Intergenic Region Internal Ribosome Entry Site Research Articles

Initiation of translation on nedicistrovirus and related intergenic region IRESs by their factor-independent binding to the P site of 80S ribosomes.

Initiation of translation on many viral mRNAs occurs by noncanonical mechanisms that involve 5' end-independent binding of ribosomes to an internal ribosome entry site (IRES). The ∼190-nt-long intergenic region (IGR) IRES of dicistroviruses such as cricket paralysis virus (CrPV) initiates translation without Met-tRNAi Met or initiation factors. Advances in metagenomics have revealed numerous dicistrovirus-like genomes with shorter, structurally distinct IGRs, such as nedicistrovirus (NediV) and Antarctic picorna-like virus 1 (APLV1). Like canonical IGR IRESs, the ∼165-nt-long NediV-like IGRs comprise three domains, but they lack key canonical motifs, including L1.1a/L1.1b loops (which bind to the L1 stalk of the ribosomal 60S subunit) and the apex of stem-loop V (SLV) (which binds to the head of the 40S subunit). Domain 2 consists of a compact, highly conserved pseudoknot (PKIII) that contains a UACUA loop motif and a protruding CrPV-like stem--loop SLIV. In vitro reconstitution experiments showed that NediV-like IRESs initiate translation from a non-AUG codon and form elongation-competent 80S ribosomal complexes in the absence of initiation factors and Met-tRNAi Met Unlike canonical IGR IRESs, NediV-like IRESs bind directly to the peptidyl (P) site of ribosomes leaving the aminoacyl (A) site accessible for decoding. The related structures of NediV-like IRESs and their common mechanism of action indicate that they exemplify a distinct class of IGR IRES.

Isolation and characterization of a novel cripavirus, the first Dicistroviridae family member infecting the cotton mealybug Phenacoccus solenopsis.

A new virus belonging to the family Dicistroviridae was identified in the hibiscus-infesting cotton mealybug Phenacoccus solenopsis. Using high-throughput sequencing (HTS) on an Illumina HiSeq platform, a single contig of the complete genome sequence was assembled. The authenticity of the sequence obtained by HTS was validated by RT-PCR and Sanger sequencing of the amplicons, which was also employed for the 3' untranslated region (UTR). The 5' UTR was sequenced using a rapid amplification of cDNA ends kit. A large segment encompassing the whole genome was amplified by RT-PCR using viral RNA extracted from mealybugs. A whole-genome nucleotide sequence comparison showed 89% sequence identity to aphid lethal paralysis virus (ALPV), covering a short segment of 44 bp. Pairwise amino acid sequence comparisons of the protein encoded by open reading frame (ORF) 2 with its counterparts in the GenBank database, showed less than 40% identity to several members of the genus Cripavirus, including ALPV. Phylogenetic analysis based on the deduced amino acid sequence of the ORF 2 protein showed that the new virus grouped with members of the genus Cripavirus. The intergenic region (IGR) internal ribosome entry site (IRES) showed the conserved nucleotides of a type I IGR IRES and had two bulge sites, three pseudoknots, and two stem-loops. Virus morphology visualized by transmission electron microscopy demonstrated spherical particles with a diameter of ~30 nm. This virus was the only arthropod virus identified in the sampled mealybugs, and the purified virus was able to infect cotton mealybugs. To the best of our knowledge, this is the first report of a Dicistroviridae family member infecting P. solenopsis, and we have tentatively named this virus Phenacoccus solenopsis virus (PhSoV).

Evaluation of the safety profile of ssRNA adjuvant derived from the IGR IRES region of CrPV

Abstract Adjuvants enhance the efficacy of vaccines by stimulating immune response relate gene expression and pathways. However, development of an appropriate adjuvant for a specific vaccine type is difficult, because of their unwanted systemic and local adverse effects. Despite intensive efforts for more than 80 years, there are only a few approved adjuvants. Poly I:C, a double stranded RNA was experimentally used as an adjuvant since 1970s. Although poly I:C dramatically stimulates the innate immune response, which is a prerequisite of a good adjuvant, inducing effective immune response while avoiding adverse events is a major challenge. In this study, we evaluated the safety and toxicological profile of a high-dose (200ug/mouse) single stranded RNA (ssRNA) derived from the intergenic region (IGR) internal ribosome entry site (IRES) of cricket paralysis virus (CrPV) as a novel adjuvant in a mouse model. The results indicate that there were no significant differences in body weight and food intake pattern in mice immunized with ssRNA adjuvant and the control group. Hematological parameters like ALT and AST levels appeared to be normal in both treated and control group. Histology of organs such as liver, spleen, lung, and kidney appeared to be normal. There was no evidence of tissue damage in mice treated with ssRNA adjuvant and control group. IgE levels, which is marker of autoimmune disease, is also not different compared with treated group and control group. In addition, the adjuvant effect is similar in both high-dose and low-dose (20ug/mouse) treated groups. In conclusion, novel ssRNA adjuvant exhibited a safe toxicological profile in mouse model. The results obtained may be utilized for conducting further clinical trials in humans.

Functional Insights into the Adjacent Stem-Loop in Honey Bee Dicistroviruses That Promotes Internal Ribosome Entry Site-Mediated Translation and Viral Infection

All viruses must successfully harness the host translational apparatus and divert it towards viral protein synthesis. Dicistroviruses use an unusual internal ribosome entry site (IRES) mechanism whereby the IRES adopts a three-pseudoknot structure that accesses the ribosome tRNA binding sites to directly recruit the ribosome and initiate translation from a non-AUG start site. A subset of dicistroviruses, including the honey bee Israeli acute paralysis virus (IAPV), encode an extra stem-loop (SLVI) 5' -adjacent to the IGR IRES. Previously, the function of this additional stem-loop is unknown. Here, we provide mechanistic and functional insights into the role of SLVI in IGR IRES translation and in virus infection. Biochemical analyses of a series of mutant IRESs demonstrated that SLVI does not function in ribosome recruitment but is required for proper ribosome positioning on the IRES to direct translation. Using a chimeric infectious clone derived from the related Cricket paralysis virus, we showed that the integrity of SLVI is important for optimal viral translation and viral yield. Based on structural models of ribosome-IGR IRES complexes, the SLVI is predicted to be in the vicinity of the ribosome E site. We propose that SLVI of IAPV IGR IRES functionally mimics interactions of an E-site tRNA with the ribosome to direct positioning of the tRNA-like domain of the IRES in the A site.IMPORTANCEViral internal ribosome entry sites are RNA elements and structures that allow some positive-sense monopartite RNA viruses to hijack the host ribosome to start viral protein synthesis. We demonstrate that a unique stem-loop structure is essential for optimal viral protein synthesis and for virus infection. Biochemical evidence shows that this viral stem-loop RNA structure impacts a fundamental property of the ribosome to start protein synthesis.

Molecular analysis of the factorless internal ribosome entry site in Cricket Paralysis virus infection.

The dicistrovirus Cricket Paralysis virus contains a unique dicistronic RNA genome arrangement, encoding two main open reading frames that are driven by distinct internal ribosome entry sites (IRES). The intergenic region (IGR) IRES adopts an unusual structure that directly recruits the ribosome and drives translation of viral structural proteins in a factor-independent manner. While structural, biochemical, and biophysical approaches have provided mechanistic details into IGR IRES translation, these studies have been limited to in vitro systems and little is known about the behavior of these IRESs during infection. Here, we examined the role of previously characterized IGR IRES mutations on viral yield and translation in CrPV-infected Drosophila S2 cells. Using a recently generated infectious CrPV clone, introduction of a subset of mutations that are known to disrupt IRES activity failed to produce virus, demonstrating the physiological relevance of specific structural elements within the IRES for virus infection. However, a subset of mutations still led to virus production, thus revealing the key IRES-ribosome interactions for IGR IRES translation in infected cells, which highlights the importance of examining IRES activity in its physiological context. This is the first study to examine IGR IRES translation in its native context during virus infection.

Commandeering the Ribosome: Lessons Learned from Dicistroviruses about Translation.

To replicate, all viruses depend entirely on the enslavement of host cell ribosomes for their own advantage. To this end, viruses have evolved a multitude of translational strategies to usurp the ribosome. RNA-based structures known as internal ribosome entry sites (IRESs) are among the most notable mechanisms employed by viruses to seize host ribosomes. In this article, we spotlight the intergenic region IRES from the Dicistroviridae family of viruses and its importance as a model for IRES-dependent translation and in understanding fundamental properties of translation.

Temporal Regulation of Distinct Internal Ribosome Entry Sites of the Dicistroviridae Cricket Paralysis Virus.

Internal ribosome entry is a key mechanism for viral protein synthesis in a subset of RNA viruses. Cricket paralysis virus (CrPV), a member of Dicistroviridae, has a positive-sense single strand RNA genome that contains two internal ribosome entry sites (IRES), a 5′untranslated region (5′UTR) and intergenic region (IGR) IRES, that direct translation of open reading frames (ORF) encoding the viral non-structural and structural proteins, respectively. The regulation of and the significance of the CrPV IRESs during infection are not fully understood. In this study, using a series of biochemical assays including radioactive-pulse labelling, reporter RNA assays and ribosome profiling, we demonstrate that while 5′UTR IRES translational activity is constant throughout infection, IGR IRES translation is delayed and then stimulated two to three hours post infection. The delay in IGR IRES translation is not affected by inhibiting global translation prematurely via treatment with Pateamine A. Using a CrPV replicon that uncouples viral translation and replication, we show that the increase in IGR IRES translation is dependent on expression of non-structural proteins and is greatly stimulated when replication is active. Temporal regulation by distinct IRESs within the CrPV genome is an effective viral strategy to ensure optimal timing and expression of viral proteins to facilitate infection.

A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation

Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs.

Switch from cap- to factorless IRES-dependent 0 and +1 frame translation during cellular stress and dicistrovirus infection.

Internal ribosome entry sites (IRES) are utilized by a subset of cellular and viral mRNAs to initiate translation during cellular stress and virus infection when canonical cap-dependent translation is compromised. The intergenic region (IGR) IRES of the Dicistroviridae uses a streamlined mechanism in which it can directly recruit the ribosome in the absence of initiation factors and initiates translation using a non-AUG codon. A subset of IGR IRESs including that from the honey bee viruses can also direct translation of an overlapping +1 frame gene. In this study, we systematically examined cellular conditions that lead to IGR IRES-mediated 0 and +1 frame translation in Drosophila S2 cells. Towards this, a novel bicistronic reporter that exploits the 2A “stop-go” peptide was developed to allow the detection of IRES-mediated translation in vivo. Both 0 and +1 frame translation by the IGR IRES are stimulated under a number of cellular stresses and in S2 cells infected by cricket paralysis virus, demonstrating a switch from cap-dependent to IRES-dependent translation. The regulation of the IGR IRES mechanism ensures that both 0 frame viral structural proteins and +1 frame ORFx protein are optimally expressed during virus infection.

Characterizing IGR IRES-mediated translation initiation for use in yeast cell-free protein synthesis

Eukaryotic cell-free protein synthesis (CFPS) systems are limited, in part, by inefficient translation initiation. Here, we report three internal ribosome entry site (IRES) sequences from the Dicistroviridae family that are highly active in yeast CFPS. These include the intergenic region (IGR) IRES from cricket paralysis virus (CrPV), plautia stali intestine virus (PSIV) and Solenopsis invicta virus 1 (SINV1). Optimization of combined transcription and translation (Tx/Tl) CFPS reactions primed with linear DNA containing the CrPV IGR IRES resulted in batch synthesis yields of 0.92 ± 0.17 μg/mL luciferase. Further template engineering, such as including the first 12 nt of native CrPV gene, increased yields to 2.33 ± 0.11 μg/mL. We next observed that the inclusion of a 50 nt poly(A) to the 3' end of the IGR IRES-mediated message increased yields an additional 81% to 4.33 ± 0.37 μg/mL, without any effect on mRNA stability or copy number. This was surprising because the CrPV IGR IRES requires no known translation initiation factors. Lastly, we investigated a method to inhibit background expression through competitive inhibition by supplying the reaction with 5' cap structure analog. This study highlights the crucial role translation initiation plays in yeast CFPS and offers a simple platform to study IRES sequences.

Cell‐free protein expression based on extracts from CHO cells

Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974; McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 µg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the specific labeling of integral membrane proteins and the cell-free production of posttranslationally modified proteins.

Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

Internal ribosome entry site (IRES) RNAs are elements of viral or cellular mRNAs that bypass steps of canonical eukaryotic cap-dependent translation initiation. Understanding of the structural basis of IRES mechanisms is limited, partially due to a lack of high-resolution structures of IRES RNAs bound to their cellular targets. Prompted by the universal phylogenetic conservation of the ribosomal P site, we solved the crystal structures of proposed P site binding domains from two intergenic region IRES RNAs bound to bacterial 70S ribosomes. The structures show that these IRES domains nearly perfectly mimic a tRNA • mRNA interaction. However, there are clear differences in the global shape and position of this IRES domain in the intersubunit space compared to those of tRNA, supporting a mechanism for IRES action that invokes hybrid state mimicry to drive a noncanonical mode of translocation. These structures suggest how relatively small structured RNAs can manipulate complex biological machines.

Manipulation of ribosomes by viral RNAs

Viruses employ a variety of strategies to manipulate the basic molecular machinery of their hosts and to coordinate important events during infection. In many cases, virally encoded RNAs are at the heart of these strategies. One example of this are in viral internal ribosome entry sites (IRES) RNAs, which recruit, position, and activate the host cell's translation machinery to the viral RNA. The correct functioning of IRESs is critical for the successful infection of these viruses, which include hepatitis C virus, polio, HIV‐1, and others. In addition, IRESs are used by some cellular mRNAs and are thus are a potentially important mechanism of post‐transcriptional gene regulation. The intergenic region (IGR) IRESs of the Dicistroviridae viruses use the most streamlined IRES mechanism identified to date, binding directly to the ribosome and manipulating it into initiating translation of the viral message. As such, the IGR IRESs have been dubbed “molecular hijackers.” We are using the IGR IRESs as a model to understand some of the basic molecular tenets of IRES RNA structure, IRES‐ribosome interactions and IRES function, as well as to understand the basic workings of the ribosome that are potentially important for post‐transcription regulation, in general. We have developed a detailed model for the mechanism of the IGR IRESs using results from cryo‐EM, X‐ray crystallography, and other structural, functional, and biophysical methods. I will present new data from both structural and functional studies that test our mechanistic model and also suggest new ideas for how these RNA‐based molecular hijackers work.

RPS25 is essential for translation initiation by the Dicistroviridae and hepatitis C viral IRESs.

Most eukaryotic mRNAs are translated using a cap-dependent mechanism of translation. However, approximately 10% of mammalian mRNAs initiate translation using a cap-independent mechanism that is not well understood. These mRNAs contain an internal ribosome entry site (IRES) located in the 5' untranslated region. The cricket paralysis virus (CrPV) intergenic region IRES (IGR IRES) functions in yeast, mammals, and plants, and does not require any translation initiation factors. We used yeast genetics to understand how ribosomes are recruited directly to the mRNA by an IRES. We found that Rps25p has an essential role in CrPV IGR IRES activity in yeast and mammalian cells but not in cap-dependent translation. Purified 40S ribosomal subunits lacking Rps25 are unable to bind to the IGR IRES in vitro. The hepatitis C virus (HCV) IRES also requires Rps25, demonstrating the function of Rps25 is conserved across IRES types. Yeast strains lacking Rps25 exhibit only slight defects in global translation, readthrough, ribosome biogenesis, and programmed ribosomal frameshifting. This work is the first demonstration of a ribosomal protein that is specifically required for IRES-mediated translation initiation. Our findings provide us with the beginnings of a model for the molecular interactions of an IRES with the ribosome.

Host and Viral Translational Mechanisms during Cricket Paralysis Virus Infection

The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2alpha phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2alpha by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2alpha phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2alpha phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5' untranslated region (5'UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5'UTR and IGR IRES.

Translation initiation factors are not required for Dicistroviridae IRES function in vivo

The cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry site (IRES) uses an unusual mechanism of initiating translation, whereby the IRES occupies the P-site of the ribosome and the initiating tRNA enters the A-site. In vitro experiments have demonstrated that the CrPV IGR IRES is able to bind purified ribosomes and form 80S complexes capable of synthesizing small peptides in the absence of any translation initiation factors. These results suggest that initiation by this IRES is factor-independent. To determine whether the IGR IRES functions in the absence of initiation factors in vivo, we assayed IGR IRES activity in various yeast strains harboring mutations in canonical translation initiation factors. We used a dicistronic reporter assay in yeast to determine whether the CrPV IGR IRES is able to promote translation sufficient to support growth in the presence of various deletions or mutations in translation initiation factors. Using this assay, we have previously shown that the CrPV IGR IRES functions efficiently in yeast when ternary complexes (eIF2*GTP*initiator tRNA(met)) are reduced. Here, we demonstrate that the CrPV IGR IRES activity does not require the eukaryotic initiation factors eIF4G1 or eIF5B, and it is enhanced when eIF2B, the eIF3b subunit of eIF3, or eIF4E are impaired. Taken together, these data support a model in which the CrPV IGR IRES is capable of initiating protein synthesis in the absence of any initiation factors in vivo, and suggests that the CrPV IGR IRES initiates translation by directly recruiting the ribosomal subunits in vivo.

Examining the relative activity of several dicistrovirus intergenic internal ribosome entry site elements in uninfected insect and mammalian cell lines

Comparisons of the relative activities of 11 intergenic region (IGR) internal ribosome entry site (IRES) elements of insect dicistrovirus with 5' IRES elements of the hepatitis C and encephalomyocarditis viruses were performed in insect and mammalian cells. Dual luciferase assays were performed to determine the most effective dicistrovirus IGR IRES in the lepidopteran cell lines Sf9 (Spodoptera frugiperda) and BmN (Bombyx mori), and the dipteran cell lines S2 (Drosophila melanogaster) and ATC-10 (Aedes aegypti). Evaluation of dual luciferase expression from DNA plasmids and in vitro-transcribed RNA revealed apparent splicing with certain IRES elements. Though IRES activity depended upon the cell line examined, the black queen cell and Drosophila C dicistrovirus intergenic IRES elements were most effective for coupled gene expression in the diverse insect cell lines examined.

Comparing the three-dimensional structures of Dicistroviridae IGR IRES RNAs with other viral RNA structures

The intergenic region (IGR) internal ribosome entry site (IRES) RNAs do not require any of the canonical translation initiation factors to recruit the ribosome to the viral RNA, they eliminate the need for initiator tRNA, and they begin translation from the A-site. The function of these IRESs depends on a specific three-dimensional folded RNA structure. Thus, a complete understanding of the mechanisms of action of these IRESs requires that we understand their structure in detail. Recently, the structures of both domains of the IGR IRES RNAs were solved by X-ray crystallography, providing the first glimpse into an entire IRES RNA structure. Here, I present an analysis of these structures, emphasizing how the structures explain many aspects of IGR IRES function, discussing how these structures have similarities to motifs found in other viral RNAs, and illustrating how these structures give rise to new mechanistic hypotheses.

TRNA–mRNA mimicry drives translation initiation from a viral IRES

Internal ribosome entry site (IRES) RNAs initiate protein synthesis in eukaryotic cells by a noncanonical cap-independent mechanism. IRESes are critical for many pathogenic viruses, but efforts to understand their function are complicated by the diversity of IRES sequences as well as by limited high-resolution structural information. The intergenic region (IGR) IRESes of the Dicistroviridae viruses are powerful model systems to begin to understand IRES function. Here we present the crystal structure of a Dicistroviridae IGR IRES domain that interacts with the ribosome's decoding groove. We find that this RNA domain precisely mimics the transfer RNA anticodon-messenger RNA codon interaction, and its modeled orientation on the ribosome helps explain translocation without peptide bond formation. When combined with a previous structure, this work completes the first high-resolution description of an IRES RNA and provides insight into how RNAs can manipulate complex biological machines.

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.