We utilized the nedicistrovirus (NediV) intergenic region (IGR) internal ribosomal entry site (IRES)-mediated, initiation factor-independent translation initiation system and determined high-resolution structures of 80S ribosome complexes with the NediV IRES in various functional states, including binary complexes, aminoacyl-transfer RNA (tRNA)-bound complexes, and complexes with elongation factor eEF2. In binary complexes, the NediV IRES primarily occupies the ribosomal P site, exhibiting conformational flexibility and engaging the ribosome at multiple interaction sites. Upon translocation, the IRES undergoes structural rearrangements, including destabilization of its PKI domain, facilitating the transition to canonical elongation. Crucially, we captured an eEF2-bound complex, along with an eEF1A-bound failed decoding complex featuring a mismatched tRNA, the latter representing the first instance of a canonical elongation complex visualized in the presence of a natural, hydrolysable nucleotide and without the addition of any trapping agents. These findings provide a comprehensive structural overview of IGR IRES-mediated translation initiation and its transition to elongation, revealing key mechanistic details of viral translation and proofreading.

SARS-CoV-2 nonstructural protein 1 (Nsp1) binds to 40S ribosomal subunits and induces host protein synthesis shut off by inhibiting translation initiation and triggering endonucleolytic cleavage of cellular mRNAs. Irrespective of the mode of initiation, Nsp1-mediated cleavage is induced by the cooperative action of the N-terminal domain of Nsp1, the RRM domain of eIF3g and 40S subunits. Using in vitro reconstitution, we determined that cleavage occurs by transesterification following intramolecular nucleophilic attack of the 2'OH of the ribose on the adjacent phosphodiester bond yielding 5'OH and 2',3'-cyclic phosphate termini. Cleavage requires a guanosine ~10-22 nucleotides from the 5' end of mRNA, occurs within a narrow window upstream of this G, is most efficient between nucleotides at positions -6/-7 and -7/-8 relative to G, and shows a preference for Pu at positions -7 or -8 which provides the 2'OH for the nucleophilic attack. Zero-length UV cross-linking of Nsp1 to nucleotides at positions -1 and -2 suggests that the critical guanosine may be recognized by Nsp1. Resistance to Nsp1-mediated cleavage of SARS-CoV-2 mRNA was ensured both by the relatively long distance between its G23G24 and the 5'end and by the preceding oligoPy stretch lacking purines at positions -7 or -8 upstream of G23G24.

Viral internal ribosomal entry sites (IRESs) are highly structured cis-acting RNAs that mediate end-independent initiation of translation. Their origin remains obscure. The simplest IRESs (type 6) occur in the intergenic region of Dicistroviridae genomes (order Picornavirales), consist of two pseudoknots, and initiate translation by factor-independent binding to ribosomes. Larger variants contain a third pseudoknot that modifies the mechanism of IRES function by engaging with the ribosomal head and promoting binding to the ribosomal aminoacyl site. Metagenomic analyses undertaken to identify structurally distinct type 6 IRESs identified subsets ranging from ∼120-260 nt in length. They differ by the cumulative addition of structural elements, suggesting an accretion mechanism for the structural elaboration of IRESs. Insertions occurred at specific loci, possibly reflecting non-templated nucleotide insertion during replication, and form additional subdomains. Biochemical analysis showed that these novel classes of type 6 IRES all bound directly to the ribosomal peptidyl site. Identification of chimeric IRESs implicates recombinational exchange of domains as a second mechanism for the diversification of IRES structure. Recombination likely also accounts for the presence of type 6 IRESs at the 5'-end of dicistrovirus-like genomes and in families other than Dicistroviridae, including Marnaviridae (order Picornavirales) and Tombusviridae (order Tolivirales).

Members of the Picornaviridae family of viruses have single-stranded positive-sense RNA genomes, and are conventionally considered to contain a single open reading frame (ORF) preceded by a long 5′ untranslated region (UTR). The 5′UTR contains an internal ribosomal entry site (IRES) that mediates end-independent initiation of translation of ORF1, resulting in synthesis of a polyprotein that is proteolytically processed to yield the individual structural and nonstructural proteins. However, recent studies indicate that the dogma that picornavirus genomes contain a single ORF (ORF1) is an oversimplification, and that members of many picornavirus genera contain an additional ORF that is translated to yield an accessory protein that may influence pathogenicity and tissue specificity. This review summarizes the structural organization of picornavirus genomes that contain a second ORF. Most commonly, these ORFs overlap the 5′-end of ORF1, but they may also be wholly distinct from ORF1 or located entirely within it in a different reading frame. Ribosomal access to these alternate ORFs has been shown or is hypothesized to depend on a remarkable variety of non-canonical translation mechanisms, including ribosomal recruitment by a second IRES, leaky scanning after IRES-mediated internal ribosomal entry, cap-independent initiation, frame-shifting and termination–reinitiation.

Abstract The encephalomyocarditis virus (EMCV) internal ribosomal entry side (IRES) and other Type 2 IRESs favor translation of the viral genome during infection. The domains H-L of these IRESs specifically interact with the cellular translation initiation factors eIF4G/eIF4A through their essential JK domain. However, the JK domain is not sufficient for IRES activity, which also strictly requires the preceding domain I of unknown function. To identify interactions that drive ribosomal attachment to eIF4G/eIF4A-bound Type 2 IRESs, we determined the cryo-EM structure of 48S initiation complexes formed on the EMCV IRES. The apical cloverleaf of domain I contacts ribosomal proteins uS13 and uS19 via its subdomain Id, whereas the essential GNRA tetraloop in subdomain Ic interacts with the TψC domain of initiator tRNA. The IRES-tRNA interaction also provides a mechanism for release of the IRES after eIF2 is replaced by eIF5B during subunit joining to allow attachment of 60S subunits. Functional assays supported the exceptional role of these interactions for initiation on this IRES. The strong conservation of the apex of domain I amongst Type 2 IRESs suggests that the reported interactions provide a common general mechanism of ribosomal attachment on them all.

The encephalomyocarditis virus (EMCV) IRES and other Type 2 IRESs comprise domains H-L and specifically interact with eIF4G/eIF4A through their essential JK domain. However, the JK domain is not sufficient for IRES function, which also requires the preceding domain I of unknown function. To identify interactions that drive ribosomal recruitment of eIF4G/eIF4A-bound Type 2 IRESs, we determined the cryo-EM structure of 48S initiation complexes formed on the EMCV IRES. It revealed that the apical domain I cloverleaf contacts ribosomal proteins uS13 and uS19 via its Id subdomain and that the essential GNRA tetraloop in subdomain Ic interacts directly with the TψC domain of initiator tRNA. Functional assays supported the exceptional role of these interactions for initiation on this IRES. The strong conservation of primary and secondary structures of the apex of domain I among Type 2 IRESs suggests that the reported interactions are a common essential feature of them all.

The papers introduced in the Commentary present new insights and review aspects of current knowledge concerning the competition between viruses and their hosts for the cellular translation apparatus. Viruses depend on this apparatus and utilize diverse mechanisms to usurp it for the translation of viral mRNAs and to suppress synthesis of cellular proteins. Virus-induced modification of translation factors, selective abrogation of mRNA binding to ribosomes and degradation of cellular mRNAs all impair elements of the innate immune response, thereby undermining host defenses against infection. Various cellular mechanisms prevent translation of viral mRNAs, by modifying components of the translation apparatus to effect a generalized shut-off of translation or by binding of host proteins to viral mRNAs to induce their degradation or to prevent their engagement with the translation apparatus. Viruses have in turn evolved countermeasures to evade these defenses, for example by encoding proteins that impair the activity of host factors or via alterations in the sequence and structure of viral mRNAs. Such changes enable viral mRNAs to avoid recognition by host factors or to support translation initiation by specialized mechanisms that involve only a subset of the factors that are required by cellular mRNAs.

When a cancer cell no longer makes a particular functioning protein, it can become dependent on another protein for survival. The identification of one such relationship has uncovered a therapeutic target. Using the strategy of synthetic lethality to target cancer cells.

Examination of all publicly available Oryctolagus cuniculus (rabbit) ribosome cryo-EM structures reveals numerous confusing inconsistencies. First, there are a plethora of single nucleotide differences among the various rabbit 28S and 18S rRNA structures. Second, two nucleotides are absent from the NCBI Reference Sequence for the 18S rRNA gene. Moving forward, we propose using the Broad Institute's rabbit whole genome shotgun sequence and numbering to reduce modeling ambiguity and improve consistency between ribosome models.

Polypeptides arising from interrupted translation undergo proteasomal degradation by the ribosome-associated quality control (RQC) pathway. The ASC-1 complex splits stalled ribosomes into 40S subunits and nascent chain-tRNA-associated 60S subunits (60S RNCs). 60S RNCs associate with NEMF that promotes recruitment of the RING-type E3 ubiquitin (Ub) ligase Listerin (Ltn1 in yeast), which ubiquitinates nascent chains. RING-type E3s mediate the transfer of Ub directly from the E2~Ub conjugate, implying that the specificity of Ub linkage is determined by the given E2. Listerin is most efficient when it is paired with promiscuous Ube2D E2s. We previously found that TCF25 (Rqc1 in yeast) can impose K48-specificity on Listerin paired with Ube2D E2s. To determine the mechanism of TCF25's action, we combined functional biochemical studies and AlphaFold3 modeling and now report that TCF25 specifically interacts with the RING domain of Listerin and the acceptor ubiquitin (UbA) and imposes K48-specificity by orienting UbA such that its K48 is directly positioned to attack the thioester bond of the Ube2D1~Ub conjugate. We also found that TCF25 itself undergoes K48-specific ubiquitination by Listerin suggesting a mechanism for the reported upregulation of Rqc1 in the absence of Ltn1 and the observed degradation of TCF25 by the proteasome in vivo.

Viral internal ribosomal entry sites (IRESs) form several classes that use distinct mechanisms to mediate end-independent initiation of translation. The origin of viral IRESs is a longstanding question. The simplest IRESs comprise tandem pseudoknots and occur in the intergenic region (IGR) of Dicistroviridae genomes (order Picornavirales ). Larger IGR IRESs contain additional elements that determine specific properties such as binding to the head of the ribosoma l 40S subunit. Metagenomic analyses reported here identified novel groups of structurally distinct IGR-like IRESs. The smallest of these (∼120nt long) comprise three pseudoknots and bind directly to the ribosomal P site. Others are up to 260nt long: insertions occurred at specific loci, possibly reflecting non-templated nucleotide insertion during replication. Various groups can be arranged in order, differing by the cumulative addition of single structural elements, suggesting an accretion mechanism for the structural elaboration of IRESs. Identification of chimeric IRESs implicates recombinational exchange of domains as a second mechanism for the diversification of IRES structure. Recombination likely also accounts for the presence of IGR-like IRESs at the 5’-end of some dicistrovirus-like genomes (e.g. Hangzhou dicistrovirus 3) and in the RNA genomes of Tombusviridae (order Tolivirales ), Marnaviridae (order Picornavirale s), and the ‘Ripiresk’ picorna-like clade (order Picornavirale s).

Caliciviruses have positive-sense RNA genomes, typically with short 5′-untranslated regions (5′UTRs) that precede the long open reading frame 1 (ORF1). Exceptionally, some avian caliciviruses have long 5′UTRs containing a picornavirus-like internal ribosomal entry site (IRES), which was likely acquired by horizontal gene transfer. Here, we identified numerous additional avian calicivirus genomes with IRESs, predominantly type 2, and determined that many of these genomes contain a ~200–300 codon-long ORF (designated ORF1*) that overlaps the 5′-terminal region of ORF1. The activity of representative type 2 IRESs from grey teal calicivirus (GTCV) and Caliciviridae sp. isolate yc-13 (RaCV1) was confirmed by in vitro translation. Toeprinting showed that in cell-free extracts and in vitro reconstituted reactions, ribosomal initiation complexes assembled on the ORF1* initiation codon and at one or two AUG codons in ORF1 at the 3′-border and/or downstream of the IRES. Initiation at all three sites required eIF4A and eIF4G, which bound to a conserved region of the IRES; initiation on the ORF1* and principal ORF1 initiation codons involved eIF1/eIF1A-dependent scanning from the IRES’s 3′-border. Initiation on these IRESs was enhanced by the IRES trans-acting factors (ITAFs) Ebp1/ITAF45, which bound to the apical subdomain Id of the IRES, and PTB (GTCV) or PCBP2 (RaCV1).

Ribosomal stalling induces the ribosome-associated quality control (RQC) pathway targeting aberrant polypeptides. RQC is initiated by K63-polyubiquitination of ribosomal protein uS10 located at the mRNA entrance of stalled ribosomes by the E3 ubiquitin ligase ZNF598 (Hel2 in yeast). Ubiquitinated ribosomes are dissociated by the ASC-1 complex (ASCC) (RQC-Trigger (RQT) complex in yeast). A cryo-EM structure of the ribosome-bound RQT complex suggested the dissociation mechanism, in which the RNA helicase Slh1 subunit of RQT (ASCC3 in mammals) applies a pulling force on the mRNA, inducing destabilizing conformational changes in the 40S subunit, whereas the collided ribosome acts as a wedge, promoting subunit dissociation. Here, using an in vitro reconstitution approach, we found that ribosomal collision is not a strict prerequisite for ribosomal ubiquitination by ZNF598 or for ASCC-mediated ribosome release. Following ubiquitination by ZNF598, ASCC efficiently dissociated all polysomal ribosomes in a stalled queue, monosomes assembled in RRL, in vitro reconstituted 80S elongation complexes in pre- and post-translocated states, and 48S initiation complexes, as long as such complexes contained ≥ 30-35 3'-terminal mRNA nt. downstream from the P site and sufficiently long ubiquitin chains. Dissociation of polysomes and monosomes both involved ribosomal splitting, enabling Listerin-mediated ubiquitination of 60S-associated nascent chains.

Internal ribosomal entry sites (IRESs) engage with the eukaryotic translation apparatus to promote end-independent initiation. We identified a conserved class of ∼150 nt long intergenic region (IGR) IRESs in dicistrovirus genomes derived from members of the phyla Arthropoda, Bryozoa, Cnidaria, Echinodermata, Entoprocta, Mollusca and Porifera. These IRESs, exemplified by Wenling picorna-like virus 2, resemble the canonical cricket paralysis virus (CrPV) IGR IRES in comprising two nested pseudoknots (PKII/PKIII) and a 3'-terminal pseudoknot (PKI) that mimics a tRNA anticodon stem-loop base-paired to mRNA. However, they are ∼50 nt shorter than CrPV-like IRESs, and PKIII is an H-type pseudoknot that lacks the SLIV and SLV stem-loops that are primarily responsible for the affinity of CrPV-like IRESs for the 40S ribosomal subunit and that restrict initial binding of PKI to its aminoacyl (A) site. Wenling-class IRESs bound strongly to 80S ribosomes but only weakly to 40S subunits. Whereas CrPV-like IRESs must be translocated from the A site to the peptidyl (P) site by elongation factor 2 for elongation to commence, Wenling-class IRESs bound directly to the P site of 80S ribosomes, and decoding begins without a prior translocation step. A chimeric CrPV clone containing a Wenling-class IRES was infectious, confirming that the IRES functioned in cells.

Initiation of translation on many viral mRNAs occurs by noncanonical mechanisms that involve 5′ end-independent binding of ribosomes to an internal ribosome entry site (IRES). The ∼190-nt-long intergenic region (IGR) IRES of dicistroviruses such as cricket paralysis virus (CrPV) initiates translation without Met-tRNA i Met or initiation factors. Advances in metagenomics have revealed numerous dicistrovirus-like genomes with shorter, structurally distinct IGRs, such as nedicistrovirus (NediV) and Antarctic picorna-like virus 1 (APLV1). Like canonical IGR IRESs, the ∼165-nt-long NediV-like IGRs comprise three domains, but they lack key canonical motifs, including L1.1a/L1.1b loops (which bind to the L1 stalk of the ribosomal 60S subunit) and the apex of stem–loop V (SLV) (which binds to the head of the 40S subunit). Domain 2 consists of a compact, highly conserved pseudoknot (PKIII) that contains a UACUA loop motif and a protruding CrPV-like stem­–loop SLIV. In vitro reconstitution experiments showed that NediV-like IRESs initiate translation from a non-AUG codon and form elongation-competent 80S ribosomal complexes in the absence of initiation factors and Met-tRNA i Met . Unlike canonical IGR IRESs, NediV-like IRESs bind directly to the peptidyl (P) site of ribosomes leaving the aminoacyl (A) site accessible for decoding. The related structures of NediV-like IRESs and their common mechanism of action indicate that they exemplify a distinct class of IGR IRES.

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on β-globin, EMCV IRES and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nucleotides downstream from the mRNA entrance indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of Nsp1-NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.