Cap-independent Translational Enhancer Research Articles

Mechanistic differences in eukaryotic initiation factor requirements for eIF4GI-driven cap-independent translation of structured mRNAs

Protein translation is globally downregulated under stress conditions. Many proteins that are synthesized under stress conditions use a cap-independent translation initiation pathway. A subset of cellular mRNAs that encode for these proteins contain stable secondary structures within their 5’ untranslated region (5’UTR), and initiate cap-independent translation using elements called Cap-Independent Translation Enhancers (CITEs) or Internal Ribosome Entry Sites (IRESs) within their 5’UTRs. The interaction among initiation factors such as eIF4E, eIF4A and eIF4GI, especially in regulating the eIF4F complex during non-canonical translation initiation of different 5’UTR mRNAs, is poorly understood. Here, equilibrium-binding assays, circular dichroism studies and in vitro translation assays were employed to elucidate the recruitment of these initiation factors to the highly structured 5’UTRs of fibroblast-growth factor 9 (FGF-9) and hypoxia inducible factor 1 subunit alpha (HIF-1α) encoding mRNAs. We showed that eIF4A and eIF4E enhanced eIF4GI’s binding affinity to the uncapped 5’UTR of HIF-1α mRNA, inducing conformational changes in the protein/RNA complex. In contrast, these factors have no effect on the binding of eIF4GI to the 5’UTR of FGF-9 mRNA. Recently, Izidoro, M. S. et al. reported that the interaction of 42nt unstructured RNA to human eIF4F complex is dominated by eIF4E and ATP-bound state of eIF4A. Here we show that structured 5’UTR mRNA binding mitigates this requirement. Based on these observations, we describe two possible cap-independent translation mechanisms for FGF-9 and HIF-1α encoding mRNAs employed by cells to mitigate cellular stress conditions.

Modeling the structure and DAP5-binding site of the FGF-9 5'-UTR RNA utilized in cap-independent translation.

Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5'-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.

Structure of saguaro cactus virus 3′ translational enhancer mimics 5′ cap for eIF4E binding

The genomes of several plant viruses contain RNA structures at their 3' ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5' cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5' cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3' CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5' cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE-eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5' cap for eIF4E binding. Such 5' cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.

Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messengerRNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

Conserved Structure Associated with Different 3'CITEs Is Important for Translation of Umbraviruses.

The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3' structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3' cap-independent translation enhancers (3'CITEs), as umbraviruses can have different 3'CITEs in the central region of their lengthy 3'UTRs, and most also have a particular 3'CITE (the T-shaped structure or 3'TSS) near their 3' ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3'CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in Nicotiana benthamiana. Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3'CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3'CITEs that differentially affect the structure and translation of different umbraviruses.

Novel 3' Proximal Replication Elements in Umbravirus Genomes.

The 3' untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3'UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3' cap-independent translation enhancers (3'CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3'TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3' terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed "Trio", that are exclusively found in a subset of umbraviruses and are located just upstream of the 3'TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3' terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3'CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.

Significance of the Translation Initiation Machinery Recruiting Structures in the Blackcurrant Reversion Virus RNA 1

Unlike most eukaryotic translation which is initiated by the 5′‐m7GpppN cap (5′cap), the two genomic RNAs (RNA 1 and 2) from the Blackcurrant Reversion virus (BRV) do not have a 5′ cap but rather has special region in 3′ untranslated region (UTR) called a cap‐independent translation enhancer (CITE). CITE RNA structures bind translation initiation machinery and delivers that machinery to the 5′ UTR, typically via an RNA kissing‐loop interaction. Our research focuses on the specific RNA structures called translation initiation machinery recruiting structures (TIMRS) in the BRV RNA 1 3′ CITE and 5′ UTR, and their significance in facilitating translation by delivering translation machinery to the 5′ end of the RNA. Using site‐directed mutagenesis to alter the TIMRS and a luciferase reporter to measure protein production of the mutants, we are analyzing the relationship between structure and function. Thus far we have found altering the TIMRS in the 3′ CITE structure does show a difference in protein production compared to the wild type, which indicates the structures' significance and their contribution in translation efficiency. We believe the TIMRS is responsible for recruiting translation machinery or possibly the ability to “kiss” to 5′ UTR stem loop. This study can aid in understanding not only plant viruses but other animal or human RNA viruses that utilize 3′ or 5′ structures to regulate cap‐independent translation.

Analyzing the 5′‐3′ Kissing‐Loop Interaction Sequence for Cap‐Independent Translation Initiation of Blackcurrant Reversion Virus

Translation regulation commonly involves the 5′‐m7GpppN cap which binds the initiation factor 4E bound to recruit the other initiation factors and the small ribosomal subunit. Yet some viruses, such as the Blackcurrant Reversion virus (BRV), have no 5′‐cap but are still able to translate using alternative mechanisms such as 3′ cap independent translation enhancers (3′ CITE). The 3′ CITEs in BRV’s bipartite genome are predicted to work like 4E by recruiting initiation factors and bringing them to the 5′ end for initiation of translation though a 5′‐3′ RNA kissing interaction, called the kissing‐loop interaction sequence (KIS). Our goal is to verify whether or not the KIS between the 5′ UTR and 3′ CITE exists to promote translation initiation. Using site‐directed mutagenesis and in vitro transcription, we created a series of KIS mutations (including compensatory mutations). Our preliminary in vitro luciferase translation experiments indicate the putative KIS between the 5′ UTR and 3′ CITE may not be important for translation initiation, however, we are currently working to verify these results and identify alternative KISs between the 5′ and 3′ ends. Although many CITEs use 5′‐3′ RNA kissing interactions to deliver translation machinery to the 5′ end of the genome, it is not required for CITE function. The BRV CITE may function through a protein bridge or some other unknown mechanism.

Structural characterization of a new subclass of panicum mosaic virus-like 3' cap-independent translation enhancer.

Canonical eukaryotic mRNA translation requires 5′cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5′cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3′UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson–Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson–Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions.

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs.

Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5′ end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5′ end, even if located in 3′ untranslated regions (3′ UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular “translation factories”, which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles’ heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.

Efficient translation of Eggplant mottled dwarf nucleorhabdovirus N and X genes requires both 5\u2032 and 3\u2032 UTRs

BackgroundCircularization of RNA mediated by association of translation factors and RNA elements in 5′ and 3′ UTRs is a common feature for translation control in eukaryotes. There is no information about translation in plant rhabdoviruses and little information is known in animal rhabdoviruses.MethodsThe role of 5′ and 3′ UTRs in two genes of EMDV in translation were studied using luciferase constructs and RNA structures of these sequences were analyzed by SHAPE and Inline probing.ResultsWe have found that efficient translation of N and X mRNAs of nucleorhabdovirus Eggplant mottled dwarf virus (EMDV) requires elements present in both 5′ and 3′ UTRs. Luciferase reporter constructs containing precise 5′ and 3′ UTRs of the N and X genes had substantially higher translational activity compared with constructs containing only the 5′ or 3′ UTR. The 3′UTR of carmovirus Turnip crinkle virus, which contains a well-characterized cap-independent translation enhancer, was unable to complement the lack of EMDV 3′ UTR. Addition of cap analog to luciferase constructs containing the UTRs of the N gene did not restore translation, and translation of the reporter construct in the absence of the 5′ cap was higher than the capped construct. No RNA-RNA interactions between 5′ and 3′ UTRs were detected by EMSA or in-line cleavage structural assays. Deletion of 11 nucleotides from the 3′ terminus negated the synergistic activity of the 3′UTR.ConclusionsThe results with RNA-RNA interaction suggesting that translational synergy between the UTRs may utilize alternative means. Mutation analysis in 3′UTR suggesting that the polyadenylation signal sequence contained in this location may play a critical role in translation.

Articles of Significant Interest in This Issue

Opium poppy mosaic virus (OPMV) is a single-stranded plus-sense RNA virus that does not encode a capsid protein and requires a helper virus. Ilyas et al. (e02109-20) found that OPMV generates two similarly sized subgenomic RNAs (sgRNAs), one of which is synthesized by the replicase and is the template for translation of viral movement proteins. The other is generated by an exoribonuclease-resistant sequence, a mechanism that normally produces small, noncoding sgRNAs. However, this sgRNA codes for a 30-kDa protein with a functional role in OPMV accumulation. The authors also determined that OPMV uses a Barley yellow dwarf virus-like translation element (BTE) as one of its 3′ cap-independent translation enhancers. While BTEs were thought to have little in common beyond a 17-nucleotide conserved sequence, analysis of the 14 known BTE structures showed that BTEs contain additional, strongly conserved sequences and structural features that include residues known to interact with translation initiation factor eIF4G.

Structural Analysis and Whole Genome Mapping of a New Type of Plant Virus Subviral RNA: Umbravirus-Like Associated RNAs.

We report the biological and structural characterization of umbravirus-like associated RNAs (ulaRNAs), a new category of coat-protein dependent subviral RNA replicons that infect plants. These RNAs encode an RNA-dependent RNA polymerase (RdRp) following a −1 ribosomal frameshift event, are 2.7–4.6 kb in length, and are related to umbraviruses, unlike similar RNA replicons that are related to tombusviruses. Three classes of ulaRNAs are proposed, with citrus yellow vein associated virus (CYVaV) placed in Class 2. With the exception of CYVaV, Class 2 and Class 3 ulaRNAs encode an additional open reading frame (ORF) with movement protein-like motifs made possible by additional sequences just past the RdRp termination codon. The full-length secondary structure of CYVaV was determined using Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing and phylogenic comparisons, which was used as a template for determining the putative structures of the other Class 2 ulaRNAs, revealing a number of distinctive structural features. The ribosome recoding sites of nearly all ulaRNAs, which differ significantly from those of umbraviruses, may exist in two conformations and are highly efficient. The 3′ regions of Class 2 and Class 3 ulaRNAs have structural elements similar to those of nearly all umbraviruses, and all Class 2 ulaRNAs have a unique, conserved 3′ cap-independent translation enhancer. CYVaV replicates independently in protoplasts, demonstrating that the reported sequence is full-length. Additionally, CYVaV contains a sequence in its 3′ UTR that confers protection to nonsense mediated decay (NMD), thus likely obviating the need for umbravirus ORF3, a known suppressor of NMD. This initial characterization lays down a road map for future investigations into these novel virus-like RNAs.

A translation enhancer element from black beetle virus engages yeast eIF4G1 to drive cap-independent translation initiation

Cap-independent translation initiation plays crucial roles in fine-tuning gene expression under global translation shutdown conditions. Translation of uncapped or de-capped transcripts can be stimulated by Cap-independent translation enhancer (CITE) elements, but the mechanisms of CITE-mediated translation initiation remain understudied. Here, we characterized a short 5ʹ-UTR RNA sequence from black beetle virus, BBV-seq. Mutational analysis indicates that the entire BBV-seq is required for efficient translation initiation, but this sequence does not operate as an IRES-type module. In yeast cell-free translation extracts, BBV-seq promoted efficient initiation on cap-free mRNA using a scanning mechanism. Moreover, BBV-seq can increase translation efficiency resulting from conventional cap-dependent translation initiation. Using genetic approaches, we found that BBV-seq exploits RNA-binding properties of eIF4G1 to promote initiation. Thus, BBV-seq constitutes a previously uncharacterized short, linear CITE that influences eIF4G1 to initiate 5′ end-dependent, cap-independent translation. These findings bring new insights into CITE-mediated translational control of gene expression.

Short and Sweet: Viral 5`-UTR as a Canonical and Non-Canonical Translation Initiation Switch.

The replication of viruses requires host cell functions, specifically for protein synthesis, as viruses lack their own translational machinery. Failure to translate viral mRNAs and generate viral proteins would affect the propagation and evolution of a virus. Thus, independently of their size, complexity, and genomes, viruses evolved sophisticated molecular mechanisms to hijack the translational apparatus of a host in order to recruit ribosomes for efficient protein production. One of the prevalent mechanisms of translation regulation utilized by viruses is non-canonical translation initiation. It is often governed by the 5’-untranslated regions (5’-UTRs) present upstream of a protein-coding sequence in viral mRNAs, such as internal ribosome entry sites (IRESs) and capindependent translation enhancers (CITEs). Viruses can also utilize canonical translation initiation factors of a host in non-canonical ways. Understanding strategies and mechanisms used by viruses to generate proteins is an important task, as it might help develop new therapeutic interventions. We previously have demonstrated that mRNA from the genome of the black beetle virus (BBV) of the Nodaviridae family contains short and unstructured 5’-UTR, which governs translation initiation as a CITE and as a canonical translational enhancer. In this Commentary, we summarize cap-dependent and cap-independent translation initiation mechanisms and further elaborate on the unique ability of the BBV mRNA 5’-UTR to switch between these two modes of translation initiation in the context of the viral life cycle. Medical implications in treating the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection by targeting viral 5’-UTRs are also discussed.

Investigation of Novel RNA Elements in the 3'UTR of Tobacco Necrosis Virus-D.

RNA elements in the untranslated regions of plus-strand RNA viruses can control a variety of viral processes including translation, replication, packaging, and subgenomic mRNA production. The 3′ untranslated region (3′UTR) of Tobacco necrosis virus strain D (TNV-D; genus Betanecrovirus, family Tombusviridae) contains several well studied regulatory RNA elements. Here, we explore a previously unexamined region of the viral 3′UTR, the sequence located upstream of the 3′-cap independent translation enhancer (3′CITE). Our results indicate that (i) a long-range RNA–RNA interaction between an internal RNA element and the 3′UTR facilitates translational readthrough, and may also promote viral RNA synthesis; (ii) a conserved RNA hairpin, SLX, is required for efficient genome accumulation; and (iii) an adenine-rich region upstream of the 3′CITE is dispensable, but can modulate genome accumulation. These findings identified novel regulatory RNA elements in the 3′UTR of the TNV-D genome that are important for virus survival.

Eukaryotic Translation Initiation Factors Shape RNA Viruses Resistance in Plants

Viruses are representative of a global threat to agricultural production. Genetic resistance is the preferred strategy for the control of viral infection and against loss of crop yield. Viral protein synthesis requires host cellular factors for translating their viral RNAs, and for regulating their replication and cell to cell systemic movement. Therefore, the viruses are dependent on cellular translation factors. Mutations in the gene encoding eIF4E and eIF4G or their isoforms, eIFiso4E, eIFiso4G and eIF2Bβ have been mapped as a source of plant potyvirus while other genus of plant virus recessive resistance genes in many species are originated from these loci. Some of other plant translation factors, such as eIF3, eIF4A-like helicases, eEF1A and eEF1B, which are required in interacting with viral RNAs and regulating various aspects of the infection cycle, have also been identified. Here, we summarized the mechanisms utilized by RNA viruses of eukaryotic plants and the essential roles of eIFs in virus infection. Moreover, we discussed the potential of eIFs as a target gene in the development of genetic resistance to viruses for crop improvement. This review highlighted newly revealed examples of abnormal translational strategies and provided insights into natural host resistance mechanisms that have been linked to 3′ cap-independent translational enhancer activity.

Structural Importance of Genomic RNA1 3′ CITE in Blackcurrant Reversion Nepovirus Protein Synthesis

Canonical translation initiation and regulation involve end‐to‐end interactions between initiation factors bound to a 7‐methyl guanosine triphosphate cap at the 5′ end and at a poly(A) tail, at the 3′ end of the messenger RNA. This characteristic is also prevalent in many RNA viruses such as the Blackcurrant Reversion Nepovirus (BRV), which has two bipartite genomic RNAs (RNA1 and RNA2) that contain the 3′ poly(A) tail but lack the 5′ cap. The mechanism implemented by BRV during translation initiation is not clearly defined; however, in other cap‐less, positive‐sense RNA plant viruses, structured RNA in the 3′ UTR binds translation initiation factors and delivers the machinery to the 5′ end via proposed RNA‐RNA kissing‐loop interactions. This non‐canonical (cap‐independent) mechanism is directly related to the primary, secondary and higher‐order RNA structure called a cap‐independent translation enhancer (CITE) in the 3′ UTR. Considering the end‐to‐end communication and recruitment of translation factors is dependent upon RNA structure, our goal is to determine the secondary and putative tertiary structure of the 3′ CITE found in the genomic RNA1 in BRV, and correlate the structure to translation initiation function. With this in mind, we identified a set of functionally‐deficient mutants using a luciferase reporter translation assay. We then probed our purified wild‐type and mutant RNAs with a series of covalent nucleobase and sugar backbone modifiers to identify the secondary (and potential tertiary) structure of each RNA. Linking the location of specific modifications to the efficiency of the translation process, our findings show that mutation of a conserved uracil within a highly stable G+C‐rich stem‐loop decreases translation by approximately 40%, but does not significantly alter the structure of the CITE RNA. However, mutating half of the stem to its Watson‐Crick complement significantly alters the RNA1 CITE structure and decreases translation by approximately 80%. From these results, we propose a model whereby the structure and stability of the stem is required to present the loop sequence (including the conserved U) for translation factor recruitment to the viral RNA. Additionally, this data indicates the locations and RNA structures within the CITE directly involved in translation machinery recruitment, allowing for a better understanding and comparison of the tendencies displayed by BRV as well as other positive‐sense RNA plant viruses.Support or Funding InformationColorado‐Wyoming Alliance for Minority Participation in STEM Research Grant MSU Denver College of Letters, Arts and Sciences Dean's Seed Grant MSU Denver Undergraduate Research Conference Student GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structure‐Function Analysis of the 3′ CITE from the Blackcurrant Reversion Nepovirus Genomic RNA 2 (BRV2)

Canonical eukaryotic translation initiation is enhanced by interactions between the 5′ 7‐methylguanosine cap (m7G) and the 3′ poly(A) tail, which exist on opposite ends of the mRNA molecule in the untranslated regions (UTRs). Viruses, however, hijack translation components using non‐canonical methods; typically differing in the structure of their 5′ and/or 3′ UTRs. Blackcurrant reversion nepovirus (BRV), a virus that infects the blackcurrant plant, has a genome consisting of two positive‐sense RNAs (RNA1 and RNA2). Neither RNA has a 5′ m7G cap, yet they both have a poly(A) tail. Structures within the 3′ UTRs of RNA1 and RNA2 called cap‐independent translation enhancers (3′ CITEs), are hypothesized to interact with a region of their 5′ UTRs. These distinct structural interactions facilitate the recruitment of essential initiation factors needed for translation, but this exact mechanism is not well understood. It is thought that the 5′ and 3′ UTRs possess conserved and complementary sequences which facilitate interaction. This study seeks to elucidate the specific secondary structure of the RNA2 3′ CITE and to correlate such to its role in translation initiation. Using in silico modeling of folded RNA2 and in vitro RNA structural probing, we determined a preliminary secondary structure for the 3′ CITE. We also identified structural changes when the putative 5′ ‐ 3′ UTR complementarity is disrupted. Using a luciferase reporter gene flanked by the RNA2 3′ CITE and wild‐type BRV2 5′ UTR, we show how the putative 5′ ‐ 3′ UTR complementarity is required for the quantity of and the rate at which luciferase protein is made. We conclude that the 3′ CITE is important for promoting translation initiation and the putative 5′ ‐ 3′ UTR complementarity between the ends of the viral RNA is necessary but not sufficient for protein production. The structure‐function relationship of the BRV RNA2 3′ CITE will lead to a clearer understanding of how BRV can be controlled to prevent Blackcurrant Reversion Disease (BRD) and shed light on vital processes that govern this unique class of nepoviruses.Support or Funding InformationColorado‐Wyoming Alliance for Minority Participation in STEMMSU Denver College of Letters, Arts and Sciences Dean's Seed GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Unusual dicistronic expression from closely spaced initiation codons in an umbravirus subgenomic RNA.

Translation commencing at closely spaced initiation codons is common in RNA viruses with limited genome space. In the subgenomic RNA (sgRNA) of Pea enation mosaic virus 2, two closely spaced, out-of-frame start codons direct synthesis of movement/stability proteins p26 and p27. Efficient translation from AUG26/AUG27 is dependent on three 3′-proximal cap-independent translation enhancers (3′CITEs), whereas translation of the genomic (gRNA) requires only two. Contrary to strictly scanning-dependent initiation at the gRNA, sequence context of AUG26/AUG27 does not conform with Kozak requirements and insertion of efficient upstream AUGs had pronounced effects for AUG26 but only moderate effects for AUG27. Insertion of a hairpin within an extended 5′ UTR did not significantly impact translation from AUG26/AUG27. Furthermore, AUG27 repressed translation from upstream AUG26 and this effect was mitigated when inter-codon spacing was reduced. Addition of a stable hairpin to the very 5′ end of the sgRNA severely restricted translation, testifying that this 3′CITE-driven initiation is 5′ end-dependent. Similar to gRNA, sgRNA reporter transcripts were nearly exclusively associated with light polysomes and 3′CITE-promoted long-distance interaction connecting the sgRNA ends affected the number of templates translated and not the initiation rate. We propose a non-canonical, 3′CITE-driven mechanism for efficient dicistronic expression from umbravirus sgRNAs.