Medicine, Faculty of
Non UBC
Biochemistry and Molecular Biology, Department of
DSpace
Viruses 16 (5): 695 (2024)
University of British Columbia. Life Sciences Institute
Chen, Yihang
Chapagain, Subash
Chien, Jodi
Pereira, Higor Sette
Patel, Trushar R.
Inoue-Nagata, Alice K.
Jan, Eric
The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.
Citation: Chen, Y.; Chapagain, S.;Chien, J.; Pereira, H.S.; Patel, T.R.;Inoue-Nagata, A.K.; Jan, E.Factor-Dependent Internal RibosomeEntry Site and -1 ProgrammedFrameshifting Signal in theBemisia-Associated Dicistrovirus 2.Viruses 2024, 16, 695. https://doi.org/10.3390/v16050695Academic Editor: Bo LiangReceived: 4 February 2024Revised: 25 April 2024Accepted: 26 April 2024Published: 28 April 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).virusesArticleFactor-Dependent Internal Ribosome Entry Site and -1Programmed Frameshifting Signal in the Bemisia-AssociatedDicistrovirus 2Yihang Chen 1, Subash Chapagain 1, Jodi Chien 1, Higor Sette Pereira 2 , Trushar R. Patel 2 ,Alice K. Inoue-Nagata 3 and Eric Jan 1,*1 Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia,Vancouver, BC V6T 1Z3, Canada; yihangchen1013@gmail.com (Y.C.); chsubash@student.ubc.ca (S.C.);chienjodi@gmail.com (J.C.)2 Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University ofLethbridge, Lethbridge, AB T1K 3M4, Canada; higor.pereira@uleth.ca (H.S.P.); trushar.patel@uleth.ca (T.R.P.)3 Embrapa Vegetables, Brasília 70770-901, Brazil; alice.nagata@embrapa.br* Correspondence: ej@mail.ubc.caAbstract: The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiationmechanism: the IRES recruits ribosomes directly without using protein factors and initiates translationfrom a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically,the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regionsthat interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. Wepreviously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here,using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would directthe translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGRcan support IRES translation in vitro but does so through a mechanism that is not typical of knownfactorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon.Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4Ainhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependentmechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops;however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinctconformations for translation initiation. In summary, we have provided evidence of the first -1 FSframeshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thushighlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.Keywords: virus; frameshifting; RNA; IRES1. IntroductionRNA viruses carry genomes with limited coding capacity. As such, a key essential stepin the viral replication cycle is commandeering the host translational machinery to directviral protein synthesis. In eukaryotes, protein synthesis is initiated via the cap-dependentscanning model, whereby ~10 core translation factors mediate recruitment of the small40S ribosomal subunit to the 5′cap and scanning [1]. RNA viruses have adapted uniquestrategies to recruit and manipulate the host ribosome, some of which utilize an internalribosome entry site (IRES), which is a cis-acting RNA element, typically structured, thatcan mediate ribosome recruitment in a 5′-end independent manner and using a subsetof translation initiation factors and/or other cellular proteins. Currently, viral IRESsViruses 2024, 16, 695. https://doi.org/10.3390/v16050695 https://www.mdpi.com/journal/virusesViruses 2024, 16, 695 2 of 20can be grouped into six distinct classes based on the conservation of RNA structure andfactor requirements; however, with the expanding viromes identified via metagenomicapproaches [2,3], it is anticipated that many more classes have yet to be discovered.The dicistrovirus intergenic IRES studied to date are the most streamlined translationmechanism to date [4]. Dicistrovirus contains a ~8–10 kb viral RNA genome encodingtwo main open reading frames, ORF1 and ORF2. ORF1 encodes the viral non-structuralproteins such as replicase, protease, and RNA helicase, whereas ORF2 encodes the structuralproteins. The ORFs are translated by distinct IRESs, the 5′UTR and the IGR IRES. The5′UTR IRES utilizes a Type III-like IRES mechanism requiring several translation factors [5].The model IGR IRES, exemplified by that in cricket paralysis virus (CrPV), adopts a triple-pseudoknot RNA structure that can directly recruit the 40S ribosomal subunit, and thenthe 60S, to form the 80S ribosome and initiate translation from a non-AUG codon. SomeIGR IRESs can recruit preformed 80S to direct translation, including a fraction of CrPV IGRIRES [6–8]. Cryo-EM studies of IRES:ribosome complexes captured in the translocationcycles revealed that distinct domains of the IRES interact with specific parts of the ribosomein order to manipulate the ribosome into a primed elongation mode of translation [9–13].Within the IGR IRESs, there are distinct subtypes with specific RNA structures; they adopta similar RNA structure as the CrPV IGR IRES to direct translation [13,14]. However, it isevident that there may be other more atypical IGR IRES mechanisms within dicistroviridae.The IGR from Halastavi árva virus (HalV), isolated from the intestinal contents of freshwatercarp (Cyprinus carpio) [15], was identified to contain novel IRES features [7]; the HaIV IGRIRES lacks key domains including PKIII, SLIV, and SLV. Moreover, the HaIV IGR IRESdoes not recruit the 40S subunit but instead binds to preformed 80S ribosomes to drivetranslation. The discovery of HaIV IGR IRES and other similar IRESs leads to the possibilityof alternative IGR IRES mechanisms within the family Dicistroviridae.Virome metagenomics studies have identified novel dicistrovirus-like genomes, whichprovide an opportunity to address the repertoire of IRES mechanisms that drive proteinsynthesis in this viral family [2,3]. A recent report by Hedil et al. identified a noveldicistrovirus genome with unique features. Bemisia-associated dicistrovirus 2 (BaDV-2)was isolated from the sweet potato whitefly Bemisia tabaci [16]. The 8012 nt genomicsequence of BaDV-2 contains (Accession MN231041) three ORFs (ORF1a, ORF1b, andORF2) with a 5′UTR, an intergenic region (IGR), a 3′UTR, and a poly(A) tail. ORF1acontains an RNA helicase (nt 1780-2121) and a 3C cysteine protease motif (nt 3541-3654).ORF1b is in -1 frame and contains an RdRp motif (nt 4047-5261). ORF2 contains twopicornavirus capsid protein-like Rhv motifs (nt 5893 to 6177 and 6730–7002, respectively), acalicivirus coat-protein motif (nt 7021 to 7209), and a CrPV-capsid protein-like motif (nt 7336to 7977). A putative -1 frameshifting signal (-1 FS) was proposed to drive the translationof the full-length ORF1 polyprotein, consisting of a slippery sequence (GUCUUUU, nt3780-6) and a putative pseudoknot, whereas a putative IGR IRES was proposed to drivethe translation of ORF2.In this study, we demonstrated that the BaDV-2 IGR supports IRES activity using abicistronic reporter in vitro in insect Spodoptera frugiperda (Sf) 21 and rabbit reticulocytelysate (RRL) translation extracts. Deletion and mutagenesis analyses mapped the BaDV-2IRES activity within a 140-nucleotide element containing a predicted stem-loop. Moreover,the IRES is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respec-tively, indicating that this IRES is factor-dependent. Finally, we showed that the BADV-2putative -1 FS uses a stem-loop structure and slippery sequence to support 4% and 8% -1frameshifting in Sf-21 and RRL, respectively. In summary, we have provided evidence of anovel IRES and -1 FS mechanism in the dicistrovirus family, thus highlighting the diversityof viral strategies to direct viral protein synthesis.Viruses 2024, 16, 695 3 of 202. Materials and Methods2.1. Cell CultureDrosophila Schneider 2 cell line (S2) cells (Invitrogen) were maintained in Shields andSang M3 insect medium (Sigma-Aldrich, St. Louis, MI, USA) supplemented with 10% fetalbovine serum at 25 ◦C.2.2. PlasmidsBaDV-2 IGR IRES fragment (nucleotides 5325 to nucleotides 5694, Accession No.MN231041) (Twist Biosciences) was cloned into bicistronic and monocistronic luciferasereporter plasmids using Gibson assembly. Deletion mutations and specific nucleotidemutations were generated by PCR-based methods. BaDV-2 IGR IRES with a downstreamP2A peptide sequence (Twist Biosciences) was cloned into the CrPV infectious clone [17],replacing the CrPV IGR IRES.2.3. In Vitro TranscriptionMonocistronic, bicistronic, and infectious clones were linearized by NarI, BamHI,and Eco53KI (NEB) restriction enzymes, respectively. RNA was transcribed using T7RNA polymerase and subsequently purified with an RNeasy kit (Qiagen). 5′capping andpolyadenylation were performed post-transcriptionally (CellScript). The RNA integrity andpurity was confirmed by denaturing agarose gel analysis, and concentration was measuredwith a spectrophotometer (Nanodrop).2.4. TransfectionS2 cells (3 × 106 cells) were transfected with bicistronic or infectious clone RNA (3 µg)using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). Formonitoring reporter RNA translation under CrPV infection, S2 cells were transfected withbicistronic RNA for 1 h, followed by infection with CrPV (MOI = 20) for 4.5 h. For infectiousclone transfection experiments, S2 cells were transfected and then harvested at the indicatedtimes and lysed in 1× passive lysis buffer (Promega, Tokyo, Japan). Lysates were clearedand protein concentration was measured by Bradford assay (Bio-Rad, Hercules, CA, USA).Western blotting was performed using an anti-VP2 antibody.2.5. In Vitro Translation ReactionsIn vitro transcribed bicistronic (1 µg) and infectious clone (2 µg) RNAs were incubatedin Sf-21 cell extract (Promega) or rabbit reticulocyte lysate (RRL) (Promega) for 2 h or45 min, respectively, in the presence of [35S]-methionine/cysteine. Inhibitors (hippuristanolor NSC119889) at appropriate concentrations were added to the reaction 5 min prior tothe addition of RNA. Reactions were either loaded and resolved on 15% SDS-PAGE gelsand analyzed by phosphorimager analysis (Typhoon, Amersham, Chicago, IL, USA) oranalyzed for enzymatic luciferase activity (Promega) using a luminometer (Turner DesignsTD-20/20, San Jose, CA, USA).2.6. Ribosome Filter Binding AssayThe 40S and 60S ribosomal subunits were purified as previously described [18]. RNAwas dephosphorylated by FastAP thermosensitive alkaline phosphatase (Thermo FisherScientific) then labeled with T4 polynucleotide kinase (NEB) and [γ32P]-ATP. [γ32P]-RNA(final concentration: 0.5 nM) was heated at 65 ◦C for 3 min prior to the addition of 1× bufferE (final concentration: 20 mM Tris pH 7.5, 100 mM KCl, 2.5 mM MgOAc, 0.25 mM Sper-midine and 2 mM DTT) and gently cooled to room temperature for 20 min, allowingfor proper folding. The RNA was then incubated with purified 40S ribosomal subunitsfrom 0.1 nM to 100 nM for 5 min, followed by 60S ribosomal subunits from 0.15 nM to150 nM for 15 min, with 50 ng/mL in vitro transcribed noncompetitor RNA preparedfrom pcDNA3 (linearized with EcoRV) to prevent non-specific binding [19]. Reactionswere loaded to the Bio-Dot filtration apparatus (Bio-Rad) with nitrocellulose and nylonViruses 2024, 16, 695 4 of 20membranes (Zeta-Probe, Bio-Rad). The membranes were then washed three times with1× buffer E, then dried and imaged by phosphoimager analysis (Amershan Typhoon,ImageQuant™ 800). The fraction bound and the dissociation constant (KD) were calculatedas previously described [18].2.7. RNA Purification and Size Exclusion Chromatography with Multi-Angle Light Scattering(SEC-MALS)In vitro transcribed BaDV-2 IGR IRES RNA was purified by size exclusion chromatog-raphy (SEC) with an ÄKTA pure fast protein liquid chromatography (FPLC) (Global LifeScience Solutions, New York, NY, USA) as previously described [20]. RNA-containingfractions were then pooled and concentrated by ethanol precipitation with resuspension inHEPES folding buffer (final concentration: 50 mM HEPES, 150 mM NaCl, 15 mM MgCl2,3% Glycerol, and at pH 7.4) for SEC-MALS analysis and small-angle X-ray scattering(SAXS) analysis at the concentration of 2 mg/mL. SEC-MALS analysis was performed aspreviously described with the Optilab Refractive Index System (Wyatt Technology) [20].2.8. Small-Angle X-ray Scattering (SAXS) and Atomic Structure CalculationSAXS was performed as previously described by D’Souza et al. [20]. Briefly, SAXSdata for BaDV-2 “minimal” IRES RNA were collected at 2 mg/mL using HEPES foldingbuffer. Samples were run at Diamond Light Source Ltd. synchrotron (Didcot, UK) onthe B21 SAXS beamline, with a high-performance liquid chromatography (HPLC) systemattached upstream to ensure the monodispersity [21]. A specialized flow cell was employedin conjunction with an inline Agilent 1200 HPLC system (Agilent Technologies, Stockport,UK). Then, 50 µL of RNA samples were injected onto a Shodex KW403-4F (Showa DenkoAmerica Inc., New York, NY, USA) size exclusion column with a flow rate maintained at0.160 mL/minute. The eluted samples were exposed to X-rays with 3 s exposure time and600 frames. Analysis of scattering data was performed using the ATSAS suite as previouslydescribed [22,23]. Twenty RNA models were generated using DAMMIN, averaged andfiltered to a single representative using DAMAVER.We utilized MC-Fold [24] to anticipate multiple low-energy secondary structures forBaDV-2 IRES RNA. This was performed to produce secondary structure predictions fordownstream use. In each case, we chose the structures with the lowest energy as inputfiles for MC-Sym, an application that facilitates the reconstruction of three-dimensionalstructures using known structures based on fragments [24]. Using MC-Sym, we computed100 all-atom structures for each RNA and minimized them using MC-Sym’s implementedprotocols. These minimized structures were then compared to experimental SAXS datawith CRYSOL based on minimized structures, to determine Rg and the goodness-of-fit parameter (χ2) [25]. To subject the minimized structures to CRYSOL, we used theprotocols implemented in MC-Sym. Finally, we ranked the MC-Sym-derived structureswith low-resolution structures based on their χ2 values and aligned them using the programSUPCOMB [26].2.9. SHAPE ProbingFor SHAPE-MaP analysis of the “minimal” IRES, in vitro transcribed RNA for BaDV-2IRES was used as described [27]. Briefly, 500 ng of RNA was heated to 95◦ for 3 min,followed by the addition of Buffer E (final concentration of 20 mM Tris, pH 7.5, 0.1 MKCl, pH 7.0, 2.5 mM MgOAc, 0.25 mM spermidine and 2 mM dithiothreitol (DTT)) andincubated at 30 ◦C for 20 min. Folded RNA was modified by adding N-methylisatoicanhydride (NMIA) dissolved in DMSO (final concentration 5 mM) and incubated for45 min at 30 ◦C. Control reactions containing only DMSO (no NMIA), as well as additionaldenaturing control (DC) reactions were performed in parallel. The RNA was recovered byethanol precipitation.RT Primer extension of modified and control RNAs was performed with primer5′ -CCTTCACTTTTAACATGGTTGGCCTG-3′ using Superscript III Reverse TranscriptaseViruses 2024, 16, 695 5 of 20(SSIII, Thermo Fisher Scientific) with Mn2+, followed by second-strand cDNA synthe-sis (NEB #E6111). The ds cDNA was then used for subsequent library preparation andsequencing. Libraries were sequenced on an Illumina MiSeq platform following the man-ufacturer’s standard cluster generation and sequencing protocols (UBC Sequencing andBioinformatics Consortium).SHAPE-Mapper 2.0 was used to analyze the sequencing data as described [28].RNAstructure [29] was used to predict and model the secondary structure using theSHAPE-MaP data.3. Results3.1. BaDV-2 Contains a Programmed -1 Frameshift (-1 FS) SignalA pseudoknot structure with an upstream putative slippery sequence “GUCUUUU”,which are characteristics of a -1 programmed frameshift signal (FS), was proposed withinthe BaDV-2 genome [16,30] (Figure 1A). To determine whether this element can supportframeshifting, we cloned the putative -1 FS element (nucleotides 3742 to 3884, AccessionMN231041) into a bicistronic reporter between the Renilla (RLuc, 0 frame) and Fireflyluciferase (FLuc, -1 frame) open reading frames (Figure 1B). We cloned sequences upstreamand downstream of the predicted -1 FS signal to ensure the entire element is present. Wealso generated an in-frame control reporter whereby the RLuc and FLuc ORFs are in thesame frame and a mutation (UUU3783-85 to CCC) (SS MUT) that disrupts the putativeslippery sequence from “GUCUUUU” to “GUCCCCU” (Figure 1B).Bicistronic reporter RNAs were incubated in Sf-21 insect cell lysate or rabbit reticu-locyte lysate (RRL) and monitored luciferase activities and expression by incorporationof [35S]-methionine/cysteine (Figure 1C,D). FLuc activity was detected in Sf21 and RRLreactions with the bicistronic reporter containing the wild-type BADV-2 -1 FS. Moreover, afusion RLuc-FLuc protein product (~100 kDa) was detected, supporting that -1 frameshift-ing occurred (Figure 1C,D, lane 2). An RLuc protein was also detected, indicating that afraction of translating ribosomes did not undergo frameshifting (Figure 1C,D, lane 2). Asexpected, the in-frame control reporter RNA resulted in robust expression of the 100 kDaRLuc-FLuc fusion protein and loss of RLuc expression (Figure 1C,D, lane 3). Based onthe relative luciferase expression, the BADV-2 -1 FS signal activity occurred at 4 and8% efficiency in Sf-21 and RRL, respectively. Mutant bicistronic RNAs containing either apremature stop codon at the end of RLuc (Figure 1C,D, lane 1) or the slippery sequencemutant (SS Mut, UUU3783-5CCC) (Figure 1C,D, lane 4) abolished FLuc expression andactivity, thus supporting the presence of a -1 FS signal. These results demonstrated that theBaDV-2 genome contains a bona fide -1 FS signal.3.2. Characterization of the BADV-2 -1 FS SignalThe proposed BaDV-2 -1 FS signal contains a putative pseudoknot structure sevennucleotides downstream of the slippery sequence [31]. To delineate the minimal BADV-2-1 FS signal, systematic 3-end deletions were created (Figure 2A). Progressive 3′ dele-tions significantly inhibited -1 FS activity, including 3′DEL 3 and 3′DEL 2 (Figure 2).These results suggested that the elements downstream of the putative -1 FS element maypromote frameshifting.Viruses 2024, 16, 695 6 of 20Viruses 2024, 16, x FOR PEER REVIEW 6 of 21 Figure 1. In vitro translation assays of BaDV-2 -1 FS in Sf-21 and RRL. (A) Simplified schematic view of the BaDV-2 genome. (B) The predicted secondary structure of BaDV-2 -1 frameshifting signal and schematic view of the bicistronic reporter with RLuc and FLuc. The slippery sequence is shown in red letters and the amino acids decoded in the -1 and 0 frames are shown in black letters. Bicistronic reporter RNAs containing wild-type or mutant BaDV-2 -1 FSS and in-frame controls were incubated in (C) Sf-21 or (D) RRL extracts. “RLuc Stop” contains a premature stop codon at the end of RLuc; “RLuc_FS_-1FLuc” contains the wild-type -1 FS with FLuc in -1 frame; “SS Mut” contains a mutant slippery sequence (UUU3783-5CCC); “0FLuc” is the in-frame control in which the RLuc and FLuc are both in the 0-reading frame. (left) Reactions were resolved on SDS-PAGE gel and monitored by phosphorimager analysis. Shown is a representative SDS-PAGE gel from at least three independent experiments. (right) Luciferase activities were measured and calculated as a ratio of FLuc/RLuc and normalized to the in-frame control (0FLuc). Groups were compared to 0FLuc. A one-way ANOVA statistical test was used to determine the p values (** p < 0.01). Shown are averages from at least three independent experiments ± standard deviation. Figure 1. In vitro translation assays of BaDV-2 -1 FS in Sf-21 and RRL. (A) Simplified schematic viewof the BaDV-2 genome. (B) The predicted secondary structure of BaDV-2 -1 frameshifting signal andschematic view of the bicistronic reporter with RLuc and FLuc. The slippery sequence is shown inred letters and the amino acids decoded in the -1 and 0 frames are shown in black letters. Bicistronicreporter RNAs containing wild-type or mutant B DV-2 -1 FSS and in-frame controls were inc batedin (C) Sf-21 or (D) RRL extracts. “RLuc Stop” contains a premature stop codon at the end of RLuc;“RLuc_FS_-1FLuc” contains the wild-type -1 FS with FLuc in -1 frame; “SS Mut” contains a mutantslippery sequence (UUU3783-5CCC); “0FLuc” is the in-frame control in which the RLuc and FLucare both in the 0-reading frame. (left) Reactions were resolved on SDS-PAGE gel and monitored byphosphorimager analysis. Shown is a representative SDS-PAGE gel from at least three independentexperiments. (right) Luciferase activiti s were measured and calculated as a ratio of FLuc/RLuc andnormalized to th in-frame co trol (0FLuc). Groups were compared to 0FLuc. A one-way ANOVAstatistical test was used to determine the p values (** p < 0.01). Shown are averages from at least threeindependent experiments ± standard deviation.Viruses 2024, 16, 695 7 of 20To determine whether the predicted stem-loop and pseudoknot are important forBaDV-2 -1 FS, we generated mutations that would disrupt base-pairing and tested-1 FSactivity in RRL (Figure 2C). Mutations that disrupt the bottom base-paired domain ofthe stem-loop (M1, M2) abolished -1 FS activity to the same extent as the SS MUT; how-ever, compensatory mutations (M1+M2) that restored base-pairing did not rescue activitycompletely, suggesting that the nucleotide identities may be important (Figure 2D). Mu-tations that disrupt the upper base-paired region (M4) inhibited -1 FS activity; however,the corresponding disrupted base-paired region (M5) had only a minor effect (~84% ofthe wild type) (Figure 2D). Compensatory mutations (M4 + M5) rescued -1 FS activity towild-type or higher levels, suggesting that nucleotide identity of ACC (nt 3804 to 3806)may be important (Figure 2D). Of note, mutant (M5), which would disrupt the predictedpseudoknot base-pairing, had only a minor effect on -1 FS activity, thus indicating thatthe putative pseudoknot may not be critical for frameshifting (Figure 2D). Mutations thatdisrupt the apical loop (M3) also did not affect -1 FS activity (Figure 2D). In summary, wehave demonstrated that the BaDV-2 genome contains a bona fide -1 FS signal and identifiedseveral key nucleotides and a stem-loop that are required for activity.3.3. BaDV-2 IGR Supports Internal Ribosome EntryWe investigated whether the BaDV-2 IGR supports IRES activity. Our initial analysispredicted an atypical RNA structure that had features that were similar to known dicistro-virus IRES structures [16]. The predicted start site based on the proposed IRES structurestarts at a GCG codon (nucleotide 5668 to 5670). We cloned the IGR of BaDV-2 (nucleotide5325 to 5694) into the intergenic region of the RLuc-FLuc bicistronic reporter construct(Figure 3A). IRES activity was calculated as a ratio of FLuc/RLuc, where RLuc serves as anormalization control across experiments. We incubated in vitro transcribed RNAs intoSf-21 extracts and monitored scanning-dependent RLuc translation and IRES-mediatedFLuc translation. As expected, CrPV IGR IRES directed FLuc expression and activity,whereas a mutant CrPV IGR IRES that disrupts PKI base-pairing (CC6214-5 to GG mutant)abolished IRES activity (Figure 3A). The BaDV-2 IGR also resulted in FLuc expression,~1.5-fold translation compared to the wild-type CrPV IGR IRES (Figure 3A). To furtherconfirm that the BaDV-2 IGR can support IRES activity and that the IRES is not linked totranslation of RLuc, we added a strong hairpin loop within the 5′UTR upstream of RLuc toabolish scanning-dependent translation (Figure 3B). We also inserted the strong hairpinwithin the 5′UTR upstream of a BaDV-2 IRES-FLuc monocistronic reporter (Figure 3C).In reactions containing the bicistronic RNAs with the 5′UTR hairpin loop, only FLuc, butnot RLuc, was translated, indicating the hairpin blocks’ scanning-dependent translationof RLuc (Figure 3B,C). Further, addition of an upstream hairpin within the BaDV-2 IRESmonocistronic RNA still resulted in FLuc activity. These results demonstrated conclusivelythat the BaDV-2 IGR contains an IRES.3.4. “Mimimal” BADV-2 IGR That Directs IRES ActivityThe initial predicted BaDV-2 IRES structure adopts several stem-loop and pseudoknotstructures that resembled the dicistrovirus IGR IRES such as the CrPV IGR IRES [16]. Aseries of mutations was generated that disrupt predicted base-pairing, and BaDV-2 IRESactivity was examined in vitro (Figure S1A). In summary, the majority of mutants didnot affect BaDV-2 IRES activity (Figure S1B), suggesting an alternative RNA structure forBaDV-2 IRES translation initiation.We next generated a series of 5′ and 3′ deletion mutations within the IGR region topinpoint the minimal sequence for BaDV-2 IRES translation. Initially, nucleotides 5325 to5694 of BaDV-2 was cloned and showed IRES activity, which we denote as wild type (full-length) (Figure 4). Compared to the wild-type BaDV-2 IRES, 5′ deletions from nucleotide5325–5421 (5′DEL 2) but not from nucleotide (nt) 5325–5366 (5′Del 1) inhibited IRES activity(Figure 4A), thus setting the boundaries from the 5′end of the BaDV-2 IRES. Using a similarapproach from the 3′end, the majority of 3′ deletions from 5506–5694 (3′DEL 1-5) resulted inViruses 2024, 16, 695 8 of 20IRES activity as similar to or higher as compared to the wild-type IRES activity (Figure 4B).The only exception was 3′DEL 3, which showed ~30% of wild-type IRES activity (Figure 4B).To further pinpoint the minimal IRES, we generated combinations of 5′ and 3′ deletions.Combining 5′DEL 1 and 3′DEL 4 or 3′DEL 5 still showed IRES activity, whereas combining5′DEL 2 and 3′DEL 4 inhibited IRES activity to ~40% of wild-type IRES activity (Figure 4B).Thus, the deletion analysis showed that the “minimal” BaDV-2 IRES is contained within a140-nucleotide region from nt 5366–5506.Viruses 2024, 16, x FOR PEER REVIEW 7 of 21 3.2. Characterization of the BADV-2 -1 FS Signal The proposed BaDV-2 -1 FS signal contains a putative pseudoknot structure seven nucleotides downstream of the slippery sequence [31]. To delineate the minimal BADV-2 -1 FS signal, systematic 3-end deletions were created (Figure 2A). Progressive 3′ deletions significantly inhibited -1 FS activity, including 3′DEL 3 and 3′DEL 2 (Figure 2). These re-sults suggested that the elements downstream of the putative -1 FS element may promote frameshifting. To determine whether the predicted stem-loop and pseudoknot are important for BaDV-2 -1 FS, we generated mutations that would disrupt base-pairing and tested-1 FS activity in RRL (Figure 2C). Mutations that disrupt the bottom base-paired domain of the stem-loop (M1, M2) abolished -1 FS activity to the same extent as the SS MUT; however, compensatory mutations (M1+M2) that restored base-pairing did not rescue activity com-pletely, suggesting that the nucleotide identities may be important (Figure 2D). Mutations that disrupt the upper base-paired region (M4) inhibited -1 FS activity; however, the c r-responding disrupted base-paired region (M5) had only a minor effect (~84% of the wild type) (Figure 2D). Compensatory mutations (M4 + M5) rescued -1 FS activity to wild-type or higher levels, suggesting t at nucleotide id ntity of ACC (nt 3804 to 3806) may be im-portant (Figure 2D). Of note, mutant (M5), hich would disrupt the predicted pseudoknot base-pairing, had only a minor effect on -1 FS activity, thus indicating that the putative pseudoknot may not be critical for frameshifting (Figure 2D). Mutations that disrupt the apical loop (M3) also did not affect -1 FS activity (Figure 2D). In summary, we have demonstrated that the BaDV-2 genome contains a bona fide -1 FS signal and identified several key nucleotides and a stem-loop that are required for activity. Figure 2. Structural analysis of BaDV-2 -1 FS element. (A) Schematic of deletion mutants surroundingthe putative BaDV-2 -1 FS. The slippery sequence is shown in red letters. (B) In vitro transcribedRNAs were incubated in RRL and luciferase activities were measured and normalized to wild-typeBaDV-2 -1 FS (BaDV-2 FS WT). Groups were compared to the wild-type BaDV-2 -1 FS. (C) Schematicview of BaDV-2 -1 FS mutations. (D) In vitro transcribed RNAs were incubated in RRL, and luciferaseactivities were measured and normalized to wild-type BaDV-2 -1 FSS (BaDV-2 WT). The top reddotted line represents Fluc/RLuc ratio of the wild-type -1 FS activity normalized to 1. The lowerred dot line represents the background characterized by a mutation in the slippery site (SS MUT,UUU3783-5CCC) that abolishes -1 FS activity. Groups were compared to BaDV-2 WT. A one-wayANOVA statistical test was used to determine the p values (* p < 0.05, ** p < 0.01). “n.s.” denotes thedifference is not significant between the experimental groups and WT (p > 0.05). Shown are averagesfrom at least three independent experiments ± standard deviation.Viruses 2024, 16, 695 9 of 20Viruses 2024, 16, x FOR PEER REVIEW 9 of 21 Figure 3. BaDV-2 IGR supports internal ribosome entry. (A) Bicistronic reporter RNAs containing either the CrPV IGR or the BaDV-2 IGR, (B) bicistronic RNAs with a 5′UTR strong hairpin, or (C) monocistronic reporter RNAs with a 5′UTR hairpin and the BaDV-2 IGR (top schematics) were in-cubated in Sf-21 extracts. In vitro transcribed RNAs were analyzed by agarose gel analysis (middle). Translation of FLuc and RLuc was measured by either radioisotope [35S]-methionine/cysteine incor-poration followed by SDS-PAGE and phosphorimager analysis or luciferase assays (graphs shown bottom). A paired t-test was used to determine the p values. ** p < 0.01. “n.s.” denotes the difference is not significant between the control groups and the experimental groups (p > 0.05). Shown are representative SDS-PAGE gels and the averages from at least three independent experiments ± standard deviation. 3.4. “Mimimal” BADV-2 IGR That Directs IRES Activity The initial predicted BaDV-2 IRES structure adopts several stem-loop and pseu-doknot structures that resembled the dicistrovirus IGR IRES such as the CrPV IGR IRES [16]. A series of mutations was generated that disrupt predicted base-pairing, and BaDV-2 IRES activity was examined in vitro (Figure S1A). In summary, the majority of mutants did not affect BaDV-2 IRES activity (Figure S1B), suggesting an alternative RNA structure for BaDV-2 IRES translation initiation. We next generated a series of 5′ and 3′ deletion mutations within the IGR region to pinpoint the minimal sequence for BaDV-2 IRES translation. Initially, nucleotides 5325 to 5694 of BaDV-2 was cloned and showed IRES activity, which we denote as wild type (full-length) (Figure 4). Compared to the wild-type BaDV-2 IRES, 5′ deletions from nucleotide 5325–5421 (5′DEL 2) but not from nucleotide (nt) 5325–5366 (5′Del 1) inhibited IRES activ-ity (Figure 4A), thus setting the boundaries from the 5′end of the BaDV-2 IRES. Using a similar approach from the 3′end, the majority of 3′ deletions from 5506–5694 (3′DEL 1-5) resulted in IRES activity as similar to or higher as compared to the wild-type IRES activity Figure 3. BaDV-2 IGR supports internal ribosome entry. (A) Bicistronic reporter RNAs contain-ing either the CrPV IGR or the BaDV-2 IGR, (B) bicistronic RNAs with a 5′UTR strong hairpin, or(C) monocistronic reporter RNAs with a 5′UTR hairpin and the BaDV-2 IGR (top schematics) wereincubated in Sf-21 extracts. In vitro transcribed RNAs were analyzed by agarose gel analysis (mid-dle). Translation of FLuc and RLuc was measured by either radioisotope [35S]-methionine/cysteineincorporation followed by SDS-PAGE and phosphorimager analysis or luciferase assays (graphsshown bottom). A paired t-test was used to determine the p values. ** < 0.01. “n.s.” de-notes the d ff rence is not significant between the control groups and the exp rimental groups(p > 0.05). Shown are representative SDS-PAGE gels and the averages from at least three independentexperiments ± standard deviation.3.5. Structural Analysis of the BaDV-2 “Minimal” IRESSmall-angle X-Ray scattering (SAXS) is a method to resolve the size and shape ofmonodispersed macromolecules [32]. A prerequisite for SAXS is a pure, monodispersedsample that displays similar hydrodynamic properties [33]. We first purified BaDV-2 “mini-mal” IRES RNA made from T7 promoter by FPLC Superdex 200 (S200). The elution profileof the RNAs is as shown in Figure 5A for pure monomeric BaDV-2 IRES RNA collectedat approximately 12 to 13 mL. Purified RNA was analyzed on SEC-MALS to determinethe absolute molecular weight. SEC-MALS confirmed BaDV-2 IGR IRES had a molecularweight of 47.53 (±0.79%) kDa (Figure 5B), consistent with the predicted molecular weight(49.22 kDa) and indicating it is a monomer in solution. Next, we assessed BaDV-2 IRESstructural envelope using SAXS. The absence of aggregation in the solution is indicatedby the linearity observed in the Guinier plots (Figure 5C). The low angle region can beused to calculate Rg at inverted space, which is equal to 49.63 Å. The Guinier plot pro-vided reciprocal information, which was then converted into a real-space electron pairedViruses 2024, 16, 695 10 of 20distribution using indirect Fourier transformations, resulting in the derivation of the P(r)plot (Figure 5D). The P(r) plot indicates that BaDV-2 “minimal” IRES assumes an elon-gated structure, with a Dmax of approximately 160 Å and a real-space Rg calculated at49.87 ± 0.22 Å, as observed in the Gaussian distribution. Here, we showed a representativemodel in Figure 5C, in which the light blue is the SAXS low-resolution structure, and the su-perimposed model is the lowest energy atomistic structure determined; the correspondingsecondary structure model in represented in Figure 5E.To examine the secondary structure predicted by the SAXS IRES model, we re-examined the IRES translational activities by generating new mutations at key positionsthat would be predicted to alter base-pairing (Figure 5F, bottom).Mutation of UAGU to GUUA (M4), which would disrupt an apical stem-loop reducedIRES translation by ~40%, whereas all other mutations that disrupted other predictedstem-loops did not disrupt IRES translation.To further examine the structure of the BADV-2 IRES, we performed SHAPE structuralprobing (SHAPE-MaP) (Figure S3). SHAPE reactivities of each nucleotide was mappedonto the BADV-2 IRES. In general, the majority of nucleotides showed high reactivitiesthroughout the IRES. The UAGU (nt 5447–5450), which when mutated to GUUA (M4)(Figure 5, bottom) leads to a decrease in translation by 40%, showed high SHAPE reac-tivity (Figure S3). In summary, the mutational analyses and structural probing analysesstrongly hinted that the BaDV-2 IRES may adopt an unstructured RNA element thatdirects translation.3.6. BaDV-2 IGR Does Not Support IRES Activity in S2 CellsNext, we examined whether the BaDV-2 IGR IRES functions in mock- and dicistrovirus-infected S2 cells. We tested in both mock and infected cells as it has been shown that IREStranslation is stimulated under virus infection [34]. We transfected in vitro transcribedbicistronic RNAs containing wild-type or mutant CrPV IRES or full-length wild-type,“minimal” or the 3′deletion (3′ DEL 5) mutant BaDV-2 IRES) into S2 cells, then infected withCrPV (MOI = 20) and measured FLuc activity at 6 h after transfection. In CrPV-infected cells,only the reporter RNAs containing the CrPV IRES displayed FLuc activity, whereas reporterRNAs containing the mutant CrPV IRES or any of the BaDV-2 IRES did not, suggestingthat the BaDV-2 IRES translation is not supported in S2 cells. (Figure 6A).We reasoned that the BaDV-2 IRES may only be functional under a viral genomecontext. We previously showed that the heterologous dicistrovirus IRESs can supportIRES translation and infection using a chimeric CrPV infectious clone [18,35]. Briefly, wereplaced the CrPV IGR IRES with the BaDV-2 IGR IRES (full length, nt 5325 to 5694) in theCrPV infectious clone (Figure 6B). We also generated a clone with an inserted P2A “stop-go”peptide site between BaDV-2 IGR IRES and ORF2 to ensure proper expression of CrPVORF2 [36]. We first determined whether the BaDV-2 IRES could support translation of theinfectious clone in Sf-21 extracts. Our results showed that the CrPV but not BaDV-2 IGRIRES supported translation of ORF2 proteins, as evidenced of VP2 expression (Figure 6C).To determine whether the CrPV-BaDV-2 chimeric clone is infectious, we transfected in vitrotranscribed infectious clone RNAs into S2 cells and then followed VP2 expression byimmunoblotting at 144 h after transfection. We previously showed that detection of VP2after transfection of CrPV infectious clone RNA reflects productive CrPV infection in S2cells [17]. VP2 was detected in S2 cells transfected with wild-type CrPV clone but not amutant CrPV clone containing a stop codon in ORF1 or with the CrPV-BaDV-2 IGR IRESchimeric clone (both with and without P2A peptide site) (Figure 6D). Moreover, cytopathiceffects were observed in S2 cells transfected with the wild-type CrPV clone but not with theother clones. In summary, the BaDV-2 IGR IRES is not active in Drosophila cells or underCrPV infection.Viruses 2024, 16, 695 11 of 20Viruses 2024, 16, x FOR PEER REVIEW 10 of 21 (Figure 4B). The only exception was 3′DEL 3, which showed ~30% of wild-type IRES ac-tivity (Figure 4B). To further pinpoint the minimal IRES, we generated combinations of 5′ and 3′ deletions. Combining 5′DEL 1 and 3′DEL 4 or 3′DEL 5 still showed IRES activity, whereas combining 5′DEL 2 and 3′DEL 4 inhibited IRES activity to ~40% of wild-type IRES activity (Figure 4B). Thus, the deletion analysis showed that the “minimal” BaDV-2 IRES is contained within a 140-nucleotide region from nt 5366–5506. Figure 4. Deletion analysis of BaDV-2 IGR IRES. (A) Schematic of the initial predicted structureof the BaDV-2 IGR IRES. The red AUG denotes the predicted start codon. (B) 5′ and 3′ deletionswere generated within the BaDV-2 IGR. “▲” = DEL = deletion. (C) Bicistronic RNAs containingthe corresponding deletion mutants were incubated in Sf-21 extracts and RLuc and FLuc activitiesmeasured and normalized to the wild-type BaDV-2 IRES. The red line denotes the FLuc:RLuc ratio ofthe wild type IRES for comparison. A one-way ANOVA statistical test was used to determine thep value and thus the significance levels. Groups were compared to WT BaDV. ** p < 0.01. “n.s.”denotes the difference is not significant between the experimental groups and WT (p > 0.05). Shownare the averages from at least three independent experiments ± standard deviation.Viruses 2024, 16, 695 12 of 20Viruses 2024, 16, x FOR PEER REVIEW 12 of 21 Figure 5. Structural analysis of BaDV-2 IGR IRES. (A) FPLC profiles associated with BaDV-2 IGR “minimal” IRES IVT RNA purification. (B) SEC-MALS traces of the peak from the BaDV-2 IRES RNA run and the absolute molecular weight across them. (C) The absence of aggregation and the presence of a liner Guinier region in the sample indicates its purity, which enables the calculation of Rg from the low-angle region data and signifies the homogeneity of the sample. (D) Normalized pair distance distribution plots for BaDV-2 IRES RNA permit the determination of Rg derived from the SAXS dataset and including each molecule’s Dmax. P(r) versus radial distance plot indicates an elongated RNA structure in solution. (E) Predicted BaDV-2 IRES RNA structure model from SAXS data (light blue) overlapped with atomistic structure, represented in ribbons. (F) The secondary structural model of BaDV-2 IRES corresponding to the predicted model as shown in (E). The red letters denote the predicted AUG start codon. Mutation analysis of BaDV-2 “minimal” IRES with bicistronic reporter Sf-21 translation assays were performed and the results (M1-7) were measured Figure 5. Structural analysis of BaDV-2 IGR IRES. (A) FPLC profiles associated with BaDV-2 IGR“minimal” IRES IVT RNA purification. (B) SEC-MALS traces of the peak from the BaDV-2 IRESRNA run and the ab olute molecular weight across them. (C) Th absence of aggregation and thepresence of a liner Guinier region in the sample indicates its purity, which enables the calculationof Rg from the low-angle region data and signifies the homogeneity of the sample. (D) Normalizedpair distance distribution plots for BaDV-2 IRES RNA permit the determination of Rg derived fromthe SAXS dataset and including each molecule’s D ax. P(r) versus radial distance plot indicates anelongated RNA structure in solution. (E) Predicted BaDV-2 IRES RNA structure model from SAXSdata (light blue) overlapped with atomistic structure, represented in ribbons. (F) The secondarystructural model of BaDV-2 IRES corresponding to the predicted model as shown in (E). The redletters denote the predicted AUG start codon. Mutation analysis of BaDV-2 “minimal” IRES withbicistronic reporter Sf-21 translation assays were performed and the results (M1-7) were measuredby luciferase assays and normalized to wild-type (WT) BaDV-2 IGR IRES “minimal” IRES (bottom).Groups were compared to WT BaDV-2 IRES (red dotted line). An ANOVA statistical test was used todetermine the p value and thus the significance levels. * p < 0.05. “n.s.” denotes the difference is notsignificant between the experimental groups and WT (p > 0.05). Shown are the averages from at leastthree independent experiments ± standard deviation.Viruses 2024, 16, 695 13 of 20iruses 2024, 16, x FOR PEER REVIEW 14 of 21 Figure 6. BaDV-2 IGR IRES activity in infectious clone and in cells. (A) Bicistronic reporters were transfected into S2 cells followed by mock infection or CrPV infection (MOI = 20). Cells were col-lected 6 h post transfection. Bicistronic RNAs tested contain the wild-type CrPV, mutant CrPV (CC6214-5 to GG to disrupt PKI base-pairing), wild-type BaDV-2 full-length IGR IRES (WT BaDV-2), BaDV-2 3′ DEL 5, and BaDV “minimal” IRES (double deletion at both 3′ and 5′ end. Luciferase activities were measured and normalized to wild-type CrPV with mock infection. Red and black dots represent data points from three independent experiments. A paired t-test was used to deter-mine the p value and thus the significance levels. “n.s.” denotes the difference is not significant be-tween the control groups and the experimental groups (p > 0.05). Shown are the averages from at least three independent experiments ± standard deviation. (B) Schematic of infectious clone CrPV (CrPV, CrPV_IGR_IRES) and chimeric clones with CrPV IGR IRES replaced with BaDV-2 IGR IRES (BaDV-2 IGR IRES) and with P2A-site added after BaDV-2 IGR IRES (BaDV-2 IGR IRES+ P2A). (C) Infectious clone RNAs were incubated in Sf-21 extracts containing [35S]-methionine/cysteine and then monitored by SDS-PAGE analysis. Mock = mock transfection; OS = ORF1Stop; CrPV = pCrPV; BaDV = BaDV-2 IGR IRES; P2A = BaDV-2 IGR IRES + P2A. Shown is a representative gel from at least three independent experiments. (D) In vitro transcribed infectious clone RNAs were trans-fected into S2 cells for 144 h. VP2 expression was detected by immunoblotting. Shown are a repre-sentative SDS PAGE gel and the averages from at least three independent experiments ± standard deviation. 3.7. BaDV-2 IGR IRES Does Not Bind to Purified Ribosomes A unique feature of dicistrovirus IGR IRESs is its ability to recruit ribosomes directly without the aid of initiation factors [37]. The CrPV IGR IRES can assemble ribosomes by first recruiting 40S subunits followed by 60S subunit joining; however, ~8% of the time, the CrPV IRES can also bind to pre-formed 80S ribosomes [8,38]. We assessed ribosomal binding to the IRES using an established filter binding assay [35]. Incubating increasing concentration of purified salt-washed ribosomes with wild-type CrPV IRES resulted in CrPV IRES: ribosome complexes with an apparent KD of 1.4 ± 0.3 nM (Figure 7), which is BInfectious clone RNA in sf21 cell lysateA FLucCrPVOSMock BaDV P2A754535252015CrPVOSMock BaDV P2A604535 VP2TubulinC Infectious clone RNA in S2 cellsMockCrPV BaDV-2MT WT WT 3 del 5 Minimal Infection010203040FLucCrPV IGR IRESBaDV-2 IGR IRESBaDV-2 IGR IRES + P2A DORF2 polyprotein 3DVP2n.s.n.s.n.s.n.s.n.s.. .transfected into S2 cells followed by mock infection or CrPV infection (MOI = 20). Cells were collected6 h post transfection. Bicistronic RNAs tested contain the wild-type CrPV, mutant CrPV (CC6214-5 toGG to disrupt PKI base-pairing), wild-type BaDV-2 full-length IGR IRES (WT BaDV-2), BaDV-2 3′DEL 5, and BaDV “minimal” IRES (double deletion at both 3′ and 5′ end. Luciferase activities weremeasured and normalized to wild-type CrPV with mock infection. Red and black dots represent datapoints from three independent experiments. A paired t-test was used to determine the p value andthus the significance levels. “n.s.” denotes the difference is not significant between the control groupsand the experim tal groups (p > 0.05). Shown are the averages from at least three independentexperiments ± standard deviation. (B) Sch matic of infectious clone CrPV (CrPV, CrP _IGR_I )and chimeric clones with CrPV IGR IRES replaced with BaDV-2 IGR IRES (BaDV-2 IGR IRES) andwith P2A-site added after BaDV-2 IGR IRES (BaDV-2 IGR IRES+ P2A). (C) Infectious clone RNAs wereincubated in Sf-21 extracts containing [35S]-methionine/cysteine and then monitored by SDS-PAGEanalysis. Mock = mock transfection; OS = ORF1Stop; CrPV = pCrPV; BaDV = BaDV-2 IGR IRES;P2A = BaDV-2 IGR IRES + P2A. Shown is a representative gel from at least three independentexperiments. (D) In vitro transcribed infectious clone RNAs were transfected into S2 cells for 144 h.VP2 expression was detected by immunoblotting. Shown are a representative SDS PAGE gel and theaverages from at least three independent experiments ± standard deviation.3.7. BaDV-2 IGR IRES Does Not Bind to Purified RibosomesA unique feature of dicistrovirus IGR IRESs is its ability to recruit ribosomes directlywithout the aid of initiation factors [37]. The CrPV IGR IRES can assemble ribosomes byfirst recruiting 40S subunits followed by 60S subunit joining; however, ~8% of the time,the CrPV IRES can also bind to pre-formed 80S ribosomes [8,38]. We assessed ribosomalbinding to the IRES using an established filter binding assay [35]. Incubating increasingconcentration of purified salt-washed ribosomes with wild-type CrPV IRES resulted inViruses 2024, 16, 695 14 of 20CrPV IRES: ribosome complexes with an apparent KD of 1.4 ± 0.3 nM (Figure 7), which issimilar to previous reports [35]. By contrast, a mutant CrPV IRES containing disruptionsthat disrupt all three pseudoknot structures showed little to no 80S binding (Figure 7). TheBaDV-2 IRES did not bind to purified ribosomes (Figure 7, KD = 940.3 ± 4377 nM), thusindicating that this IRES is likely not using a factorless mechanism for translation.Viruses 2024, 16, x FOR PEER REVIEW 15 of 21 similar to previous reports [35]. By contrast, a mutant CrPV IRES containing disruptions that disrupt all three pseudoknot structures showed little to no 80S binding (Figure 7). The BaDV-2 IRES did not bind to purified ribosomes (Figure 7, KD = 940.3 ± 4377 n ), thus indicating that this IRES is likely not using a factorless echanism for translation. Figure 7. Affinity of 80S-BaDV-2 IGR IRES complexes. Filter binding assays. [32P]-BaDV-2 IGR IRES, [32P]-(CrPV) IGR IRES or mutant (∆PKI/II/III) CrPV IGR IRES were incubated with increas-ing amounts of purified salt-washed 80S. The fractions bound were quantified by filter binding assay followed by phosphorimager analysis. CrPV WT has a KD value of 1.4 nM, CrPV mutant (MT, mutations that disrupt all three pseudoknot base-pairings) has a KD value of 321.0 nM and WT BaDV-2 has a KD value of 940.3 nM. Shown are the averages from at least three independent experiments ± standard deviation. 3.8. BaDV-2 IGR IRES Initiates Translation from an AUG Start Codon Our results so far point to an atypical IRES mechanism that drives BaDV-2 IGR IRES translation. We next focused on determining the start site of the BaDV-2 IGR IRES. In-frame stop codons (SC) were inserted across the full-length BaDV-2 IGR (Figure 8A). Stop codon insertions at nt 5503, 5608, and 5668 (SC1, 2, 3) all decreased IRES activity to ~5–25% of wild-type IRES activity (Figure 8, thus suggesting that the start site is upstream of nt 5503. Moreover, these experiments are in line with the deletion analysis that the “min-imal” IRES is contained between nt 5366 and 5506. A stop codon insertion at nt 5444 (SC4) did not affect IRES activity, thus indicating that the start codon is between nts 5444 and 5503. We hypothesized that the AUG (nt 5491–5493) codon may be the initiation codon of the IRES. We mutated the AUG (nt 5491–5493) codon to UAG (M1) or GCG (M2) or deleted the AUG (DEL). Deletion of the AUG codon or mutating to UAG (M1) dramatically inhib-ited IRES translation by ~75–80% compared to wild-type IRES activity (Figure 8), albeit some residual ~20–25% IRES activity was observed. We reasoned that the residual IRES activities with the AUG codon mutations may be due to ribosomes recruited to the BaDV-2 IRES and then scanned downstream to initiate translation from the AUG codon of FLuc. To address this, we deleted the AUG codon of FLuc in combination with the deletion of the mutant AUG (nt 5491–5493) codons. Deleting the AUG codon of FLuc within the wild-type BaDV-2 IGR IRES inhibited IRES activity by ~10–15% compared to the wild-type IRES with the AUG codon of FLuc. By contrast, combining the deleted AUG codon of FLuc with deletion of AUG (nt 5491–5493) or M1 or M2 mutants abolished IRES-mediated FLuc activity completely. These results strongly demonstrated that the BaDV-2 IGR IRES initi-ates from the AUG (nt 5491–5493) codon and that a fraction of the 40S ribosomal subunits recruited to the IRES can scan to initiate translation from a downstream AUG codon. CrPV WTCrPV TM BaDV-2 80S Binding Fraction Bound1.4 321.0 940.3KD/nMRibosome ConcentrationFig re . ffi ity of 80S-BaDV-2 IGR IRES complexes. Filter binding ssays. [32P]-BaDV-2 IGRIRES, [32P]-(CrPV) IGR IRES or utant (∆PKI/II/III) CrPV IGR IRES were incubated with increas-ing amounts of purified salt-washed 80S. The fractions bound were quantified by filter bindingassay followed by phosphorimager analysis. CrPV WT has a KD value of 1.4 nM, CrPV mutant(MT, mutations that disrupt all three pseudoknot base-pairings) has a KD value of 321.0 nM andWT BaDV-2 has a KD value of 940.3 nM. Shown are the averages from at least three independentexperiments ± standard deviation.3.8. BaDV-2 IGR IRES Initiates Translation from an AUG Start CodonOur results so far point to an atypical IRES mechanism that drives BaDV-2 IGR IREStranslation. We next focused on determining the start site of the BaDV-2 IGR IRES. In-framestop codons (SC) were inserted across the full-length BaDV-2 IGR (Figure 8A). Stop codoninsertions at nt 5503, 5608, and 5668 (SC1, 2, 3) all decreased IRES activity to ~5–25% ofwild-type IRES activity (Figure 8, thus suggesting that the start site is upstream of nt 5503.Moreover, these experiments are in line with the deletion analysis that the “minimal” IRESis contained between nt 5366 and 5506. A stop codon insertion at nt 5444 (SC4) did notaffect IRES activity, thus indicating that the start codon is between nts 5444 and 5503. Wehypothesized that the AUG (nt 5491–5493) codon may be the initiation codon of the IRES.We mutated the AUG (nt 5491–5493) codon to UAG (M1) or GCG (M2) or deleted theAUG (DEL). Deletion of the AUG codon or mutating to UAG (M1) dramatically inhibitedIRES translation by ~75–80% compared to wild-type IRES activity (Figure 8), albeit someresidual ~20–25% IRES activity was observed. We r ason d that the residual IRES activitieswi h th AUG codon mutations m y be due to ribosomes recruited to th BaDV-2 IRESand then scanned downstream o initiate translation from the AUG codon of FLuc. Toaddress this, we delet d he AUG codon of FLuc in ombinat on with the deletion of themutant AUG (nt 5491–5493) codons. Deleting he AUG codon of FLuc within e wild-typeBaDV-2 IGR IRES inhibited IRES activity by ~10–15% compared to the wild-type IRESwith the AUG codon of FLuc. By contrast, combining the del t d AUG codon of FLucith deletion of ( t 91–5493) or 1 r t ts lis I - e iated cactivity completely. These results trongly demonstrated that the BaDV-2 IGR I ES init atesfrom the AUG (nt 5491–5493) codon a d that fraction of the 40S ribosomal subunitsrecruited to the IRES can scan to initiate translation from a downstream AUG codon.Viruses 2024, 16, 695 15 of 20Viruses 2024, 16, x FOR PEER REVIEW 16 of 21 Figure 8. Stop codon mutations to confirm the BaDV-2 IGR IRES initiation site. (A) Schematic of stop codon mutations along the BaDV-2 IGR IRES (upper). The red letters denote the predicted AUG start codon. In vitro translation assays were performed in Sf-21 cell lysate with the indicated muta-tions using bicistronic reporter RNAs. RLuc and FLuc activities were measured and normalized to the wild-type BaDV-2 bicistronic RNA (bottom). An ANOVA statistical test was used to determine the p value and thus the significance levels. Groups were compared to WT BaDV. ** represents p < 0.01. “n.s.” denotes the difference is not significant between the experimental groups and WT (p > 0.05). Shown are the averages from at least three independent experiments ± standard deviation. 3.9. BaDV-2 IGR IRES Translation Requires eIF4A and eIF2 To determine whether the BaDV-2 IRES uses translation factors, we tested IRES trans-lation in extract incubated with inhibitors that target specific translation initiation factors (Figure 9A). Specifically, hippuristanol inhibits eIF4A helicase activity and NSC119889 blocks eIF2 from binding to initiator Met-tRNAi [39,40]. We pre-incubated these inhibitors in RRL or Sf-21 extracts followed by addition of IRES-containing reporter RNAs. Incubat-ing with hippuristanol (10 µM and 15 µM) or NSC119889 (12.5 µM) significantly de-creased scanning-dependent RLuc translation, as expected (Figure 9B,C). By contrast, CrPV IRES-mediated FLuc translation was relatively resistant to hippuristanol or NSC119889, in line with the CrPV IRES driving factorless translation (Figure 9B,C) [41]. Interestingly, BaDV-2 IRES translation was sensitive to both drug treatments inhibiting translation as much as scanning dependent, thus demonstrating that eIF4A and eIF2 ter-nary complexes are required for BaDV-2 IRES translation. Figure 8. Stop codon mutations to confirm the BaDV-2 IGR IRES initiation site. (A) Schematic of stopcodon mutations along the BaDV-2 IGR IRES (upper). The red letters denote the predicted AUG startcodon. In vitro translation assays were performed in Sf-21 cell lysate with the indicated mutationsusing bicistronic reporter RNAs. RLuc and FLuc activities were m asured and ormalized to thewild-type BaDV-2 bicistronic RNA (bottom). An NOVA statistical test was u ed to determine thep value and thus the significan e levels. Groups were compared to WT BaDV. ** represents p < 0.01.“n.s.” denotes the difference is not significant between the experimental groups and WT (p > 0.05).Shown are the averages from at least three independent experiments ± standard deviation.3.9. BaDV-2 IGR IRES Tra slatio e ire IT deter i e hether the BaDV-2 I ES uses translation factors, we tested IRES trans-lation in extract incubated ith inhibitors that target specific translation initiation factors(Figure 9 ). Specifically, i puristanol inhibits eIF4 helicase activity and S 119889blocks eIF2 fro binding to initiator et-t i [39,40]. We pre-incubated these inhibitorsin r Sf-21 extracts f ll e by addition of IRES-containing reporter RNAs. Incubatingwith hippuristanol (10 µM and 15 µM) or NSC119889 (12.5 µM) significantly decreasedscanning-dependent RLuc translation, as expected (Figure 9B,C). By contrast, CrPV IRES-mediated FLuc translation was relatively resistant to hippuristanol or NSC119889, in linewith the CrPV IRES driving factorless translation (Figure 9B,C) [41]. Interestingly, BaDV-2IRES translation was sensitive to both drug treatments inhibiting translation as muchas scanning dependent, thus demonstrating that eIF4A and eIF2 ternary complexes arerequired for BaDV-2 IRES translation.Viruses 2024, 16, 695 16 of 20Viruses 2024, 16, x FOR PEER REVIEW 17 of 21 Figure 9. Effects of translation inhibitors on BaDV-2 IRES translation. (A) Schematic illustration of bicistronic reporter RNAs with CrPV or BaDV-2 IGR IRESs. In vitro transcribed reporter RNAs were incubated (B) in RRL with hippuristanol or (C) in Sf21 extracts with NSC119889 at the indicated concentrations. RLuc and FLuc activities were measured and normalized to DMSO-treated lysates with wild-type CrPV or BaDV-2 IGR IRES bicistronic RNAs. A one-way ANOVA statistical test was used to determine the p values. * p < 0.05, ** p < 0.01. “n.s.” denotes the difference is not significant between the control groups and the experimental groups (p > 0.05). Shown are the averages from at least three independent experiments ± standard deviation. 4. Discussion To date, the dicistrovirus family contains the most streamlined mechanisms of eu-karyotic translation utilizing an RNA structure to directly recruit ribosomes and initiate translation. Recently, variations in dicistrovirus IRESs have come to light, showing that the dicistrovirus IRESs can have distinct ribosome assembly pathways (ex. IRES binds to preformed 80S) and can initiate from ribosomal P or A sites [6,7]. Here, in this study, we focused on a mechanism of a whitefly dicistrovirus virus, BaDV-2. Like other dicistrovi-ruses, the BaDV-2 intergenic region can support IRES translation in vitro; however, sev-eral evidence demonstrate that this mechanism is distinct from other known dicistrovirus IRES mechanisms. One, the BaDV-2 IRES does not bind to ribosomes directly and likely requires a subset of translation initiation factors and other factors. Our results showed that i r 9. ff t f tr sl ti i i it r -2 IRES translation. ( ) Sc e atic ill str ti fbicistronic reporter RNAs with CrPV or BaDV-2 IGR IRESs. In vitro transcribed reporter RNAs wereincubated (B) in RRL with hippuristanol or (C) in Sf21 extracts with NSC119889 at the indicatedconcentrations. RLuc and FLuc activities were measured and normalized to DMSO-treated lysateswith wild-type CrPV or BaDV-2 IGR IRES bicistronic RNAs. A one-way ANOVA statistical test wasused to determine the p values. * p < 0.05, ** p < 0.01. “n.s.” denotes the difference is not significantbetween the control groups and the experimental groups (p > 0.05). Shown are the averages from atleast three independent experiments ± standard deviation.4. DiscussionTo date, the dicistrovirus family contains the most streamlined mechanisms of eu-karyotic translation utilizing an RNA structure to directly recruit ribosomes and initiatetranslation. Recently, variations in dicistrovirus IRESs have come to light, showing thatthe dicistrovirus IRESs can have distinct ribosome assembly pathways (ex. IRES binds topreformed 80S) and can initiate from ribosomal P or A sites [6,7]. Here, in this study, we fo-cused on a mechanism of a whitefly dicistrovirus virus, BaDV-2. Like other dicistroviruses,the BaDV-2 intergenic region can support IRES translation in vitro; however, several evi-dence demonstrate that this mechanism is distinct from other known dicistrovirus IRESmechanisms. One, the BaDV-2 IRES does not bind to ribosomes directly and likely requiresViruses 2024, 16, 695 17 of 20a subset of translation initiation factors and other factors. Our results showed that eIF2and eIF4A are required for BaDV-2 IRES translation. Two, the BaDV-2 IRES is within a 140-nucleotide element and starts translation from an AUG codon, in line with the requirementof eIF2 for IRES translation. Three, the BaDV-2 IRES likely does not utilize an RNA structureor may adopt different conformations to direct ribosome recruitment. Extensive mutationalanalysis and SAXS analysis and the requirement of the RNA helicase eIF4A support thismodel. Our results demonstrate that the BaDV-2 contains a novel IRES mechanism that isdistinct from other dicistrovirus IRES mechanisms to date and may represent a novel classof IRES.Our studies highlight the diverse IRES mechanisms that may be used within the familyDicistroviridae. It is likely that the BaDV-2 IRES evolved mechanisms that require factors inorder to adapt to and drive viral protein synthesis in BaDV-2 infection in whiteflies. Thus,there may be specific cellular infection conditions that promote BaDV-2 IRES translation.Indeed, we showed that the BaDV-2 IRES is not functional in Drosophila cells and underCrPV infection (Figure 6). However, it is interesting to note that the BADV-2 IRES translationis supported in vitro in Sf21 extracts and not in Drosophila cells, thus pointing to specifichost factors required for BADV-2 IRES translation. To date, until this study, dicistrovirusIRES mechanisms in general all utilize a factorless ribosome assembly mechanism; however,it is clear that the diverse translation mechanisms are utilized under specific dicistrovirusinfections. The BaDV-2 IRES represents one of the most divergent IRES mechanismswithin the family Dicistroviridae, which likely means that there are many alternative viraltranslation strategies that are utilized by dicistroviruses. It will be interesting to followthe evolutionary trajectory of dicistroviruses and whether they utilize a factorless IRESmechanism, a BaDV-2 IRES-like mechanism, or a yet undiscovered translation mechanism.The BaDV-2 IRES utilizes at least eIF2 and eIF4A for IRES translation. Moreover, theexact structure of BaDV-2 that supports IRES function needs further investigation. Al-though SAXS analysis predicted a structure containing several stem-loops, our mutationalanalysis and SHAPE structural probing did not support this model. The data point to anunstructured or potential remodeling of the RNA structure that supports BaDV-2 IRESfunction, which is supported by its requirement for eIF4A. This evidence suggests a mecha-nism that is a bit reminiscent of some cellular IRESs that are thought to use unstructuredelements that may be ribosome landing elements [42,43]. Further investigation into thespecific BaDV-2 IRES elements and/or RNA conformations is warranted.Our results have for the first time provided biochemical evidence of a bona fide -1 FSsignal within a dicistrovirus genome. The BaDV-2 -1 FS signal contains standard elementsof a frameshift element including a stem-loop and an upstream slippery sequence [28]. Weruled out the requirement of a predicted pseudoknot for BaDV-2 -1 FS activity in vitro. The-1 FS signal is between the 3C protease and RNA-dependent RNA polymerase (RdRP) [16].Thus, it is likely that the -1 FS signal regulates the exact stoichiometry of the RdRP to otherviral non-structural proteins for precise replication activity during the viral life cycle. Itwill be of interest to identify other -1 FS signals within discistroviruses and whether thisis more widespread within this viral family. It is noteworthy that BaDV-1 has a typicaldicistronic genome, while BaDV-2 is primarily tricistronic.The whitefly complex Bemisia tabaci is a critical agricultural pest that occurs in mosttropical and subtropical countries and affects important crops, like cotton, cassava, beans,soybeans, and vegetables [44–47]. The identification of viruses such as BaDV-1 and BaDV-2that are associated with whiteflies may provide a direction for biopesticide approachesto control these pests [16,48], for example, those baculoviruses used for controlling cater-pillars [49–51]. Understanding the molecular mechanisms, such as IRES-mediated viralprotein synthesis, will provide valuable insights into the viral infection strategies of thesepest-associated viruses that may be exploited for therapeutic approaches in agriculture.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v16050695/s1, Figure S1: Predicted secondary model of BaDV-2Viruses 2024, 16, 695 18 of 20IGR IRES; Figure S2: BaDV-2 IGR IRES activity in mock-infected and CrPV-infected cells; Figure S3:SHAPE reactivity of BaDV-2 IRES.Author Contributions: Conceptualization, Y.C. and E.J.; methodology, Y.C., S.C., J.C. and H.S.P.;validation, Y.C., S.C., J.C. and H.S.P.; formal analysis, Y.C., S.C., H.S.P., T.R.P. and E.J.; investigation,Y.C., S.C., J.C. and H.S.P.; resources, Y.C., S.C., J.C., H.S.P., T.R.P. and E.J.; writing—original draftpreparation, Y.C. and E.J.; writing—review and editing, Y.C., S.C., H.S.P., T.R.P., A.K.I.-N. and E.J.;visualization, Y.C., S.C., H.S.P., T.R.P. and E.J.; supervision, T.R.P. and E.J.; funding acquisition, T.R.P.,A.K.I.-N. and E.J. All authors have read and agreed to the published version of the manuscript.Funding: This study was supported by a CIHR Project Grant (PJT-178342) and an NSERC Discoverygrant (RGPIN-2023-03658) to E.J. H.S.P. is supported partly by NSERC CREATE Postdoctoral Fellow-ship and Canada Research Chair support to T.R.P. T.R.P. acknowledges the Canada Research Chairprogram, NSERC Research Tools and Infrastructure, and the Canada Foundation for Innovationgrants. SAXS data were collected at the B21 beamline at the DIAMOND synchrotron. A.K.I.-N. issupported by Brazilian Agricultural Research Corporation: 20.22.03.023.00.00 and Fundacao de Apoioa Pesquisa do Distrito Federal: 00193-00002409/2023-17.Institutional Review Board Statement: Not applicable.Data Availability Statement: The original contributions presented in the study are included in thearticle/supplementary material, further inquiries can be directed to the corresponding author/s.Acknowledgments: We thank the Jan lab for helpful discussions. Hippuristanol was a generousgifted by Jerry Pelletier.Conflicts of Interest: The authors declare no conflict of interest.References1. Merrick, W.C.; Pavitt, G.D. Protein Synthesis Initiation in Eukaryotic Cells. Cold Spring Harb. Perspect. Biol. 2018, 10, a033092.[CrossRef] [PubMed]2. Edgar, R.C.; Taylor, B.; Lin, V.; Altman, T.; Barbera, P.; Meleshko, D.; Lohr, D.; Novakovsky, G.; Buchfink, B.; Al-Shayeb, B.; et al.Petabase-scale sequence alignment catalyses viral discovery. Nature 2022, 602, 142–147. [CrossRef] [PubMed]3. Shi, M.; Lin, X.-D.; Tian, J.-H.; Chen, L.-J.; Chen, X.; Li, C.-X.; Qin, X.-C.; Li, J.; Cao, J.-P.; Eden, J.-S.; et al. Redefining theinvertebrate RNA virosphere. Nature 2016, 540, 539–543. [CrossRef]4. Warsaba, R.; Sadasivan, J.; Jan, E. Dicistrovirus-Host Molecular Interactions. Curr. Issues Mol. Biol. 2020, 34, 83–112. [CrossRef][PubMed]5. Gross, L.; Vicens, Q.; Einhorn, E.; Noireterre, A.; Schaeffer, L.; Kuhn, L.; Imler, J.-L.; Eriani, G.; Meignin, C.; Martin, F. TheIRES5′UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure. NucleicAcids Res. 2017, 45, 8993–9004. [CrossRef] [PubMed]6. Mis´cicka, A.; Lu, K.; Abaeva, I.S.; Pestova, T.V.; Hellen, C.U. Initiation of translation on nedicistrovirus and related intergenicregion IRESs by their factor-independent binding to the P site of 80S ribosomes. RNA 2023, 29, 1051–1068. [CrossRef] [PubMed]7. Abaeva, I.S.; Vicens, Q.; Bochler, A.; Soufari, H.; Simonetti, A.; Pestova, T.V.; Hashem, Y.; Hellen, C.U. The Halastavi árva VirusIntergenic Region IRES Promotes Translation by the Simplest Possible Initiation Mechanism. Cell Rep. 2020, 33, 108476. [CrossRef]8. Petrov, A.; Grosely, R.; Chen, J.; O’leary, S.E.; Puglisi, J.D. Multiple Parallel Pathways of Translation Initiation on the CrPV IRES.Mol. Cell 2016, 62, 92–103. [CrossRef]9. Pisareva, V.P.; Pisarev, A.V.; Fernández, I.S. Dual tRNA mimicry in the Cricket Paralysis Virus IRES uncovers an unexpectedsimilarity with the Hepatitis C Virus IRES. eLife 2018, 7, e34062. [CrossRef]10. Murray, J.; Savva, C.G.; Shin, B.-S.; E Dever, T.; Ramakrishnan, V.; Fernández, I.S.; Biology, M.L.O.M.; States, U. Structuralcharacterization of ribosome recruitment and translocation by type IV IRES. eLife 2016, 5, e13567. [CrossRef]11. Fernández, I.S.; Bai, X.-C.; Murshudov, G.; Scheres, S.H.; Ramakrishnan, V. Initiation of Translation by Cricket Paralysis VirusIRES Requires Its Translocation in the Ribosome. Cell 2014, 157, 823–831. [CrossRef] [PubMed]12. Muhs, M.; Hilal, T.; Mielke, T.; Skabkin, M.A.; Sanbonmatsu, K.Y.; Pestova, T.V.; Spahn, C.M. Cryo-EM of Ribosomal 80SComplexes with Termination Factors Reveals the Translocated Cricket Paralysis Virus IRES. Mol. Cell 2015, 57, 422–432. [CrossRef][PubMed]13. Abeyrathne, P.D.; Koh, C.S.; Grant, T.; Grigorieff, N.; Korostelev, A.A. Ensemble cryo-EM uncovers inchworm-like translocationof a viral IRES through the ribosome. eLife 2016, 5, e14874. [CrossRef] [PubMed]14. Acosta-Reyes, F.; Neupane, R.; Frank, J.; Fernández, I.S. The Israeli acute paralysis virus IRES captures host ribosomes bymimicking a ribosomal state with hybrid tRNAs. EMBO J. 2019, 38, e102226. [CrossRef] [PubMed]15. Boros, Á.; Pankovics, P.; Simmonds, P.; Reuter, G. Novel Positive-Sense, Single-Stranded RNA (+ssRNA) Virus with Di-CistronicGenome from Intestinal Content of Freshwater Carp (Cyprinus carpio). PLoS ONE 2011, 6, e29145. [CrossRef] [PubMed]Viruses 2024, 16, 695 19 of 2016. Hedil, M.; Nakasu, E.Y.; Nagata, T.; Wen, J.; Jan, E.; Michereff-Filho, M.; Inoue-Nagata, A.K. New features on the genomicorganization of a novel dicistrovirus identified from the sweet potato whitefly Bemisia tabaci. Virus Res. 2020, 288, 198112.[CrossRef]17. Kerr, C.H.; Wang, Q.S.; Keatings, K.; Khong, A.; Allan, D.; Yip, C.K.; Foster, L.J.; Jan, E. The 5′ Untranslated Region of aNovel Infectious Molecular Clone of the Dicistrovirus Cricket Paralysis Virus Modulates Infection. J. Virol. 2015, 89, 5919–5934.[CrossRef]18. Wang, X.; Vlok, M.; Flibotte, S.; Jan, E. Resurrection of a Viral Internal Ribosome Entry Site from a 700 Year Old Ancient NorthwestTerritories Cripavirus. Viruses 2021, 13, 493. [CrossRef]19. Jan, E.; Sarnow, P. Factorless Ribosome Assembly on the Internal Ribosome Entry Site of Cricket Paralysis Virus. J. Mol. Biol. 2002,324, 889–902. [CrossRef]20. D’souza, M.H.; Mrozowich, T.; Badmalia, M.D.; Geeraert, M.; Frederickson, A.; Henrickson, A.; Demeler, B.; Wolfinger, M.T.;Patel, T.R. Biophysical characterisation of human LincRNA-p21 sense and antisense Alu inverted repeats. Nucleic Acids Res. 2022,50, 5881–5898. [CrossRef]21. Cowieson, N.P.; Edwards-Gayle, C.J.C.; Inoue, K.; Khunti, N.S.; Doutch, J.; Williams, E.; Daniels, S.; Preece, G.; Krumpa, N.A.;Sutter, J.P.; et al. Beamline B21: High-throughput small-angle X-ray scattering at Diamond Light Source. J. Synchrotron Radiat.2020, 27, 1438–1446. [CrossRef] [PubMed]22. Krahn, N.; Zhang, J.; Melnikov, S.V.; Tharp, J.M.; Villa, A.; Patel, A.; Howard, R.J.; Gabir, H.; Patel, T.R.; Stetefeld, J.; et al. tRNAshape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut. Nucleic Acids Res. 2023, 52, 513–524.[CrossRef] [PubMed]23. Pereira, H.S.; Gemmill, D.L.; Siddiqui, M.Q.; Vasudeva, G.; Patel, T.R. Mapping and characterization of G-quadruplexes inmonkeypox genomes. J. Med. Virol. 2023, 95, e28783. [CrossRef] [PubMed]24. Parisien, M.; Major, F. The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data. Nature 2008, 452, 51–55.[CrossRef] [PubMed]25. Svergun, D.; Barberato, C.; Koch, M.H.J. CRYSOL—A Program to Evaluate X-ray Solution Scattering of Biological Macromoleculesfrom Atomic Coordinates. J. Appl. Crystallogr. 1995, 28, 768–773. [CrossRef]26. Kozin, M.B.; Svergun, D.I. Automated matching of high- and low-resolution structural models. J. Appl. Crystallogr. 2001, 34, 33–41.[CrossRef]27. A Wilkinson, K.; Merino, E.J.; Weeks, K.M. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): QuantitativeRNA structure analysis at single nucleotide resolution. Nat. Protoc. 2006, 1, 1610–1616. [CrossRef] [PubMed]28. Busan, S.; Weeks, K.M. Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2.RNA 2017, 24, 143–148. [CrossRef]29. Reuter, J.S.; Mathews, D.H. RNAstructure: Software for RNA secondary structure prediction and analysis. BMC Bioinform. 2010,11, 129. [CrossRef]30. Brierley, I.; Pennell, S. Structure and Function of the Stimulatory RNAs Involved in Programmed Eukaryotic-1 RibosomalFrameshifting. Cold Spring Harb. Symp. Quant. Biol. 2001, 66, 233–248. [CrossRef]31. Atkins, J.F.; Loughran, G.; Bhatt, P.R.; Firth, A.E.; Baranov, P.V. Ribosomal frameshifting and transcriptional slippage: Fromgenetic steganography and cryptography to adventitious use. Nucleic Acids Res. 2016, 44, 7007–7078. [CrossRef] [PubMed]32. Stetefeld, J.; McKenna, S.A.; Patel, T.R. Dynamic light scattering: A practical guide and applications in biomedical sciences.Biophys. Rev. 2016, 8, 409–427. [CrossRef] [PubMed]33. Dzananovic, E.; Patel, T.R.; Chojnowski, G.; Boniecki, M.J.; Deo, S.; McEleney, K.; Harding, S.E.; Bujnicki, J.M.; McKenna, S.A.Solution conformation of adenovirus virus associated RNA-I and its interaction with PKR. J. Struct. Biol. 2014, 185, 48–57.[CrossRef] [PubMed]34. Wang, Q.S.; Jan, E. Switch from Cap- to Factorless IRES-Dependent 0 and +1 Frame Translation during Cellular Stress andDicistrovirus Infection. PLoS ONE 2014, 9, e103601. [CrossRef] [PubMed]35. Au, H.H.T.; Elspass, V.M.; Jan, E. Functional Insights into the Adjacent Stem-Loop in Honey Bee Dicistroviruses That PromotesInternal Ribosome Entry Site-Mediated Translation and Viral Infection. J. Virol. 2018, 92, 10–128. [CrossRef] [PubMed]36. Wang, Q.S.; Au, H.H.; Jan, E. Methods for studying IRES-mediated translation of positive-strand RNA viruses. Methods 2013, 59,167–179. [CrossRef]37. Wilson, J.E.; Pestova, T.V.; Hellen, C.U.; Sarnow, P. Initiation of Protein Synthesis from the A Site of the Ribosome. Cell 2000, 102,511–520. [CrossRef] [PubMed]38. Jan, E.; Kinzy, T.G.; Sarnow, P. Divergent tRNA-like element supports initiation, elongation, and termination of protein biosynthe-sis. Proc. Natl. Acad. Sci. USA 2003, 100, 15410–15415. [CrossRef]39. Novac, O.; Guenier, A.-S.; Pelletier, J. Inhibitors of protein synthesis identified by a high throughput multiplexed translationscreen. Nucleic Acids Res. 2004, 32, 902–915. [CrossRef]40. Cencic, R.; Pelletier, J. Hippuristanol—A potent steroid inhibitor of eukaryotic initiation factor 4A. Translation 2016, 4, e1137381.[CrossRef]41. Jang, C.J.; Jan, E. Modular domains of the Dicistroviridae intergenic internal ribosome entry site. RNA 2010, 16, 1182–1195.[CrossRef] [PubMed]Viruses 2024, 16, 695 20 of 2042. Komar, A.A.; Hatzoglou, M. Internal Ribosome Entry Sites in Cellular mRNAs: Mystery of Their Existence. J. Biol. Chem. 2005,280, 23425–23428. [CrossRef] [PubMed]43. Mitchell, S.A.; Spriggs, K.A.; Bushell, M.; Evans, J.R.; Stoneley, M.; Le Quesne, J.P.; Spriggs, R.V.; Willis, A.E. Identification of amotif that mediates polypyrimidine tract-binding protein-dependent internal ribosome entry. Genes Dev. 2005, 19, 1556–1571.[CrossRef] [PubMed]44. Byrne, D.N.; Bellows, T.S.J. Whitefly Biology. Annu. Rev. Entomol. 1991, 36, 431–457. [CrossRef]45. Legg, J.P.; Sseruwagi, P.; Boniface, S.; Okao-Okuja, G.; Shirima, R.; Bigirimana, S.; Gashaka, G.; Herrmann, H.-W.; Jeremiah, S.;Obiero, H.; et al. Spatio-temporal patterns of genetic change amongst populations of cassava Bemisia tabaci whiteflies drivingvirus pandemics in East and Central Africa. Virus Res. 2014, 186, 61–75. [CrossRef]46. Robertson, J.L.; Stansly, P.A.; Naranjo, S.E. Bemisia: Bionomics and Management of a Global Pest. J. Econ. Èntomol. 2014, 107, 482.[CrossRef]47. Schutze, I.X.; E Naranjo, S.; Yamamoto, P.T. Impact of Bemisia tabaci MEAM1 (Hemiptera: Aleyrodidae) on Soybean Yield andQuality Under Field Conditions. J. Econ. Èntomol. 2022, 115, 757–766. [CrossRef] [PubMed]48. Nakasu, E.Y.; Hedil, M.; Nagata, T.; Michereff-Filho, M.; Lucena, V.S.; Inoue-Nagata, A.K. Complete genome sequence andphylogenetic analysis of a novel dicistrovirus associated with the whitefly Bemisia tabaci. Virus Res. 2018, 260, 49–52. [CrossRef][PubMed]49. Moscardi, F. Assessment of the application of baculoviruses for control of lepidoptera. Annu. Rev. Èntomol. 1999, 44, 257–289.[CrossRef]50. Ferreira, B.; Melo, F.; Silva, A.; Sanches, M.; Moscardi, F.; Ribeiro, B.; Souza, M. Biological and molecular characterization of twoAnticarsia gemmatalis multiple nucleopolyhedrovirus clones exhibiting contrasting virulence. J. Invertebr. Pathol. 2019, 164, 23–31.[CrossRef]51. Arthurs, S.; Dara, S.K. Microbial biopesticides for invertebrate pests and their markets in the United States. J. Invertebr. Pathol.2019, 165, 13–21. [CrossRef] [PubMed]Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individualauthor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.
Article
eng
Reviewed
Vancouver : University of British Columbia Library
Multidisciplinary Digital Publishing Institute
CC BY 4.0
Faculty
Researcher
virus
frameshifting
RNA
IRES
Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2
Text