Cap-independent Translational Enhancer Research Articles

A Dual Interaction Between the 5'- and 3'-Ends of the Melon Necrotic Spot Virus (MNSV) RNA Genome Is Required for Efficient Cap-Independent Translation.

In eukaryotes, the formation of a 5′-cap and 3′-poly(A) dependent protein–protein bridge is required for translation of its mRNAs. In contrast, several plant virus RNA genomes lack both of these mRNA features, but instead have a 3′-CITE (for cap-independent translation enhancer), a RNA element present in their 3′-untranslated region that recruits translation initiation factors and is able to control its cap-independent translation. For several 3′-CITEs, direct RNA-RNA long-distance interactions based on sequence complementarity between the 5′- and 3′-ends are required for efficient translation, as they bring the translation initiation factors bound to the 3′-CITE to the 5′-end. For the carmovirus melon necrotic spot virus (MNSV), a 3′-CITE has been identified, and the presence of its 5′-end in cis has been shown to be required for its activity. Here, we analyze the secondary structure of the 5′-end of the MNSV RNA genome and identify two highly conserved nucleotide sequence stretches that are complementary to the apical loop of its 3′-CITE. In in vivo cap-independent translation assays with mutant constructs, by disrupting and restoring sequence complementarity, we show that the interaction between the 3′-CITE and at least one complementary sequence in the 5′-end is essential for virus RNA translation, although efficient virus translation and multiplication requires both connections. The complementary sequence stretches are invariant in all MNSV isolates, suggesting that the dual 5′–3′ RNA:RNA interactions are required for optimal MNSV cap-independent translation and multiplication.

Importance of Unique Secondary Structures in Genomic RNA1 3′ CITE in Blackcurrant Reversion Nepovirus Translation

Translation initiation and regulation commonly involve end‐to‐end interactions between proteins (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. End‐to‐end communication is also prevalent in many RNA viruses, such as the Blackcurrant Reversion Nepovirus (BRV), which has two bipartite genomic RNAs (RNA1 and RNA2), both of which contain the 3′ poly(A) tail but lack the 5′ cap. The mechanism implemented by BRV during translation initiation is not clearly defined. In other cap‐less, positive‐sense RNA plant viruses, structured RNA in the 5′ and 3′ UTRs bind via proposed RNA‐RNA kissing‐loop interactions indicating that the recruitment of protein synthesis machinery is directly related to their primary, secondary and higher‐order structure. One form of non‐canonical (cap‐independent) translation involves structured RNA called the cap‐independent translation enhancers (CITEs). These components are proposed to bind to initiation factors as a 5′cap‐replacement and play an important role in the efficient translation of uncapped RNA genomes. Considering the end‐to‐end communication and recruitment of translation factors is dependent upon RNA structure, our main goal focuses on determining the secondary and possible tertiary structures of the 3′ CITE in the genomic RNAs found in the BRV, specifically RNA1. With this in mind, we have probed our purified RNA with a series of covalent nucleobase, sugar and backbone modifiers in addition to testing the function of a series of structural mutants via reporter assays. We found that the RNA1 CITE structure plays an important role in the process of viral protein synthesis by either facilitating end‐to‐end interaction with the 5′ UTR and/or directly recruiting important translation protein machinery. These findings shed light on structure‐based mechanisms used for end‐to‐end communication in non‐canonical mRNAs.Support or Funding InformationMSU Denver Colorado‐Wyoming Alliance for Minority Participation Undergraduate Research Grant, MSU Denver Dean's Grant, MSU Denver Provost GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural and Functional Diversity of Plant Virus 3'-Cap-Independent Translation Enhancers (3'-CITEs).

Most of the positive-strand RNA plant viruses lack the 5′-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5′- or 3′-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3′-end of viruses belonging to the family Tombusviridae and the genus Luteovirus. Seven classes of 3′-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3′-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5′-end by different mechanisms, often long-distance RNA–RNA interactions. As previously proposed and recently found in one case in nature, 3′-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3′-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3′-CITEs belonging to different classes.

Concerted action of two 3' cap-independent translation enhancers increases the competitive strength of translated viral genomes.

Several families of plant viruses evolved cap-independent translation enhancers (3′CITE) in the 3′ untranslated regions of their genomic (g)RNAs to compete with ongoing cap-dependent translation of cellular mRNAs. Umbravirus Pea enation mosaic virus (PEMV)2 is the only example where three 3′CITEs enhance translation: the eIF4E-binding Panicum mosaic virus-like translational enhancer (PTE) and ribosome-binding 3′ T-shaped structure (TSS) have been found in viruses of different genera, while the ribosome-binding kl-TSS that provides a long-distance interaction with the 5′ end is unique. We report that the PTE is the key translation promoting element, but inhibits translation in cis and in trans in the absence of the kl-TSS by sequestering initiation factor eIF4G. PEMV2 strongly outcompeted a cellular mRNA mimic for translation, indicating that the combination of kl-TSS and PTE is highly efficient. Transferring the 3′–5′ interaction from the kl-TSS to the PTE (to fulfill its functionality as found in other viruses) supported translationin vitro, but gRNA did not accumulate to detectable levels in protoplasts in the absence of the kl-TSS. It was shown that the PTE in conjunction with the kl-TSS did not markedly affect the translation initiation rate but rather increased the number of gRNAs available for translation. A model is proposed to explain how 3′CITE-based regulation of ribosome recruitment enhances virus fitness.

Differential use of 3’CITEs by the subgenomic RNA of Pea enation mosaic virus 2

The genomic RNA (gRNA) of Pea enation mosaic virus 2 (PEMV2) is the template for p33 and −1 frameshift product p94. The PEMV2 subgenomic RNA (sgRNA) encodes two overlapping ORFs, p26 and p27, which are required for movement and stability of the gRNA. Efficient translation of p33 requires two of three 3’ proximal cap-independent translation enhancers (3’CITEs): the kl-TSS, which binds ribosomes and engages in a long-distance interaction with the 5’end; and the adjacent eIF4E-binding PTE. Unlike the gRNA, all three 3’CITEs were required for efficient translation of the sgRNA, which included the ribosome-binding 3’TSS. A hairpin in the 5’ proximal coding region of p26/p27 supported translation by the 3’CITEs by engaging in a long-distance RNA:RNA interaction with the kl-TSS. These results strongly suggest that the 5’ ends of PEMV2 gRNA and sgRNA connect with the 3’UTR through similar long-distance interactions while having different requirements for 3’CITEs.

Unique Structures in the 3′ UTR of Blackcurrant Reversion Nepovirus Genomic RNA 1 Promote Translation Initiation

Canonical translation initiation is promoted and regulated by an interaction between proteins bound to a cap structure (a methylated guanosine triphosphate, m7G, as the 5′ nucleotide) at the 5′ end and those bound to a polyadenylate [poly(A)] tail at the 3′ end of a messenger RNA (mRNA). This end‐to‐end interaction contributes to the recruitment, positioning, and activation of the translation machinery. Blackcurrant reversion nepovirus (BRV) has two bipartite genomic RNAs (RNA 1 and RNA 2) that do not contain 5′ cap structures, but do have 3′ poly(A) tails. Hence, the mechanism of end‐to‐end interaction in BRV has not been clearly defined. In other cap‐less, positive‐sense RNA plant viruses, structured RNA in the 5′ and 3′ untranslated regions (UTRs) bind via proposed RNA‐RNA kissing‐loop interactions between a 5′ hairpin loop and a 3′ cap independent translation enhancer (CITE). Likewise, the mechanism by which the 5′ and 3′ UTRs recruit protein synthesis machinery is directly related to their primary, secondary and higher‐order structure. Considering that the end‐to‐end communication and recruitment of translation factors is dependent upon RNA structure, our main goal focuses on determining the secondary and possible tertiary structures of the 3′ CITE structure in the genomic RNAs found in the BRV. Toward that goal, we have in vitro transcribed the minimal BRV RNA 1 CITE sequence, and probed our purified RNA with a series of covalent nucleobase, sugar and backbone modifiers. Combining our results, we have found a unique secondary structure for the BRV RNA 1 CITE that differs from a previously proposed structure based upon in silico thermodynamic folding predictions. This structure likely plays an important role regulating protein synthesis of viral proteins encoded by the RNA 1 message by either facilitating end‐to‐end interaction with the 5′ UTR and/or directly recruiting important translation protein machinery.Support or Funding InformationMSU Denver College of Letters, Arts & Sciences Mini‐grant, MSU Denver Undergraduate Research Grant, MSU Denver Center for Advanced STEM Education Undergraduate Research Grant

Abstract A17: Probing a translational switch involved in tumor angiogenesis

Abstract As a tumor proliferates, it rapidly outgrows its blood supply, creating an oxygen-starved environment. Low oxygenation (hypoxia) suppresses the canonical biochemical pathway through which the translation of messenger RNAs (mRNAs) into proteins by the ribosome is typically initiated. Despite shutting down this general mechanism for initiating protein synthesis, the tumor can continue to initiate translation of a subset of mRNAs that are involved in angiogenesis and tumor survival. To explain this observation, it has been proposed that, in addition to the eukaryotic mRNA-specific 7-methyl guanosine cap at the 5' end, these pro-angiogenic mRNAs, of which Fibroblast Growth Factor-9 (FGF9) and Hypoxia Inducible Factor 1α (HIF1α) are prototypical members, contain highly stable structures in their 5' untranslated regions. These structures, also referred to as Cap Independent Translation Enhancers (CITEs), form part of a switch that activates a non-canonical pathway for initiating translation, presumably by binding directly to eukaryotic initiation factor 4G (eIF4G), thereby recruiting ribosomes in a manner distinct from the canonical 5' cap-dependent mechanism. To test this hypothesis, here we report the binding affinities of pro-angiogenic mRNAs, as well as control mRNAs lacking CITEs, to eIF4G as a measure of their ability to initiate translation in the hypoxic environment of a tumor. Understanding the dynamics of eIF4G-CITE interactions can inform the design and development of novel angiogenesis inhibitors that function by targeting these interactions. Citation Format: Maya Ramachandran, Somdeb Mitra, Ruben L. Gonzalez, Jr. Probing a translational switch involved in tumor angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A17.

More than just scanning: the importance of cap-independent mRNA translation initiation for cellular stress response and cancer.

The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds at the cap structure at the 5' end of the mRNA and scans the 5' untranslated region (5'UTR) until an initiation codon is found. However, under conditions that impair canonical cap-dependent translation, the synthesis of some proteins is kept by alternative mechanisms that are required for cell survival and stress recovery. Alternative modes of translation initiation include cap- and/or scanning-independent mechanisms of ribosomal recruitment. In most cap-independent translation initiation events there is a direct recruitment of the 40S ribosome into a position upstream, or directly at, the initiation codon via a specific internal ribosome entry site (IRES) element in the 5'UTR. Yet, in some cellular mRNAs, a different translation initiation mechanism that is neither cap- nor IRES-dependent seems to occur through a special RNA structure called cap-independent translational enhancer (CITE). Recent evidence uncovered a distinct mechanism through which mRNAs containing N 6-methyladenosine (m6A) residues in their 5'UTR directly bind eukaryotic initiation factor 3 (eIF3) and the 40S ribosomal subunit in order to initiate translation in the absence of the cap-binding proteins. This review focuses on the important role of cap-independent translation mechanisms in human cells and how these alternative mechanisms can either act individually or cooperate with other cis-acting RNA regulons to orchestrate specific translational responses triggered upon several cellular stress states, and diseases such as cancer. Elucidation of these non-canonical mechanisms reveals the complexity of translational control and points out their potential as prospective novel therapeutic targets.

Analysis of the interacting partners eIF4F and 3′‐CITE required for Melon necrotic spot virus cap‐independent translation

We have shown previously that the translation of Melon necrotic spot virus (MNSV, family Tombusviridae, genus Carmovirus) RNAs is controlled by a 3'-cap-independent translation enhancer (CITE), which is genetically and functionally dependent on the eukaryotic translation initiation factor (eIF) 4E. Here, we describe structural and functional analyses of the MNSV-Mα5 3'-CITE and its translation initiation factor partner. We first mapped the minimal 3'-CITE (Ma5TE) to a 45-nucleotide sequence, which consists of a stem-loop structure with two internal loops, similar to other I-shaped 3'-CITEs. UV crosslinking, followed by gel retardation assays, indicated that Ma5TE interacts in vitro with the complex formed by eIF4E + eIF4G980-1159 (eIF4Fp20 ), but not with each subunit alone or with eIF4E + eIF4G1003-1092 , suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G980-1159 , or through a double interaction with eIF4E and eIF4G980-1159 . Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G1003-1092 and cap-independent translation. We also developed an in vivo system to test the effect of mutations in eIF4E in Ma5TE-driven cap-independent translation, showing that conserved amino acids in a positively charged RNA-binding motif around amino acid position 228, implicated in eIF4E-eIF4G binding or belonging to the cap-recognition pocket, are essential for cap-independent translation controlled by Ma5TE, and thus for the multiplication of MNSV.

Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3´-Translational Enhancer that Communicates with the Corresponding 5´-Region through a Long-Distance RNA-RNA Interaction.

Cap-independent translational enhancers (CITEs) have been identified at the 3´-terminal regions of distinct plant positive-strand RNA viruses belonging to families Tombusviridae and Luteoviridae. On the bases of their structural and/or functional requirements, at least six classes of CITEs have been defined whose distribution does not correlate with taxonomy. The so-called TED class has been relatively under-studied and its functionality only confirmed in the case of Satellite tobacco necrosis virus, a parasitic subviral agent. The 3´-untranslated region of the monopartite genome of Pelargonium line pattern virus (PLPV), the recommended type member of a tentative new genus (Pelarspovirus) in the family Tombusviridae, was predicted to contain a TED-like CITE. Similar CITEs can be anticipated in some other related viruses though none has been experimentally verified. Here, in the first place, we have performed a reassessment of the structure of the putative PLPV-TED through in silico predictions and in vitro SHAPE analysis with the full-length PLPV genome, which has indicated that the presumed TED element is larger than previously proposed. The extended conformation of the TED is strongly supported by the pattern of natural sequence variation, thus providing comparative structural evidence in support of the structural data obtained by in silico and in vitro approaches. Next, we have obtained experimental evidence demonstrating the in vivo activity of the PLPV-TED in the genomic (g) RNA, and also in the subgenomic (sg) RNA that the virus produces to express 3´-proximal genes. Besides other structural features, the results have highlighted the key role of long-distance kissing-loop interactions between the 3´-CITE and 5´-proximal hairpins for gRNA and sgRNA translation. Bioassays of CITE mutants have confirmed the importance of the identified 5´-3´ RNA communication for viral infectivity and, moreover, have underlined the strong evolutionary constraints that may operate on genome stretches with both regulatory and coding functions.

Protein expression strategies in Tobacco necrosis virus-D

Tobacco necrosis virus (TNV-D) has a plus-strand RNA genome that is neither 5′ capped nor 3′ poly-adenylated. Instead, it utilizes a 3′ cap-independent translational enhancer (3′CITE) located in its 3′ untranslated region (UTR) for translation of its proteins. We have examined the protein expression strategies used by TNV-D and our results indicate that: (i) a base pairing interaction between conserved ACCA and UGGU motifs in the genomic 5′UTR and 3′CITE, respectively, is not required for efficient plant cell infection, (ii) similar potential 5′UTR-3′CITE interactions in the two viral subgenomic mRNAs are not needed for efficient translation of viral proteins in vitro, (iii) a small amount of capsid protein is translated from the viral genome by a largely 3′CITE-independent mechanism, (iv) the larger of two possible forms of capsid protein is efficiently translated, and (v) p7b is translated from subgenomic mRNA1 by a leaky scanning mechanism.

3′UTRs of carmoviruses

Carmovirus is a genus of small, single-stranded, positive-strand RNA viruses in the Tombusviridae. One member of the carmoviruses, Turnip crinkle virus (TCV), has been used extensively as a model for examining the structure and function of RNA elements in 3′UTR as well as in other regions of the virus. Using a variety of genetic, biochemical and computational methods, a structure for the TCV 3′UTR has emerged where secondary structures and tertiary interactions combine to adopt higher order 3-D structures including an internal, ribosome-binding tRNA-shaped configuration that functions as a 3′ cap-independent translation enhancer (3′CITE). The TCV 3′CITE also serves as a scaffold for non-canonical interactions throughout the 3′UTR and extending into the upstream open reading frame, interactions that are significantly disrupted upon binding by the RNA-dependent RNA polymerase. Long-distance interactions that connect elements in the 3′UTR with both the 5′ end and the internal ribosome recoding site suggest that 3′UTR of carmoviruses are intimately involved in multiple functions in the virus life cycle. Although carmoviruses share very similar genome organizations, lengths of 5′ and 3′UTRs, and structural features at the 3′ end, the similarity rapidly breaks down the further removed from the 3′ terminus revealing different 3′CITEs and unique virus-specific structural features. This review summarizes 20 years of work dissecting the structure and function of the 3′UTR of TCV and other carmoviruses. The astonishing structural complexity of the 3′UTRs of these simple carmoviruses provides lessons that are likely applicable to many other plant and animal RNA viruses.

The 3' untranslated region of Pea Enation Mosaic Virus contains two T-shaped, ribosome-binding, cap-independent translation enhancers.

Many plant viruses without 5' caps or 3' poly(A) tails contain 3' proximal, cap-independent translation enhancers (3'CITEs) that bind to ribosomal subunits or translation factors thought to assist in ribosome recruitment. Most 3'CITEs participate in a long-distance kissing-loop interaction with a 5' proximal hairpin to deliver ribosomal subunits to the 5' end for translation initiation. Pea Enation Mosaic Virus (PEMV) contains two adjacent 3'CITEs in the center of its 703-nucleotide 3' untranslated region (3'UTR), the ribosome-binding, kissing-loop T-shaped structure (kl-TSS) and eukaryotic translation initiation factor 4E-binding Panicum mosaic virus-like translation enhance (PTE). We now report that PEMV contains a third, independent 3'CITE located near the 3' terminus. This 3'CITE is composed of three hairpins and two pseudoknots, similar to the TSS 3'CITE of the carmovirus Turnip crinkle virus (TCV). As with the TCV TSS, the PEMV 3'TSS is predicted to fold into a T-shaped structure that binds to 80S ribosomes and 60S ribosomal subunits. A small hairpin (kl-H) upstream of the 3'TSS contains an apical loop capable of forming a kissing-loop interaction with a 5' proximal hairpin and is critical for the accumulation of full-length PEMV in protoplasts. Although the kl-H and 3'TSS are dispensable for the translation of a reporter construct containing the complete PEMV 3'UTR in vitro, deleting the normally required kl-TSS and PTE 3'CITEs and placing the kl-H and 3'TSS proximal to the reporter termination codon restores translation to near wild-type levels. This suggests that PEMV requires three 3'CITEs for proper translation and that additional translation enhancers may have been missed if reporter constructs were used in 3'CITE identification. Importance: The rapid life cycle of viruses requires efficient translation of viral-encoded proteins. Many plant RNA viruses contain 3' cap-independent translation enhancers (3'CITEs) to effectively compete with ongoing host translation. Since only single 3'CITEs have been identified for the vast majority of individual viruses, it is widely accepted that this is sufficient for a virus's translational needs. Pea Enation Mosaic Virus possesses a ribosome-binding 3'CITE that can connect to the 5' end through an RNA-RNA interaction and an adjacent eukaryotic translation initiation factor 4E-binding 3'CITE. We report the identification of a third 3'CITE that binds weakly to ribosomes and requires an upstream hairpin to form a bridge between the 3' and 5' ends. Although both ribosome-binding 3'CITEs are critical for virus accumulation in vivo, only the CITE closest to the termination codon of a reporter open reading frame is active, suggesting that artificial constructs used for 3'CITE identification may underestimate the number of CITEs that participate in translation.

Identification of short untranslated regions that sufficiently enhance translation in high-quality wheat germ extract

High-quality wheat germ extract (hqWGE) is very useful for the high-yield production of various types of protein. The most important key to high productivity is the design of mRNA templates. Although the design has been refined for straightforward and efficient translation in hqWGE, there is still room for improvement in untranslated regions (UTRs), especially the 3′ UTR length, because a long, cumbersome 3′ UTR is commonly used for translation enhancement. Here we examined some short viral 3′ cap-independent translation enhancers (3′ CITEs) to identify effective ones for efficient translation in hqWGE. We then combined the most effective 3′ CITE and a 5′ enhancer to further increase the translation efficiency. mRNA with the optimal short 3′ and 5′ UTRs, both of whose length was less than 150nt, exhibited a productivity of 1.4mg/mL in prolonged large-scale protein synthesis in hqWGE, which was comparable to that of control mRNA with a commonly-used long 3′ UTR (∼1200nt).

Position of the kissing-loop interaction associated with PTE-type 3′CITEs can affect enhancement of cap-independent translation

The Panicum mosaic virus-like translation enhancer (PTE) functions as a cap-independent translation enhancer (3′CITE) in members of several Tombusviridae genera including 7/19 carmoviruses. For nearly all PTE, a kissing-loop connects the element with a hairpin found in several conserved locations in the genomic RNA (5′ terminal hairpin or ~100nt from the 5′ end) and small subgenomic RNA (~63nt from the 5′ end). Moving the interaction closer to the 5′ end in reporter mRNAs using Saguaro cactus virus (SCV) sequences had either a minimal or substantial negative effect on translation. Movement of the kissing loop from position 104 to the SCV 5′ terminal hairpin also reduced translation by 4-fold. These results suggest that relocating the PTE kissing loop closer to the 5′ end reduces PTE efficiency, in contrast to results for the Barley yellow dwarf BTE and Tomato bushy stunt virus Y-shaped 3′CITEs, suggesting that different 3′CITEs have different bridging requirements.

Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking.

Many plant viruses depend on functional RNA elements, called 3'-UTR cap-independent translation enhancers (3'-CITEs), for translation of their RNAs. In this manuscript we provide direct proof for the existing hypothesis that 3'-CITEs are modular and transferable by recombination in nature, and that this is associated with an advantage for the created virus. By characterizing a newly identified Melon necrotic spot virus (MNSV; Tombusviridae) isolate, which is able to overcome eukaryotic translation initiation factor 4E (eIF4E)-mediated resistance, we found that it contains a 55 nucleotide insertion in its 3'-UTR. We provide strong evidence that this insertion was acquired by interfamilial recombination with the 3'-UTR of an Asiatic Cucurbit aphid-borne yellows virus (CABYV; Luteoviridae). By constructing chimeric viruses, we showed that this recombined sequence is responsible for resistance breaking. Analysis of the translational efficiency of reporter constructs showed that this sequence functions as a novel 3'-CITE in both resistant and susceptible plants, being essential for translation control in resistant plants. In conclusion, we showed that a recombination event between two clearly identified viruses from different families led to the transfer of exactly the sequence corresponding to a functional RNA element, giving rise to a new isolate with the capacity to infect an otherwise nonsusceptible host.

3′ Cap-Independent Translation Enhancers of Plant Viruses

In the absence of a 5' cap, plant positive-strand RNA viruses have evolved a number of different elements in their 3' untranslated region (UTR) to attract initiation factors and/or ribosomes to their templates. These 3' cap-independent translational enhancers (3' CITEs) take different forms, such as I-shaped, Y-shaped, T-shaped, or pseudoknotted structures, or radiate multiple helices from a central hub. Common features of most 3' CITEs include the ability to bind a component of the translation initiation factor eIF4F complex and to engage in an RNA-RNA kissing-loop interaction with a hairpin loop located at the 5' end of the RNA. The two T-shaped structures can bind to ribosomes and ribosomal subunits, with one structure also able to engage in a simultaneous long-distance RNA-RNA interaction. Several of these 3' CITEs are interchangeable and there is evidence that natural recombination allows exchange of modular CITE units, which may overcome genetic resistance or extend the virus's host range.

Preparation of biologically active Arabidopsis ribosomes and comparison with yeast ribosomes for binding to a tRNA-mimic that enhances translation of plant plus-strand RNA viruses

Isolation of biologically active cell components from multicellular eukaryotic organisms often poses difficult challenges such as low yields and inability to retain the integrity and functionality of the purified compound. We previously identified a cap-independent translation enhancer (3′CITE) in the 3′UTR of Turnip crinkle virus (TCV) that structurally mimics a tRNA and binds to yeast 80S ribosomes and 60S subunits in the P-site. Yeast ribosomes were used for these studies due to the lack of methods for isolation of plant ribosomes with high yields and integrity. To carry out studies with more natural components, a simple and efficient procedure has been developed for the isolation of large quantities of high quality ribosomes and ribosomal subunits from Arabidopsis thaliana protoplasts prepared from seed-derived callus tissue. Attempts to isolate high quality ribosomes from wheat germ, bean sprouts, and evacuolated protoplasts were unsuccessful. Addition of purified Arabidopsis 80S plant ribosomes to ribosome-depleted wheat germ lysates resulted in a greater than 1200-fold enhancement in in vitro translation of a luciferase reporter construct. The TCV 3′CITE bound to ribosomes with a three to sevenfold higher efficiency when using plant 80S ribosomes compared with yeast ribosomes, indicating that this viral translational enhancer is adapted to interact more efficiently with host plant ribosomes.

Tombusvirus Y-Shaped Translational Enhancer Forms a Complex with eIF4F and Can Be Functionally Replaced by Heterologous Translational Enhancers

Certain plus-strand RNA plant viruses that are uncapped and nonpolyadenylated rely on RNA elements in their 3' untranslated region, termed 3'-cap-independent translational enhancers (3'CITEs), for efficient translation of their proteins. Here, we have investigated the properties of the Y-shaped class of 3'CITE present in the tombusvirus Carnation Italian ringspot virus (CIRV). While some types of 3'CITE have been found to function through recruitment of translation initiation factors to the viral genome, no trans-acting translation-related factors have yet been identified for the Y-shaped 3'CITE. Our results indicate that the CIRV 3'CITE complexes with eIF4F and eIFiso4F, with the former mediating translation more efficiently than the latter. In nature, some classes of 3'CITE are present in several different viral genera, suggesting that these elements hold a high degree of modularity. Here, we test this concept by engineering chimeric viruses containing heterologous 3'CITEs and show that the Y-shaped class of 3'CITE in CIRV can be replaced by two alternative types of 3'CITE, i.e., a Panicum mosaic virus-like 3'CITE or an I-shaped 3'CITE, without any major loss in in vitro translation or replication efficiency in protoplasts. The heterologous 3'CITEs also mediated whole-plant infections of Nicotiana benthamiana, where distinct symptoms were observed for each of the alternative 3'CITEs and 3'CITE evolution occurred during serial passaging. Our results supply new information on Y-shaped 3'CITE function and provide insights into 3'CITE virus-host compatibilities.

Poly(A)-Binding Protein Facilitates Translation of an Uncapped/Nonpolyadenylated Viral RNA by Binding to the 3′ Untranslated Region

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.