Isolation and characterization of a novel bacteriophage WO from Allonemobius socius crickets in Missouri.

Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOAu of Drosophila simulans (94.46% identity) and WOCin2USA1 of the cherry fruit fly, Rhagoletis cingulata (99.33% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40-62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.

Virus Prospecting in Crickets-Discovery and Strain Divergence of a Novel Iflavirus in Wild and Cultivated Acheta domesticus.

Orthopteran insects have high reproductive rates leading to boom-bust population dynamics with high local densities that are ideal for short, episodic disease epidemics. Viruses are particularly well suited for such host population dynamics, due to their supreme ability to adapt to changing transmission criteria. However, very little is known about the viruses of Orthopteran insects. Since Orthopterans are increasingly reared commercially, for animal feed and human consumption, there is a risk that viruses naturally associated with these insects can adapt to commercial rearing conditions, and cause disease. We therefore explored the virome of the house cricket Acheta domesticus, which is both part of the natural Swedish landscape and reared commercially for the pet feed market. Only 1% of the faecal RNA and DNA from wild-caught A. domesticus consisted of viruses. These included both known and novel viruses associated with crickets/insects, their bacterial-fungal microbiome, or their plant food. Relatively abundant among these viral Operational Taxonomic Units (OTUs) was a novel Iflavirus, tentatively named Acheta domesticus Iflavirus (AdIV). Quantitative analyses showed that AdIV was also abundant in frass and insect samples from commercially reared crickets. Interestingly, the wild and commercial AdIV strains had short, extremely divergent variation hotspots throughout the genome, which may indicate specific adaptation to their hosts' distinct rearing environments.

Detection and Characterization of Invertebrate Iridoviruses Found in Reptiles and Prey Insects in Europe over the Past Two Decades.

Invertebrate iridoviruses (IIVs), while mostly described in a wide range of invertebrate hosts, have also been repeatedly detected in diagnostic samples from poikilothermic vertebrates including reptiles and amphibians. Since iridoviruses from invertebrate and vertebrate hosts differ strongly from one another based not only on host range but also on molecular characteristics, a series of molecular studies and bioassays were performed to characterize and compare IIVs from various hosts and evaluate their ability to infect a vertebrate host. Eight IIV isolates from reptilian and orthopteran hosts collected over a period of six years were partially sequenced. Comparison of eight genome portions (total over 14 kbp) showed that these were all very similar to one another and to an earlier described cricket IIV isolate, thus they were given the collective name lizard-cricket IV (Liz-CrIV). One isolate from a chameleon was also subjected to Illumina sequencing and almost the entire genomic sequence was obtained. Comparison of this longer genome sequence showed several differences to the most closely related IIV, Invertebrateiridovirus6 (IIV6), the type species of the genus Iridovirus, including several deletions and possible recombination sites, as well as insertions of genes of non-iridoviral origin. Three isolates from vertebrate and invertebrate hosts were also used for comparative studies on pathogenicity in crickets (Gryllusbimaculatus) at 20 and 30 °C. Finally, the chameleon isolate used for the genome sequencing studies was also used in a transmission study with bearded dragons. The transmission studies showed large variability in virus replication and pathogenicity of the three tested viruses in crickets at the two temperatures. In the infection study with bearded dragons, lizards inoculated with a Liz-CrIV did not become ill, but the virus was detected in numerous tissues by qPCR and was also isolated in cell culture from several tissues. Highest viral loads were measured in the gastro-intestinal organs and in the skin. These studies demonstrate that Liz-CrIV circulates in the pet trade in Europe. This virus is capable of infecting both invertebrates and poikilothermic vertebrates, although its involvement in disease in the latter has not been proven.

Diagnostic protocols for the detection of Acheta domesticus densovirus (AdDV) in cricket frass.

The European house cricket (Acheta domesticus) is a species of interest for the emerging insect-as-food industry. Acheta domesticus densovirus (AdDV) is a member of the Parvoviridae virus family which infects A. domesticus, causing widespread mortality and even extinction of local cricket populations. Despite the well-known detrimental effects of AdDV in commercial rearing of A. domesticus there are no optimized protocols to accurately and non-destructively detect and quantify the virus. This study establishes a new protocol for the detection of AdDV in faecal material from A.domesticus. The protocol includes methodological improvements, such as upgrading from conventional PCR to quantitative real-time PCR and is much more sensitive than previously published protocols. Moreover, this study shows that cricket faeces are a suitable, non-destructive sample substrate to infer reliably if a cricket population is infected with AdDV or not. Early detection of lethal or economic threats, such as disease-causing viruses, is an essential part of commercial cricket management as well as for monitoring the risk of spread to wild cricket populations or to (human) consumers.

Characterization of viral RNA splicing using whole-transcriptome datasets from host species.

RNA alternative splicing (AS) is an important post-transcriptional mechanism enabling single genes to produce multiple proteins. It has been well demonstrated that viruses deploy host AS machinery for viral protein productions. However, knowledge on viral AS is limited to a few disease-causing viruses in model species. Here we report a novel approach to characterizing viral AS using whole transcriptome dataset from host species. Two insect transcriptomes (Acheta domesticus and Planococcus citri) generated in the 1,000 Insect Transcriptome Evolution (1KITE) project were used as a proof of concept using the new pipeline. Two closely related densoviruses (Acheta domesticus densovirus, AdDNV, and Planococcus citri densovirus, PcDNV, Ambidensovirus, Densovirinae, Parvoviridae) were detected and analyzed for AS patterns. The results suggested that although the two viruses shared major AS features, dramatic AS divergences were observed. Detailed analysis of the splicing junctions showed clusters of AS events occurred in two regions of the virus genome, demonstrating that transcriptome analysis could gain valuable insights into viral splicing. When applied to large-scale transcriptomics projects with diverse taxonomic sampling, our new method is expected to rapidly expand our knowledge on RNA splicing mechanisms for a wide range of viruses.

Assessment of a cricket, Acheta domesticus, bioassay for Chequa Iflavirus and bunya-like virus from redclaw crayfish Cherax quadricarinatus.

Chequa iflavirus and a bunya-like virus infect redclaw crayfish (Cherax quadricarinatus) and they may cause mortality reaching 20-40% after about three weeks after a stress event. To complete River's postulates for viruses, virus-free animals are needed. Due to a lack of chequa iflavirus and bunya-like virus-free crayfish (testing shows>85% infection rate) coupled with the facts that iflavirus and bunyaviruses are found in insects and that crickets had been successful alternate hosts for crustacean viruses before, Acheta domesticus was trialled asa bioassay animal. There was no significant difference (P>0.05) in mortality rates between uninfected control crickets and infected crickets. Reverse transcriptase polymerase chain reaction for both viruses failed to find any trace of the RNA viruses in fed or inoculated crickets after 30days. The search for an alternative bioassay host will have to be widened.

The IRES5'UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure.

Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5' untranslated region (5'UTR) IRES5'UTR mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRESIGR is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRESIGR has been extensively studied, in contrast the IRES5'UTR remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES5'UTR promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES5'UTR recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2'-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5'UTR and of the minimal IRES5'UTR. The IRES5'UTR contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.

Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single-molecule fluorescence microscopy.

Protein synthesis is a complex multistep process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single-molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labeling of the ribosomes are much more complicated. Here, we use a noncanonical translation initiation based on internal ribosome entry sites (IRES), and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of cricket paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones, suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility of studying both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.

A viral aphrodisiac in the cricket Gryllus texensis.

We identified the insect iridovirus IIV-6/CrIV as a pathogen of the cricket Gryllus texensis using electron microscopy (EM) and polymerase chain reaction (PCR) analysis. EM showed that the virus attacks the fat body, an organ important for protein production, immune function and lipid storage. During infection the fat body hypertrophied, but egg production withered, leaving the lateral oviducts empty of eggs; the females were effectively sterile. EM of the testis of infected males suggests that the testis was not invaded by the virus, although sperm taken from the spermatophores of infected males showed little or no motility. Nevertheless, males and females continued to mate when infected. In fact, infected males were quicker to court females than uninfected controls. The virus benefits from the continued sexual behaviour of its host; transmission studies show that the virus can be spread through sexual contact. Sickness behaviour, the adaptive reduction of feeding and sexual behaviour that is induced by an activated immune system, was absent in infected crickets. Total haemolymph protein was reduced, as was phenoloxidase activity, suggesting a reduction in immune protein production by the fat body. The evidence suggests that during IIV-6/CrIV infection, the immune signal(s) that induces sickness behaviour is absent. Curtailment of a host's sickness behaviour may be necessary for any pathogen that is spread by host sexual behaviour.

Suppression of Penaeus merguiensis densovirus following oral delivery of live bacteria expressing dsRNA in the house cricket (Acheta domesticus) model.

Penaeus merguiensis densovirus (PmergDNV) is a serious pathogen of the banana prawn, Penaeus merguiensis leading to at least 28% production loss due to reduced growth rates and mortality of juveniles. In the present study, we reduced PmergDNV titres and subsequent mortality by feeding Acheta domesticus (previously determined as an appropriate animal model for P. merguiensis) with dsRNA specific to the capsid protein by mixing it into their food. Feeding A. domesticus with PmergDNV-specific dsRNA in advance of viral challenge increased their longevity, decreased mortality by 84.4% and reduced viral loads 24-fold below the threshold level required for mortality. Mortalities and viral loads were significantly (both P < 0.001) lower in treatments challenged with PmergDNV following exposure to bacterially expressed PmergDNV-dsRNA. This is the first study to demonstrate gene silencing via RNAi against PmergDNV in vivo through oral administration of live bacteria expressing dsRNA in a model system.

The Acheta domesticus densovirus, isolated from the European house cricket, has evolved an expression strategy unique among parvoviruses.

The Acheta domesticus densovirus (AdDNV), isolated from crickets, has been endemic in Europe for at least 35 years. Severe epizootics have also been observed in American commercial rearings since 2009 and 2010. The AdDNV genome was cloned and sequenced for this study. The transcription map showed that splicing occurred in both the nonstructural (NS) and capsid protein (VP) multicistronic RNAs. The splicing pattern of NS mRNA predicted 3 nonstructural proteins (NS1 [576 codons], NS2 [286 codons], and NS3 [213 codons]). The VP gene cassette contained two VP open reading frames (ORFs), of 597 (ORF-A) and 268 (ORF-B) codons. The VP2 sequence was shown by N-terminal Edman degradation and mass spectrometry to correspond with ORF-A. Mass spectrometry, sequencing, and Western blotting of baculovirus-expressed VPs versus native structural proteins demonstrated that the VP1 structural protein was generated by joining ORF-A and -B via splicing (splice II), eliminating the N terminus of VP2. This splice resulted in a nested set of VP1 (816 codons), VP3 (467 codons), and VP4 (429 codons) structural proteins. In contrast, the two splices within ORF-B (Ia and Ib) removed the donor site of intron II and resulted in VP2, VP3, and VP4 expression. ORF-B may also code for several nonstructural proteins, of 268, 233, and 158 codons. The small ORF-B contains the coding sequence for a phospholipase A2 motif found in VP1, which was shown previously to be critical for cellular uptake of the virus. These splicing features are unique among parvoviruses and define a new genus of ambisense densoviruses.

Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics.

The European house cricket, Acheta domesticus L., is highly susceptible to A. domesticus densovirus (AdDNV). Commercial rearings of crickets in Europe are frequently decimated by this pathogen. Mortality was predominant in the last larval stage and young adults. Infected A. domesticus were smaller, less active, did not jump as high, and the adult females seldom lived more than 10-14 days. The most obvious pathological change was the completely empty digestive caecae. Infected tissues included adipose tissue, midgut, epidermis, and Malpighian tubules. Sudden AdDNV epizootics have decimated commercial mass rearings in widely separated parts of North America since the autumn of 2009. Facilities that are producing disease-free crickets have avoided the importation of crickets and other non-cricket species (or nonliving material). Five isolates from different areas in North America contained identical sequences as did AdDNV present in non-cricket species collected from these facilities. The North American AdDNVs differed slightly from sequences of European AdDNV isolates obtained in 1977, 2004, 2006, 2007 and 2009 and an American isolate from 1988. The substitution rate of the 1977 AdDNV 5kb genome was about two nucleotides per year, about half of the substitutions being synonymous. The American and European AdDNV strains are estimated to have diverged in 2006. The lepidopterans Spodoptera littoralis and Galleria mellonella could not be infected with AdDNV. The Jamaican cricket, Gryllus assimilis, and the European field cricket, Gryllus bimaculatus, were also found to be resistant to AdDNV.

Lateral phage transfer in obligate intracellular bacteria (wolbachia): verification from natural populations.

Lateral transfer of mobile DNA is a hallmark of bacteria with a free-living replicative stage; however, its significance in obligate intracellular bacteria and other heritable endosymbionts remains controversial. Comparative sequence analyses from laboratory stocks infected with Wolbachia pipientis provide some of the most compelling evidence that bacteriophage WO-B transfers laterally between infections of the same insect host. Lateral transfer between coinfections, however, has been evaluated neither in natural populations nor between closely related Wolbachia strains. Here, we analyze bacterial and phage genes from two pairs of natural sympatric field isolates, of Gryllus pennsylvanicus field crickets and of Neochlamisus bebbianae leaf beetles, to demonstrate WO-B transfers between supergroup B Wolbachia. N. bebbianae revealed the highest number of phage haplotypes yet recorded, hinting that lab lines could underestimate phage haplotype variation and lateral transfer. Finally, using the approximate age of insect host species as the maximum available time for phage transfer between host-associated bacteria, we very conservatively estimate phage WO-B transfer to occur at least once every 0-5.4 My within a host species. Increasing discoveries of mobile elements, intragenic recombination, and bacterial coinfections in host-switching obligate intracellular bacteria specify that mobile element transfer is common in these species.

RNA interference reduces PmergDNV expression and replication in an in vivo cricket model.

RNA interference (RNAi) is an attractive anti-viral preventative because it allows interference with the expression of a viral gene in a highly sequence-specific manner. Thus, essential viral genes can be targeted by design, with little or no risk of undesired off-target effects. To investigate if stealth RNAis can mediate a sequence-specific anti-viral effect against PmergDNV, adult Acheta domesticus were injected with 5 microg of stealth RNAi or control stealth RNAi, targeting the capsid protein. Twenty-four hours post-injection, crickets were challenged with PmergDNV. Mortality was monitored for 14 days and real-time reverse transcriptase PCR was used to enumerate the number of copies of PmergDNV in cricket tissues. Whilst statistically not significant, trends in mortality suggest crickets injected with RNAi targeting PmergDNV had the lowest mortality rate (11.5%) compared to crickets injected with control dsRNAi (33%) and PmergDNV alone (25%). Crickets challenged with specific dsRNAi had statistically significantly reduced PmergDNV titres by one log (3.58 x 10(2)) compared to crickets challenged with PmergDNV alone (3.42 x 10(3)). Interestingly, even the control dsRNAi was capable of reducing PmergDNV titres by one log (3.95 x 10(2)), but did not produce an inhibitory effect quite as strong as the targeted dsRNAi for the capsid protein of PmergDNV. The introduction of dsRNAi corresponding to the capsid protein of PmergDNV, was effective in reducing viral replication in Acheta domesticus. Administration of PmergDNV-specific dsRNAis may provide an efficient counter measure against PmergDNV in prawns.

The use of insects as a bioassay for Penaeus merguiensis densovirus (PmergDNV).

The lack of available cell lines has hampered the study of viral diseases in crustaceans. This is particularly important for aquaculture which has been plagued by viral diseases since its rapid expansion to meet with the growing demand for seafood products. This study was designed to find an alternative bioassay to cell lines by investigating the use of insects as potential animal models for Penaeus merguiensis densovirus (PmergDNV). Acheta domesticus (house cricket) and Tenebrio molitor (mealworms) were challenged with approximately 1x10(6) virions of PmergDNV by inoculation. PmergDNV was detected in 20% of Tenebrio molitor and 86.6% of Acheta domesticus challenged with PmergDNV. During a subsequent time course experiment, there was a non significant increase in PmergDNV titres (10(4-5) virions), reaching a maximum peak at day 5 (10(6) copies). A threshold of PmergDNV DNA level equal to or greater than 10(3) virions was necessary for mortality in Acheta domesticus. As the inoculum increased from 10(3) DNA copies to 10(4), 10(5), 10(6), mortality increased from 20% to 60%, 80% and 100%, respectively. This is the first evidence that insects may be directly used to study viruses from crustaceans and concludes Acheta domesticus may be used as a potential model to study Penaeus merguiensis densovirus.

Experimental infection of crickets (Gryllus bimaculatus) with an invertebrate iridovirus isolated from a high-casqued chameleon (Chamaeleo hoehnelii).

Invertebrate iridoviruses (IIV) have been a regular problem for insect breeders. They have also recently been isolated from various lizard species. An iridovirus isolated from several tissues of a high-casqued chameleon (Chamaeleo hoehnelii) was identified as an IIV on the basis of electron microscopy (EM), sequencing of a portion of the major capsid protein (MCP) gene, and restriction endonuclease analysis of viral DNA. The pathogenicity of this isolate for crickets of the species Gryllus bimaculatus was tested by using 3 experimental infection studies. The mortality rates in the infected crickets ranged between 20% and 35%. The fat bodies of the crickets were examined on cell culture, with a nested PCR targeting the MCP gene, histologically, with in situ hybridization and by EM. Nested PCR was the most sensitive method for detecting IIV in the fat-body samples. Virus was re-isolated from several fat-body samples. In some fat bodies of infected crickets, massive arrays of viruses could be detected by EM. These findings support the hypothesis that IIV from insects are able to infect reptiles.

Ectropis obliqua picorna-like virus IRES-driven internal initiation of translation in cell systems derived from different origins.

Ectropis obliqua picorna-like virus (EoPV) is an insect RNA virus that causes a lethal granulosis infection of larvae of the tea looper (Ectropis obliqua). An internal ribosome entry site (IRES) mediates translation initiation of EoPV RNA. Here, bicistronic constructs were used to examine the 5' untranslated region (UTR) of EoPV for IRES activity. The capacities of the EoPV 5' UTR IRES and another insect virus IRES, the cricket paralysis virus intergenic region IRES, to mediate internal translation initiation in a variety of translation systems were also compared. The results demonstrated that the EoPV IRES functioned efficiently not only in mammalian cell-derived systems, but also in an insect cell-derived translation system. However, it functioned inefficiently in a plant cell-derived translation system. This study reveals the host preferences of the EoPV IRES and important differences in IRES function between the EoPV IRES and other characterized picorna-like insect viral IRESs.

The genome of Gryllus bimaculatus nudivirus indicates an ancient diversification of baculovirus-related nonoccluded nudiviruses of insects.

The Gryllus bimaculatus nudivirus (GbNV) infects nymphs and adults of the cricket Gryllus bimaculatus (Orthoptera: Gryllidae). GbNV and other nudiviruses such as Heliothis zea nudivirus 1 (HzNV-1) and Oryctes rhinoceros nudivirus (OrNV) were previously called "nonoccluded baculoviruses" as they share some similar structural, genomic, and replication aspects with members of the family Baculoviridae. Their relationships to each other and to baculoviruses are elucidated by the sequence of the complete genome of GbNV, which is 96,944 bp, has an AT content of 72%, and potentially contains 98 predicted protein-coding open reading frames (ORFs). Forty-one ORFs of GbNV share sequence similarities with ORFs found in OrNV, HzNV-1, baculoviruses, and bacteria. Most notably, 15 GbNV ORFs are homologous to the baculovirus core genes, which are associated with transcription (lef-8, lef-9, lef-4, vlf-1, and lef-5), replication (dnapol), structural proteins (p74, pif-1, pif-2, pif-3, vp91, and odv-e56), and proteins of unknown function (38K, ac81, and 19kda). Homologues to these baculovirus core genes have been predicted in HzNV-1 as well. Six GbNV ORFs are homologous to nonconserved baculovirus genes dnaligase, helicase 2, rr1, rr2, iap-3, and desmoplakin. However, the remaining 57 ORFs revealed no homology or poor similarities to the current gene databases. No homologous repeat (hr) sequences but fourteen short direct repeat (dr) regions were detected in the GbNV genome. Gene content and sequence similarity suggest that the nudiviruses GbNV, HzNV-1, and OrNV form a monophyletic group of nonoccluded double-stranded DNA viruses, which separated from the baculovirus lineage before this radiated into dipteran-, hymenopteran-, and lepidopteran-specific clades of occluded nucleopolyhedroviruses and granuloviruses. The accumulated information on the GbNV genome suggests that nudiviruses form a highly diverse and phylogenetically ancient sister group of the baculoviruses, which have evolved in a variety of highly divergent host orders.

Structure of the ribosome-bound cricket paralysis virus IRES RNA.

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.