F, 4A and 4B. eIF4F consists of three subunits: eIF4E (cap-binding protein), eIF4A (a DEAD-box RNA helicase, whose activity is enhanced by eIF4G and eIF4B) and eIF4G (a scaffold for eIF4E and eIF4A, which also binds eIF3). eIF4F/4A/4B cooperatively unwind the cap-proximal region of mRNA allowing 43S complexes to bind, and likely promote binding via the eIF4G–eIF3 interaction. eIF4F/4A/4B also assist 43S complexes during scanning, but scanning through highly structured 5′-UTRs requires an additional DExH-box protein, DHX29, which binds directly to 40S subunits (Pisareva et al, 2008; Abaeva et al, 2011). After a 43S complex locates the initiation codon, eIF5 and eIF5B promote hydrolysis of eIF2-bound GTP, release of eIFs from the 40S subunit and joining of a 60S subunit to form an 80S ribosome. Viruses rely on the translational apparatus of host cells, and have developed sophisticated mechanisms to suppress translation of cellular mRNAs while ensuring translation of their own. The genomes of Picornaviruses and some other positive-strand RNA viruses contain internal ribosomal entry sites (IRESs), which function with fewer initiation factors than canonical mRNAs. This enables viruses to sustain translation during infection, when the canonical mechanism of initiation is suppressed, for example, by cleavage of eIF4G by viral proteases into an N-terminal fragment that binds eIF4E and a C-terminal fragment that binds eIF4A and eIF3, and by sequestration of eIF4E (Roberts et al, 2009). Different classes of viral IRESes use different mechanisms to recruit the 40S subunit, but they are all based on non-canonical interactions of the IRES with canonical components of the translation apparatus. Thus, initiation on two distinct groups of IRES, epitomized by Hepatitis C virus (HCV) and Cricket paralysis virus (CrPV), is based on their specific interaction with the 40S subunit, resulting in its recruitment directly to the initiation codon (Jackson et al, 2010). The mechanism of initiation on the two other principal IRES groups, Type 1 and Type 2 picornavirus IRESs, which are epitomized by poliovirus (PV) and encephalomyocarditis virus (EMCV), respectively, is determined by their specific interaction with the central domain of eIF4G, which is stimulated by eIF4A (Pestova et al, 1996a, 1996b; Lomakin et al, 2000; Pilipenko et al, 2000; de Breyne et al, 2009). After binding, the eIF4G/eIF4A complex is thought to restructure the IRES to promote attachment of a 43S complex (Kolupaeva et al, 2003; de Breyne et al, 2009). Type 1 and Type 2 picornavirus IRESs are ∼450 nt long and both consists of five principal domains (designated II–VI in Type 1 and H–L in Type 2 IRESs), but they have distinct structures and their sequences are unrelated except for a few common motifs. These include a Yn-Xm-AUG motif at their 3′-border, which consists of a Yn pyrimidine tract (n=8–10 nt) separated by a spacer (m=18–20 nt) from an AUG triplet. This motif is considered to be the point of entry for 43S complexes onto Type 1 and Type 2 IRESs. However, whereas the AUG triplet of this motif in Type 2 IRESs is the initiation codon, it is silent in Type 1 IRESs, and initiation occurs ∼30–150 nt downstream (Jackson, 2005). In both Type 1 and Type 2 IRESs, the Yn-Xm-AUG motifs are preceded by domains (domain V in Type 1 IRESs and domains J–K in Type 2 IRESs) that interact specifically with eIF4G (Pestova et al, 1996b; de Breyne et al, 2009). However, the eIF4G-interacting domains of these IRESs are not homologous, and although a conserved eIF4G-binding motif occurs in domain J in all Type 2 IRESs (Clark et al, 2003; Bassili et al, 2004), no equivalent has been confirmed yet for Type 1 IRESs. The eIF4G-binding domains are preceded by the large domain IV in Type 1 IRESs and domain I in Type 2 IRESs, which are essential for IRES function, but whose roles remain unknown. These domains both contain an important apical GNRA tetraloop motif (Kaminski et al, 1994; López de Quinto and Martínez-Salas, 1997; Robertson et al, 1999). Such motifs commonly stabilize RNA structures by engaging in intramolecular packing interactions with helical RNA 'receptors' (Geary et al, 2008), but no binding target for the tetraloop of Type 1 and Type 2 IRESs has been identified yet. Importantly, in addition to canonical eIFs, all Type 1 and Type 2 IRESs also depend, albeit to different extents, on cellular IRES trans-acting factors (ITAFs). ITAFs usually contain multiple RNA-binding domains (RBDs) that interact specifically with IRESs. The requirement for ITAFs differs between and even within groups of IRESs. Thus, unr and poly(rC) binding protein 2 (PCBP2) are specific for Type 1 IRESs (Jackson, 2005), whereas ITAF45 is specific for only one member of Type 2 IRESs, the foot-and-mouth disease virus (FMDV) IRES (Pilipenko et al, 2000). Interestingly, one ITAF, pyrimidine tract-binding protein (PTB), is common to both Type 1 and Type 2 IRESs, but even then, its modes of interaction with these two types of IRESs differ. PTB's interaction with the Type 1 PV IRES primarily involves localized contacts of its RBDs 1 and 2 with the base of domain V, whereas its interaction with the Type 2 EMCV IRES is characterized by multiple dispersed contacts including those of RBDs 1 and 2 with the apex of domain K and of RBD3/RBD4 with domain H and the base of domains I and L (Kafasla et al, 2009, 2010). A long-standing hypothesis is that ITAFs function by stabilizing the optimal IRES conformation for efficient ribosomal recruitment, and consistently, recent studies revealed that cognate ITAFs promote common conformational changes in Type 2 IRESs (Yu et al, 2011). A structurally distinct picornavirus IRES, designated Type 3, occurs in the 5′-UTR of another, hepatitis A virus (Brown et al, 1991). It consists of two major domains, has no obvious homology to Type 1 and Type 2 IRESs except for a Yn-Xm-AUG motif and, in contrast to all other picornavirus IRESs, is dependent on the integrity of the eIF4F complex (Ali et al, 2001). Otherwise, it remains poorly characterized. The existence of structurally and mechanistically distinct classes of IRES raises the question of whether other unrelated IRESs are still to be identified. Here, we report that Aichivirus (AV), a member of the Kobuvirus genus of Picornaviridae that infects humans, usually subclinically, but that can lead to acute gastroenteritis (Reuter et al, 2011), contains an IRES that is structurally distinct from Type 1, Type 2 and Type 3 IRESs. In vitro reconstitution of initiation on this IRES revealed that it has some characteristics in common with Type 1 and Type 2 IRESs, and others that are unique. Results The AV IRES differs from Type 1 and Type 2 picornavirus IRESs A model of the AV 5′-UTR downstream of the previously described domains A–D (nts 1–116) (Sasaki and Taniguchi, 2003; Nagashima et al, 2005) was derived using complementary bioinformatic approaches. Probabilistic structures of domains E-Ib, Jb, Jc, K and L were obtained by applying an explicit evolutionary model (Knudsen and Hein, 2003) to aligned AV 5′-UTR sequences. A posterior decoding approach (Sato et al, 2009) confirmed these elements and identified most of the remainder of domain J. The resulting model (Figure 1A) was tested and refined by free energy minimization (e.g., Zuker, 2003). It was consistent with the pattern of cleavage of AV nts 270–800 by RNase T1 (which is specific for unpaired G residues), modification by 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulphonate (CMCT) which reacts with unpaired U and G residues, and of cleavage by RNase V1, which is specific for base-paired RNA (Figure 1A–D). The AV 5′-UTR, therefore, contains eight major secondary structure elements, designated E–L, downstream of domains A–D. Figure 1.Structure of the Aichivirus IRES. (A) Model of the secondary structure of the AV IRES (Genbank Acc. AB040749), derived as described in the text, and indicating the positions of nucleotides cleaved by RNases V1 (blue arrows) and T1 (red lozenges), or modified by CMCT (black arrows) based on data shown in (B–D). Domains are labelled E–L. The initiation codon AUG745 is circled. (B–D) Chemical (CMCT) and enzymatic (RNases V1 and T1) probing of the AV IRES in the presence and absence of PTB. Separation of lanes by white lines indicates that they were juxtaposed from the same gels. The positions of cleaved/modified nucleotides are indicated on the right using symbols as in (A). PTB-dependent changes in cleavage/modification are indicated by arrows beside the numbers. Figure source data can be found with the Supplementary Information. Download figure Download PowerPoint Picornavirus 5′-UTRs are modular, comprising elements that are required for RNA replication, followed by an IRES. To determine the 5′ border of the AV IRES, we assayed translation in rabbit reticulocyte lysate (RRL) of dicistronic mRNAs consisting of various AV 5′-UTR fragments inserted between a truncated cyclin cistron (ΔXL) and part of the AV polyprotein (Figure 2A). Deleting domains A–H (nts 1–336) reduced IRES function by only ∼25%, but its activity was decreased by over 90% by deletion of domains A–I (nts 1–430) and was abrogated by extending deletion into domain J (nts 1–450) (Figure 2B). Thus, domains I, J and K were essential for IRES function, and further analysis therefore focused on this core region and the downstream domain L containing the initiation codon. Figure 2.The 5′-border of the Aichivirus IRES and functional importance of its conserved motifs. (A) Schematic representation of dicistronic AV mRNAs containing AV 5′-UTR fragments, 5′-terminally truncated as indicated by arrows on the IRES model and inserted between truncated cyclin (ΔXL) and AV LΔVP0 reporter cistrons. (B) The effect of 5′-terminal truncations (as shown in A) on the activity of the AV 5′-UTR assayed by translation in RRL. (C) Structures of the apices of domain J of the AV IRES and of domain V of the Type 1 Coxsackievirus B3 IRES, showing the GNRA tetraloop and the positions of internal deletions made in the AV IRES. (D) Structures of the apices of domain K of the AV IRES and of domain J of the EMCV IRES, showing the conserved motif (red letters) that is required for interaction of the EMCV IRES with eIF4G. The destabilizing AGGU → UCCA mutation in the motif of the AV IRES is indicated. (E–H) The activity of monocistronic (MC) AV mRNAs comprising the entire 5′-UTR and LΔVP0 coding region and containing (E) the AGGU → UCCA mutation in the K domain conserved motif, (F) deletions in domain J as indicated in (C), and (G, H) substitutions in the GNRA tetraloop, assayed by translation in RRL. (I) Summary of the efficiency of translation of AV mRNAs with tetraloop substitutions. Download figure Download PowerPoint The structure of the AV IRES (nts 337–744) differs in many respects from those of Type 1, 2 and 3 IRESs (Brown et al, 1991; Bailey and Tapprich, 2007; Kapoor et al, 2008). Thus, AV domain I is not related to elements in any of these IRESs. Domain J consists of a long interrupted basal helix and an apical four-way helical junction (Figure 1A), similar to but smaller than domain IV in Type 1 IRESs. Its apical subdomain (Jb) also includes a GNRA tetraloop (Figure 2C), which is essential for the function of Type 1 and Type 2 IRESs. Thus, substitution of the residue at the fourth position of the GNRA motif reduced the activity of PV (Type 1) and EMCV and FMDV (Type 2) IRESs 20-fold, as did purine-to-pyrimidine substitutions at the first and third positions in Type 2 IRESs (Kaminski et al, 1994; López de Quinto and Martínez-Salas, 1997; Robertson et al, 1999). Although the apex of AV domain K contains an element identical to an apical motif in domain J of Type 2 IRESs (Figure 2D) that is essential for specific interaction with eIF4G (Clark et al, 2003; Bassili et al, 2004), these domains are otherwise unrelated, and the AV IRES also lacks an equivalent of domain K of Type 2 IRESs. Finally, while the AV initiation codon, AUG745, is preceded by a Yn motif like in Type 1/2 IRESs, in contrast to them, it is sequestered in the 5′ strand of a long, stable hairpin (Domain L). We first investigated the importance of the GNRA tetraloop-containing subdomain Jb and of the putative eIF4G-interacting motif for AV IRES function. Disruption of the eIF4G-interacting motif almost abrogated AV IRES activity (Figure 2E), indicating that it is as dependent on this motif as are Type 2 IRESs. Deletion of nts 519–541 and 508–551 at the apex of domain Jb (Figure 2C) almost abrogated translation of AV mRNA (Figure 2F, lanes 4 and 5). However, the AV IRES retained ∼20% activity following deletion of the GNRA-containing hairpin alone (nts 526–539) (Figure 2F, lane 3). Moreover, substitution of individual nucleotides at any position in the GUGA tetraloop surprisingly did not impair, and in some instances even enhanced IRES function (Figure 2G–I). Even a GUGA → CCUA mutation, which mimics an inactivating mutation in the EMCV IRES (Robertson et al, 1999), did not affect AV IRES function (Figure 2G, lane 6). Thus, although the Jb subdomain is essential for the activity of the AV IRES, its apical tetraloop does not have the same functional importance as in Type 1/2 IRESs. Factor requirements for initiation on the AV IRES We next identified initiation factors required for 48S complex formation on the AV IRES. Assembly of 48S complexes was monitored by appearance of characteristic toe-prints 15–21 nt downstream of the initiation codon, depending on the mRNA (e.g., Pestova et al, 1996a). Although efficient 48S complex formation on the AV IRES occurred in RRL in the presence of GMPPNP (Figure 3A), no 48S complexes formed in an in vitro reconstituted translation system in the presence of 40S subunits, Met-tRNAMeti and eIFs 1, 1A, 2, 3, 4A, 4B and 4F (Figure 3B). Thus, in contrast to the Type 2 EMCV IRES (Pestova et al, 1996a), canonical eIFs were not sufficient for initiation on the AV IRES. We, therefore, undertook extensive purification from RRL of additional factor(s) required by the AV IRES (Figure 3C). Figure 3.Factor requirements for initiation on the Aichivirus IRES. (A, B, D, E, G, H) Toe-printing analysis of 48S complex formation on AV MC mRNA (A) in RRL in the presence of GMPPNP, and (B, D, E, G, H) in the in vitro reconstituted system in the presence of 40S subunits, Met-tRNAMeti, eIFs, PTB1, DHX29, AGO2 and different protein fractions, as indicated. Black lozenges indicate toe-prints in domain K and in the initiation codon region that appear in the presence of PTB or PTB-containing fractions. (C) Purification scheme for PTB and DHX29. (F) The active Superdex 200 fraction resolved by SDS–PAGE, showing polypeptides identified by mass spectrometry. (I) Sensitivity of translation of DC AV mRNA (lanes 2–4) and DC CSFV mRNA containing the CSFV IRES between the upstream cyclin (XL) and downstream NS' reporters (lanes 5 and 6) to inhibition by the dominant-negative eIF4AR362Q mutant. Before addition of mRNAs, 20 μl of RRL was preincubated with indicated amounts (μg) of eIF4AR362Q. (J) Elongation competence of 48S complexes assembled with eIFs 2/3/4A/4Gm, PTB and DHX29 on AV mRNA containing a stop codon three triplets downstream of the initiation codon, assayed by toe-printing. Separation of lanes in (G, I) by white lines indicates that they were juxtaposed from the same gels. Figure source data can be found with the Supplementary Information. Download figure Download PowerPoint The activity(s) that was able to complement eIFs 1/1A/2/3/4A/4B/4F, 40S subunits and Met-tRNAMeti in promoting efficient 48S complex formation on the AV IRES, was present in the 0–40% ammonium sulphate (AS) fraction of the ribosomal salt wash (RSW). During further purification, it eluted in the 100-mM KCl flow-through fraction from DEAE, and then in the 300 mM KCl fraction from phosphocellulose (data not shown). After subsequent FPLC on MonoS, the activity eluted at 300 mM KCl (data not shown). However, after FPLC on MonoQ, none of the individual fractions exhibited full activity, and efficient 48S complex formation occurred only if the flow-through fraction and the ∼250-mM KCl elution fraction were combined (data not shown). Further chromatography of the 250-mM KCl MonoQ fraction on a Hydroxyapatite column identified its active constituent as DHX29. Together with the MonoQ flow-through fraction, recombinant DHX29 promoted efficient 48S complex formation (Figure 3D) and was therefore used in all subsequent experiments. Gel filtration on Superdex 200 of the MonoQ flow-through yielded an active fraction (Figure 3E, lanes 1–3) that contained proteins that were identified as Argonaute 2 (AGO2), RNA-activated protein kinase PKR, isoforms of PTB and elongation factor eEF1α (Figure 3F; Supplementary Tables 1–4). Recombinant PTB1 replaced the active gel-filtration fraction without loss of activity in 48S complex formation on the AV IRES when included with 40S subunits, Met-tRNAMeti, DHX29 and eIFs 1, 1A, 2, 3, 4A, 4B and 4F, whereas recombinant AGO2 and fractions containing eEF1α or PKR without PTB did not (Figure 3E, lanes 4 and 5; data not shown). Although DHX29 and PTB synergistically stimulated 48S complex formation on the IRES, the individual stimulatory activity of DHX29 was much greater than that of PTB (Figure 3G). Importantly, this is the first time that DHX29 has been reported to be required for internal ribosomal entry, and contrasts with its negative influence on the unrelated CrPV- and HCV-like IRESs (Pisareva et al, 2008). Systematic omission experiments revealed that eIF2, eIF3, eIF4A and the eIF4A-interacting central domain of eIF4G (eIF4G736−1115 designated as 'eIF4Gm') were essential for 48S complex formation on the AV IRES, eIF1 and eIF4B had a moderate stimulatory effect, whereas eIF1A and eIF4E were not required (Figure 3H). Consistent with an absolute requirement for eIF4A and eIF4Gm for 48S complex formation, AV IRES-mediated translation in RRL was very efficiently inhibited by the eIF4AR362Q dominant-negative mutant (Pause et al, 1994), whereas in control reactions, initiation on the CSFV IRES was insensitive to eIF4AR362Q (Figure 3I). Incubation with 60S subunits, eIF5, eIF5B, eEF1H, eEF2 and aa-tRNAs of 48S complexes formed on mutant AV mRNA containing a stop codon three triplets downstream of the initiation codon (Figure 3J, upper panel) in the presence of DHX29, PTB and eIFs 2/3/4A/4Gm (Figure 3J, lane 1) yielded pretermination complexes characterized by toe-prints 16–17 nt downstream of the GCA triplet preceding the stop codon (Figure 3J, lane 2), indicating that 48S complexes assembled in this way could form elongation-competent 80S ribosomes. The requirement for DHX29 results from sequestration of the initiation codon in a stable hairpin The AV initiation codon AUG745 is sequestered in a stable hairpin, domain L (Figures 1A and 4A). To investigate whether this distinctive feature of the AV IRES could account for its specific requirement for DHX29, we investigated the effect of disrupting domain L by deletion or substitution (Figure 4B) on IRES activity. Disruption of domain L enhanced the activity of the IRES during in vitro translation in RRL (Figure 4C; data not shown). Moreover, in contrast to the wt IRES, which was strictly dependent on DHX29 (Figure 4D, lanes 1 and 2), mutant IRESs with destabilizing mutations in domain L did not require DHX29 for efficient 48S complex formation (Figure 4D, lanes 4, 5, 7, 8, 10 and 11). The AV IRES' requirement for DHX29 was, therefore, determined by the need to unwind domain L. Figure 4.Conditional requirement of the Aichivirus IRES for DHX29. (A) The model of the AV IRES, with domain L in bold. (B) Structures of the L domain of the wt AV IRES and AV IRES mutants containing destabilizing deletion and substitutions in domain L. (C) The activity of MC AV mRNAs containing destabilizing substitutions in domain L (B), assayed by translation in RRL. (D) 48S complex formation on the wt AV IRES and the L domain AV IRES mutants (B) depending on the presence of DHX29, assayed by toe-printing. Black lozenges indicate toe-prints in the area of the initiation codon region of the L domain mutants. Download figure Download PowerPoint Specific functional interaction of eIF4G/eIF4A with the AV IRES Initiation on Type 1 and Type 2 IRESs is based on specific interaction of these IRESs with eIF4Gm, which is enhanced by eIF4A (Pestova et al, 1996b; Lomakin et al, 2000; de Breyne et al, 2009). Domain K of the AV IRES contains an element identical to a conserved element in domain J of Type 2 IRESs (Figure 2D) that determines their specific interaction with eIF4Gm. Disruption of this motif strongly impaired AV IRES-mediated translation (Figure 2E, lanes 2 and 3), indicating that it is similarly important for AV IRES function. We, therefore, tested whether the AV IRES also specifically interacts with eIF4Gm and whether this interaction is stimulated by eIF4A, using the directed hydroxyl radical cleavage technique, in which locally generated hydroxyl radicals cleave the IRES in the vicinity of Fe(II) tethered to unique cysteines on the surface of eIF4G via 1-(p-bromoacetamidobenzyl)-EDTA (BABE), after which cleavage sites are determined by primer extension. eIF4Gm consists of five pairs of α-helices ('HEAT-repeats') (Marcotrigiano et al, 2001; Figure 5A). We employed six single-cysteine mutants and a cysteine-less (Cys-less) eIF4Gm variant (Figure 5A) that were fully active in 48S complex formation on the EMCV IRES and that we previously used to investigate interaction of eIF4G with Type 1 and 2 IRESs (Kolupaeva et al, 2003; de Breyne et al, 2009; Yu et al, 2011). Numbering of residues in eIF4G is based on its revised sequence (NM_182917), and the cysteine residue in the D928 → D928C insertion mutant is designated as C929. Hydroxyl radicals generated from two positions (shown as coloured spheres in Figure 5A) cleaved the AV IRES in eIF4G/IRES complexes (summarized in Figure 5F). Hydroxyl radicals generated from C829, located between helices 2b and 3a, cleaved strongly at nts 690–693, moderately at nts 663–664, and weakly at nts 651–652 and 705–710, whereas C929, located after helix 4b, cleaved moderately at nts 678–680 and weakly at nts 675–676 (Figure 5C, lanes 1–3). Inclusion of eIF4A strongly enhanced cleavage of the AV IRES from both positions on the surface of eIF4Gm (Figure 5C, lanes 7–9). These data indicate that as in the case of Type 2 IRESs, eIF4Gm binds specifically to domain K of the AV IRES, with its N-terminus directed towards its base and its C-terminus directed towards its apex, which could be consistent with enhancement of this interaction by eIF4A. Figure 5.Interaction of the Aichivirus IRES with eIF4G/eIF4A. (A, B) Ribbon diagrams of (A) the HEAT-1 domain of eIF4G (PDB: 1HU3) and (B) eIF4A in the closed ATP/RNA-bound conformation (PDB: 3EX7), with spheres indicating newly introduced cysteines. (C–E) Primer extension analysis of directed hydroxyl radical cleavage of (C–E) wt and (D) AGGU → UCCA mutant AV IRESs from (C) Fe(II)-tethered eIF4Gm, in the presence/absence of eIF4A and PTB, as indicated, (D) Fe(II)-tethered eIF4Gm in the presence of eIF4A and (E) Fe(II)-tethered eIF4A in the presence of eIF4Gm. (F, G) Sites of hydroxyl radical cleavage from positions on (F) eIF4Gm and (G) eIF4A, mapped onto the secondary structures of domain K of the AV IRES. Cleavage sites are show in colours that match the colours of corresponding spheres in (A) and (B). Sites of strong cleavage are indicated by thick edging. The insert panel shows a model of the AV IRES, with domain K in bold. Download figure Download PowerPoint As in the case of Type 2 IRESs (Kolupaeva et al, 2003; Yu et al, 2011), the strongest eIF4G cleavage sites in the AV IRES (Figure 5F) overlapped with a conserved motif at the apex of domain K (Figure 2D), which is essential for efficient AV IRES-mediated translation (Figure 2E). Disruption of this motif abrogated cleavage of the IRES by Fe(II)-eIF4Gm (Figure 5D), suggesting that this mutant IRES's loss of function is due to loss of its ability to bind stably to eIF4G. We next determined the orientation of eIF4A in AV IRES/eIF4Gm/eIF4A complexes, again using directed hydroxyl radical cleavage. eIF4A consists of two RecA domains joined by a linker (Figure 5B). The eIF4A-NTD binds to the C-terminal helix of eIF4G's central domain, whereas the eIF4A-CTD binds to its N-terminal two HEAT-repeats (e.g., Marintchev et al, 2009). We employed nine single-cysteine eIF4A mutants (Figure 5B) that have previously been used to determine the position of eIF4A in the IRES/eIF4G/eIF4A complexes assembled on Type 1 and 2 IRESs (de Breyne et al, 2009; Yu et al, 2011). Hydroxyl radicals generated from three positions (shown as coloured spheres in Figure 5B) cleaved the AV IRES in IRES/eIF4G/eIF4A complexes (summarized in Figure 5G). Hydroxyl radicals from C33 and C42 in the eIF4A-NTD induced strong cleavage at nts 675–679 at the apex of domain K, and C42 also induced weaker cleavage nearby at nts 668–671 (Figure 5E, lanes 2 and 3). The overlap between the sites of cleavage induced by eIF4G-C929, eIF4A-C33 and eIF4A-C42 (Figure 5F and G) is consistent with the prox