Abaeva IS , Young C , Warsaba R , Khan N , Tran LV , Jan E , Pestova TV , Hellen CUT .

R01 AI123406 NIH HHS

	Graphical Abstract.
	Figure 1. Coherence of the Wenling group of viral genomes. (A) Phylogenetic analysis of the ORF2 capsid protein precursors from the indicated viruses, showing that the members of the Wenling group belong to a clade that is distinct from the Halárvirus clade, the Cripavirus genus, the Triatovirus genus and the two clades in the Aparavirus genus of Dicistroviridae. The numbers at the branch nodes represent the bootstrap confidence level (above 70). Bar, one amino acid substitution per site. Accession numbers are indicated in parentheses. (B) Model of the Wenling 2 IGR IRES structure, annotated to show helices (P), loops (L), single-stranded elements (S), pseudoknots (PK) I, II and III, and domains 1–3. Nucleotides are numbered at 20 nt intervals. The GCU6906–6908 triplet adjacent to PKI is the first codon in ORF2. Base pairs that are formed by compensatory substitutions in ≥22 members are boxed; base pairs that are conserved in all 26 members of the group are shaded and boxed.
	Figure 2. Chimeric CrPV infectious clone containing the Caledonia IGR IRES supports viral replication. (A) Schematic of the CrPV genome derived from an infectious clone containing either the CrPV or the Caledonia IRES. (B) Model of the Caledonia IGR IRES, labeled to show pseudoknots (PKs) I, II and III, the ORF1 stop codon (red box) and the ORF2 start codon (black box). Nucleotides are numbered at 10 nt intervals. (C) Infectious in vitro transcribed RNA was transfected into Drosophila S2 cells followed 48 h later by immunoblot detection of VP2. WT, wild-type CrPV infectious clone; ORF1/ORF2 STOP, mutant CrPV clones containing a stop codon in ORF1 or ORF2; ΔPKI, mutant IGR IRES containing mutations that disrupt PKI base pairing; CrPV–Cal, chimeric CrPV infectious clone containing an IGR IRES substituted by that of Caledonia virus. (D) CrPV and chimeric CrPV–Cal infection (MOI 5) in Drosophila S2 cells monitored by VP2 expression at the indicated hours post-infection (h.p.i.). (E) Viral yield of CrPV and chimeric CrPVx–Cal infection (MOI 0.1) at 12 and 24 h.p.i. using a log scale. Viral titers: FFU/μl. *P-value < 0.05; n = 3.
	Figure 3. Factor-independent assembly of ribosomal complexes on type 6e IRESs. Binding of rabbit 40S subunits and assembly of 80S ribosomal complexes on (A and B) P. spelaeus TSA and P. solanasi TSA2 IRESs, (C) P. karamani and Beihai 85 IRESs, (D) Caledonia beadlet anemone dicistro-like virus 1 and picornavirales Q_sR_OV_023 (picornavirales 023) IRESs and (E) picornavirales N_OV_008 and Q_sR_OV_023 (picornavirales 08 and 023) IRESs in the presence of components of the translation apparatus (A–E) at 37°C and (B) also at 20°C, as indicated. (F) Binding of rabbit and insect (S. frugiperda) 40S subunits to the P. solanasi TSA2 IRES, assembly of 80S complexes and translocation at 37°C in the presence of the indicated components. The positions of toeprints relative to the +1 nucleotide of the triplet that mimics the initiation codon are indicated to the right and left of panels, as appropriate. A dideoxynucleotide sequence generated with the same primer was run in parallel on each gel (lanes C, T, A and G) to show the corresponding viral sequences. ORF2 sequences shown below each panel are annotated to indicate the positions of the strongest set of toeprints.
	Figure 4. Factor-independent assembly of ribosomal complexes on type 6e IRESs, accessibility of the A site in IRES/80S ribosome complexes and eEF2-mediated translocation following recruitment of cognate aminoacyl-tRNA/eEF1H·GTP. (A) Binding of rabbit 40S subunits to Wenling 2 and L. antarctica TSA1 IRESs, assembly of 80S complexes and translocation in the presence of the indicated components. (B and C) eEF2-mediated ribosomal translocation in the presence of the indicated components on (B) wild-type and (C) ACU1189–1191 (Thr)→GCU (Ala) variant P. solanasi TSA2 IRESs. (D) ORF2 sequences, annotated to indicate the positions of toeprints in (A) and (B). (E) Toeprinting analysis of binding of eRF1 and eRF3 to 80S complexes assembled on P. solanasi IRES-STOP mRNA in the presence and absence of eEF2. (F and G) Influence of SERBP1 and eEF2 on the binding of ribosomes to (F) Wenling 2 and (G) P. solanasi TSA2 IRESs, assayed by toeprinting. The positions of toeprints of IRES-bound 80S complexes relative to the +1 nucleotide of the initiation codon (A) mimics UCU1026–1028 of the L. antarctica TSA1 IRES, (A and F) GCU6906–6908 of the Wenling 2 IGR IRES and (B–E, G) ACU1189–1191 of wild-type and GCU1189–1191 or UAA1189–1191 of mutant forms of the P. solanasi TSA2 IRES (A–C, E–G) before binding of factors and after (A–C) translocation or (E) binding of eRF1/eRF3 are indicated to the right and left of each panel, as appropriate. A 2 nt reverse shift in the toeprint induced by binding of eEF2 IRES/80S complexes is indicated in (F) and (G). A dideoxynucleotide sequence generated with the same primer was run in parallel on each gel (lanes C, T, A and G) to show the corresponding viral sequences. (H) Analysis of A-site accessibility in 80S ribosomes bound to Wenling 2, P. solanasi TSA2 and L. antarctica TSA1 wild-type and AUG223–225UAG mutant IRESs in the presence and absence of eEF2, assayed by mapping susceptibility to RelE cleavage. Positions of cleavages corresponding to IRES-bound ribosomal complexes are indicated to the right.
	Figure 5. Structure of the P. solanasi TSA2 IRES. (A and B) Chemical (CMCT and NAI) and enzymatic (RNase T1) probing of the IRES. A dideoxynucleotide sequence generated with the same primer was run in parallel on both gels (lanes C, T, A and G) to show the P. solanasi TSA2 sequence. The positions of cleaved/modified nucleotides are indicated on the right of each panel, with a schematic representation of the IRES showing single-stranded loops as black rectangles and base-paired elements as light blue rectangles. The initiation codon mimic ACU1189–1191 is marked on the left. (C) Model of the tertiary structure of the P. solanasi TSA2 IRES, labeled to show nucleotides and pseudoknots, and indicating the positions of nucleotides modified by CMCT (shaded circles) and NAI (open circles) or cleaved by RNase T1 (arrowheads), based on data shown in (A) and (B).
	Figure 6. Mapping the borders of the P. solanasi TSA2 IRES. Activity of the P. solanasi TSA2 IRES variants (A) with 3′-terminal deletions in supporting accurate, factor-independent binding of 80S ribosomes and (C) substitutions in the first codon of ORF2. The positions of toeprints relative to the +1 nucleotide of the initiation codon mimic ACU1189–1191 are indicated to the right and left of each panel, as appropriate. A dideoxynucleotide sequence generated with the same primer was run in parallel on each gel (lanes C, T, A and G) to show the corresponding viral sequences. (B) Structural model of the P. solanasi TSA2 IRES, annotated to show the 5′ border of deletion mutants.
	Figure 7. Mutational analysis of sequence and structural elements in the P. solanasi TSA2 IRES. (A–D) Toeprint analysis of assembly of 80S complexes on the wild-type (wt) and mutant variants of the P. solanasi TSA2 IRES. The positions of toeprints relative to the +1 nucleotide of the initiation codon mimic ACU1189–11911 are indicated to the right of each panel. Numbers below each panel refer to sites subjected to mutational analysis, as indicated in (E), which summarizes the effects of mutations on IRES function. ‘Compensated’ indicates sites at which a debilitating mutation could be compensated by second-site substitutions, and ‘Impaired’ indicates sites at which mutations that impair IRES function could not be compensated in this way. (A, B, D) Separation of lanes by white lines indicates that they were juxtaposed from the same gel.
	Figure 8. The influence of conserved structural elements in the P. solanasi TSA2 IRES on ribosome binding activity. (A) Model of the IRES, showing substitutions of nucleotides in PKI and the L1 loop, and nucleotides introduced to replace PKIII by unstructured loops. Sites of mutation are numbered as in Figure 7E. (B–E) Activity of IRES variants with substitutions (B and C) in PKI, (D) in the L1 loop and (E) of PKIII by single-stranded loops in factor-independent binding of 80S ribosomes and 40S subunits, assayed by toeprinting. The positions of toeprints relative to the +1 nucleotide of the initiation codon mimic ACU1189–1191 are indicated to the right of each panel, as appropriate.
	Figure 9. Schematic models of IGR IRES types 6a–6e. Models of (A) a type 6a IRES (e.g. cricket paralysis virus), (B) a type 6b IRES (e.g. Taura syndrome virus), (C) a type 6c IRES (e.g. Halastavi árva virus), (D) a type 6d IRES (e.g. Nedicistrovirus) and (E) a type 6e IRES (e.g. Caledonia beadlet anemone dicistro-like virus 1), labeled to indicate domain 1 (which includes PKII) colored blue, domain 2 (which includes PKIII) colored red and domain 3 (which includes PKI) colored green, stem–loops SLIII, SLIV and SLV, and the L1.1a and L1.1b loops (with conserved motifs in these loops indicated by thicker lines).

Abaeva, The Halastavi árva Virus Intergenic Region IRES Promotes Translation by the Simplest Possible Initiation Mechanism. 2020, Pubmed

Abaeva, The Halastavi árva Virus Intergenic Region IRES Promotes Translation by the Simplest Possible Initiation Mechanism. 2020, Pubmed
Acosta-Reyes, The Israeli acute paralysis virus IRES captures host ribosomes by mimicking a ribosomal state with hybrid tRNAs. 2019, Pubmed
Alkalaeva, In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3. 2006, Pubmed
Ananth, An innate twist between Crick's wobble and Watson-Crick base pairs. 2013, Pubmed
Andreev, The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome. 2008, Pubmed
Antiqueo, De novo Assembly and Analysis of Tissue-Specific Transcriptomes of the Edible Red Sea Urchin Loxechinus albus Using RNA-Seq. 2021, Pubmed , Echinobase
Arhab, Dissemination of Internal Ribosomal Entry Sites (IRES) Between Viruses by Horizontal Gene Transfer. 2020, Pubmed
Asnani, PCBP2 enables the cadicivirus IRES to exploit the function of a conserved GRNA tetraloop to enhance ribosomal initiation complex formation. 2016, Pubmed
Asnani, Widespread distribution and structural diversity of Type IV IRESs in members of Picornaviridae. 2015, Pubmed
Au, Functional Insights into the Adjacent Stem-Loop in Honey Bee Dicistroviruses That Promotes Internal Ribosome Entry Site-Mediated Translation and Viral Infection. 2018, Pubmed
Bowman, Root of the Tree: The Significance, Evolution, and Origins of the Ribosome. 2020, Pubmed
Brown, Structures of translationally inactive mammalian ribosomes. 2018, Pubmed
Brown, Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES. 2022, Pubmed
Butcher, The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks. 2011, Pubmed
Costantino, A preformed compact ribosome-binding domain in the cricket paralysis-like virus IRES RNAs. 2005, Pubmed
Dereeper, Phylogeny.fr: robust phylogenetic analysis for the non-specialist. 2008, Pubmed
Fernández, Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome. 2014, Pubmed
Finoshin, Iron metabolic pathways in the processes of sponge plasticity. 2020, Pubmed
Flis, tRNA Translocation by the Eukaryotic 80S Ribosome and the Impact of GTP Hydrolysis. 2018, Pubmed
Hatakeyama, Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts. 2004, Pubmed
Hewson, Virome Variation during Sea Star Wasting Disease Progression in Pisaster ochraceus (Asteroidea, Echinodermata). 2020, Pubmed , Echinobase
Hoang, UFBoot2: Improving the Ultrafast Bootstrap Approximation. 2018, Pubmed
Huerlimann, Multi-species transcriptomics reveals evolutionary diversity in the mechanisms regulating shrimp tail muscle excitation-contraction coupling. 2020, Pubmed
Imai, An accurately preorganized IRES RNA structure enables eIF4G capture for initiation of viral translation. 2016, Pubmed
Jackson, The mechanism of eukaryotic translation initiation and principles of its regulation. 2010, Pubmed
Jan, Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus. 2002, Pubmed
Janssen, The RNA shapes studio. 2015, Pubmed
Johnson, A transcriptome resource for the Antarctic pteropod Limacina helicina antarctica. 2016, Pubmed
Kalyaanamoorthy, ModelFinder: fast model selection for accurate phylogenetic estimates. 2017, Pubmed
Kanamori, A tertiary structure model of the internal ribosome entry site (IRES) for methionine-independent initiation of translation. 2001, Pubmed
Kerr, Molecular analysis of the factorless internal ribosome entry site in Cricket Paralysis virus infection. 2016, Pubmed
Kerr, The 5' untranslated region of a novel infectious molecular clone of the dicistrovirus cricket paralysis virus modulates infection. 2015, Pubmed
Khalturin, Polyzoa is back: The effect of complete gene sets on the placement of Ectoprocta and Entoprocta. 2022, Pubmed
Khong, Temporal Regulation of Distinct Internal Ribosome Entry Sites of the Dicistroviridae Cricket Paralysis Virus. 2016, Pubmed
Koh, Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA-like structural element in the ribosomal decoding center. 2014, Pubmed
Kolupaeva, In vitro reconstitution and biochemical characterization of translation initiation by internal ribosomal entry. 2007, Pubmed
Koonin, The Big Bang of picorna-like virus evolution antedates the radiation of eukaryotic supergroups. 2008, Pubmed
Lefébure, Less effective selection leads to larger genomes. 2017, Pubmed
Letunic, Interactive Tree Of Life (iTOL) v4: recent updates and new developments. 2019, Pubmed
Liljas, Evolutionary and taxonomic implications of conserved structural motifs between picornaviruses and insect picorna-like viruses. 2002, Pubmed
Mailliot, Viral internal ribosomal entry sites: four classes for one goal. 2018, Pubmed
Miścicka, Initiation of translation on nedicistrovirus and related intergenic region IRESs by their factor-independent binding to the P site of 80S ribosomes. 2023, Pubmed
Moniruzzaman, Virus-host relationships of marine single-celled eukaryotes resolved from metatranscriptomics. 2017, Pubmed
Muhs, Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES. 2015, Pubmed
Nakashima, Functional analysis of structural motifs in dicistroviruses. 2009, Pubmed
Neubauer, The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE. 2009, Pubmed
Nishiyama, Eukaryotic ribosomal protein RPS25 interacts with the conserved loop region in a dicistroviral intergenic internal ribosome entry site. 2007, Pubmed
Nishiyama, Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes. 2003, Pubmed
Pestova, The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection. 2002, Pubmed
Pestova, Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA. 2003, Pubmed
Petrov, Multiple Parallel Pathways of Translation Initiation on the CrPV IRES. 2016, Pubmed
Pisarev, Assembly and analysis of eukaryotic translation initiation complexes. 2007, Pubmed
Prado-Álvarez, De novo transcriptome reconstruction in aquacultured early life stages of the cephalopod Octopus vulgaris. 2022, Pubmed
Rosani, A bioinformatics approach reveals seven nearly-complete RNA-virus genomes in bivalve RNA-seq data. 2017, Pubmed
Sasaki, Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro. 1999, Pubmed
Sasaki, Methionine-independent initiation of translation in the capsid protein of an insect RNA virus. 2000, Pubmed
Sato, IPknot: fast and accurate prediction of RNA secondary structures with pseudoknots using integer programming. 2011, Pubmed
Schüler, Structure of the ribosome-bound cricket paralysis virus IRES RNA. 2006, Pubmed
Shi, The evolutionary history of vertebrate RNA viruses. 2018, Pubmed
Shi, Redefining the invertebrate RNA virosphere. 2016, Pubmed
Skabkin, Reinitiation and other unconventional posttermination events during eukaryotic translation. 2013, Pubmed
Spitale, RNA SHAPE analysis in living cells. 2013, Pubmed
Susorov, Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome. 2018, Pubmed
Sweeney, The mechanism of translation initiation on Type 1 picornavirus IRESs. 2014, Pubmed
Tate, The crystal structure of cricket paralysis virus: the first view of a new virus family. 1999, Pubmed
Trifinopoulos, W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. 2016, Pubmed
Urbarova, Ocean acidification at a coastal CO2 vent induces expression of stress-related transcripts and transposable elements in the sea anemone Anemonia viridis. 2019, Pubmed
Valles, ICTV Virus Taxonomy Profile: Dicistroviridae. 2017, Pubmed
Waldron, Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes. 2018, Pubmed
Wang, Lso2 is a conserved ribosome-bound protein required for translational recovery in yeast. 2018, Pubmed
Wilson, Initiation of protein synthesis from the A site of the ribosome. 2000, Pubmed
Wolf, Doubling of the known set of RNA viruses by metagenomic analysis of an aquatic virome. 2020, Pubmed
Wu, Abundant and Diverse RNA Viruses in Insects Revealed by RNA-Seq Analysis: Ecological and Evolutionary Implications. 2020, Pubmed
Yamamoto, Binding mode of the first aminoacyl-tRNA in translation initiation mediated by Plautia stali intestine virus internal ribosome entry site. 2007, Pubmed
Yamamoto, Ribosomal Chamber Music: Toward an Understanding of IRES Mechanisms. 2017, Pubmed
Yesselman, Sequence-dependent RNA helix conformational preferences predictably impact tertiary structure formation. 2019, Pubmed
Zell, Picorna-Like Viruses of the Havel River, Germany. 2022, Pubmed
Zhou, Virome Analysis of Normal and Growth Retardation Disease-Affected Macrobrachium rosenbergii. 2022, Pubmed
Zinoviev, Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs. 2015, Pubmed
Zinoviev, In Vitro Characterization of the Activity of the Mammalian RNA Exosome on mRNAs in Ribosomal Translation Complexes. 2020, Pubmed
Zinoviev, Two classes of EF1-family translational GTPases encoded by giant viruses. 2019, Pubmed
Zuker, Mfold web server for nucleic acid folding and hybridization prediction. 2003, Pubmed