Virus Internal Ribosome Entry Site Research Articles

A circular split nanoluciferase reporter for validating and screening putative internal ribosomal entry site elements.

Internal ribosomal entry sites (IRESs) recruit the ribosome to promote translation, typically in an m7G cap-independent manner. Although IRESs are well-documented in viral genomes, they have also been reported in mammalian transcriptomes, where they have been proposed to mediate cap-independent translation of mRNAs. However, subsequent studies have challenged the idea of these "cellular" IRESs. Current methods for screening and discovering IRES activity rely on a bicistronic reporter assay, which is prone to producing false positive signals if the putative IRES sequence has a cryptic promoter or cryptic splicing sites. Here, we report an assay for screening IRES activity using a genetically encoded circular RNA comprising a split nanoluciferase (nLuc) reporter. The circular split nLuc reporter is less susceptible to the various sources of false positives that adversely affect the bicistronic IRES reporter assay and provides a streamlined method for screening IRES activity. Using the circular split nLuc reporter, we find that nine reported cellular IRESs have minimal IRES activity. Overall, the circular split nLuc reporter offers a simplified approach for identifying and validating IRESs and exhibits reduced propensity for producing the types of false positives that can occur with the bicistronic reporter assay.

Genetic mechanisms underlying the structural elaboration and dissemination of viral internal ribosomal entry sites.

Viral internal ribosomal entry sites (IRESs) form several classes that use distinct mechanisms to mediate end-independent initiation of translation. The origin of viral IRESs is a longstanding question. The simplest IRESs comprise tandem pseudoknots and occur in the intergenic region (IGR) of Dicistroviridae genomes (order Picornavirales ). Larger IGR IRESs contain additional elements that determine specific properties such as binding to the head of the ribosoma l 40S subunit. Metagenomic analyses reported here identified novel groups of structurally distinct IGR-like IRESs. The smallest of these (∼120nt long) comprise three pseudoknots and bind directly to the ribosomal P site. Others are up to 260nt long: insertions occurred at specific loci, possibly reflecting non-templated nucleotide insertion during replication. Various groups can be arranged in order, differing by the cumulative addition of single structural elements, suggesting an accretion mechanism for the structural elaboration of IRESs. Identification of chimeric IRESs implicates recombinational exchange of domains as a second mechanism for the diversification of IRES structure. Recombination likely also accounts for the presence of IGR-like IRESs at the 5'-end of some dicistrovirus-like genomes (e.g. Hangzhou dicistrovirus 3) and in the RNA genomes of Tombusviridae (order Tolivirales ), Marnaviridae (order Picornavirale s), and the 'Ripiresk' picorna-like clade (order Picornavirale s).

Loxapine inhibits replication of hepatitis A virus in vitro and in vivo by targeting viral protein 2C.

No antiviral drugs currently are available for treatment of infection by hepatitis A virus (HAV), a causative agent of acute hepatitis, a potentially life-threatening disease. Chemical screening of a small-compound library using nanoluciferase-expressing HAV identified loxapine succinate, a selective dopamine receptor D2 antagonist, as a potent inhibitor of HAV propagation in vitro. Loxapine succinate did not inhibit viral entry nor internal ribosome entry site (IRES)-dependent translation, but exhibited strong inhibition of viral RNA replication. Blind passage of HAV in the presence of loxapine succinate resulted in the accumulation of viruses containing mutations in the 2C-encoding region, which contributed to resistance to loxapine succinate. Analysis of molecular dynamics simulations of the interaction between 2C and loxapine suggested that loxapine binds to the N-terminal region of 2C, and that resistant mutations impede these interactions. We further demonstrated that administration of loxapine succinate to HAV-infected Ifnar1-/- mice (which lack the type I interferon receptor) results in decreases in the levels of fecal HAV RNA and of intrahepatic HAV RNA at an early stage of infection. These findings suggest that HAV protein 2C is a potential target for antivirals, and provide novel insights into the development of drugs for the treatment of hepatitis A.

Virus-derived circular RNAs populate hepatitis C virus–infected cells

It is known that pre-mRNAs in eukaryotic cells can be processed to circular RNAs by a backsplicing mechanism. Circular RNAs have great stability and can sequester proteins or small RNAs to exert functions on cellular pathways. Because viruses often exploit host pathways, we explored whether the RNA genome of the cytoplasmic hepatitis C virus is processed to yield virus-derived circRNAs (vcircRNAs). Computational analyses of RNA-seq experiments predicted that the viral RNA genome is fragmented to generate hundreds of vcircRNAs. More than a dozen of them were experimentally verified by rolling-circle amplification. VcircRNAs that contained the viral internal ribosome entry site were found to be translated into proteins that displayed proviral functions. Furthermore, two highly abundant, nontranslated vcircRNAs were shown to enhance viral RNA abundance. These findings argue that novel vcircRNA molecules modulate viral amplification in cells infected by a cytoplasmic RNA virus.

Structure-based virtual screening of unbiased and RNA-focused libraries to identify new ligands for the HCV IRES model system

Using structure-based virtual screening, FRET and MST assays, novel ligands of the hepatitis C virus internal ribosome entry site were identified. This proof-of-concept study demonstrated the feasibility of RNA–ligand docking for hit identification.

Hyperglycemia facilitates EV71 replication: Insights into miR-206-mediated regulation of G3BP2 promoting EV71 IRES activity.

Background: Neurotropic virus infections actively manipulate host cell metabolism to enhance virus neurovirulence. Although hyperglycemia is common during severe infections, its specific role remains unclear. This study investigates the impact of hyperglycemia on the neurovirulence of enterovirus 71 (EV71), a neurovirulent virus relying on internal ribosome entry site (IRES)-mediated translation for replication. Methods: Utilizing hSCARB2-transgenic mice, we explore the effects of hyperglycemia in EV71 infection and elucidate the underlying mechanisms. Results: Remarkably, administering insulin alone to reduce hyperglycemia in hSCARB2-transgenic mice results in a decrease in brainstem encephalitis and viral load. Conversely, induced hyperglycemia exacerbates neuropathogenesis, highlighting the pivotal role of hyperglycemia in neurovirulence. Notably, miR-206 emerges as a crucial mediator induced by viral infection, with its expression further heightened by hyperglycemia and concurrently repressed by insulin. The use of antagomiR-206 effectively mitigates EV71-induced brainstem encephalitis and reduces viral load. Mechanistically, miR-206 facilitates IRES-driven virus replication by repressing the stress granule protein G3BP2. Conclusions: Novel therapeutic approaches against severe EV71 infections involve managing hyperglycemia and targeting the miR-206-stress granule pathway to modulate virus IRES activity.

Validating the EMCV IRES Secondary Structure with Structure-Function Analysis.

The encephalomyocarditis virus internal ribosome entry site (EMCV IRES) is a structured RNA sequence found in the 5' UTR of the genomic RNA of the encephalomyocarditis virus. The EMCV IRES structure facilitates efficient translation initiation without needing a 5' m7G cap or the cap-binding protein eIF4E. The secondary structure of IRES has been the subject of several previous studies, and a number of different structural models have been proposed. Though some domains of the IRES are conserved across the different secondary structure models, domain I of the IRES varies greatly across them. A literature comparison led to the identification of three regions of interest that display structural heterogeneity within past secondary structure models. To test the accuracy of the secondary structure models in these regions, we employed mutational analysis and SHAPE probing. Mutational analysis revealed that two helical regions within the identified regions of interest are important for IRES translation. These helical regions are consistent with only one of the structure predictions in the literature and do not form in EMCV IRES structures predicted using modern secondary structure prediction methods. The importance of these regions is further supported by multiple SHAPE protections when probing was performed after in vitro translation, indicating that these regions are involved in the IRES translation complex. This work validates a published structure and demonstrates the importance of domain I during EMCV IRES translation initiation.

RNA-based translation activators for targeted gene upregulation

Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present “translation-activating RNAs” (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research.

The intrinsically disordered region of eIF5B stimulates IRES usage and nucleates biological granule formation

Cells activate stress response pathways to survive adverse conditions. Such responses involve the inhibition of global cap-dependent translation. This inhibition is a block that essential transcripts must escape via alternative methods of translation initiation, e.g., an internal ribosome entry site (IRES). IRESs have distinct structures and generally require a limited repertoire of translation factors. Cellular IRESs have been identified in many critical cellular stress response transcripts. We previously identified cellular IRESs in the murine insulin receptor (Insr) and insulin-like growth factor 1 receptor (Igf1r) transcripts and demonstrated their resistance to eukaryotic initiation factor 4F (eIF4F) inhibition. Here, we find that eIF5B preferentially promotes Insr, Igf1r, and hepatitis C virus IRES activity through a non-canonical mechanism that requires its highly charged and disordered N terminus. We find that the N-terminal region of eIF5B can drive cytoplasmic granule formation. This eIF5B granule is triggered by cellular stress and is sufficient to specifically promote IRES activity.

The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs.

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

Analysis of the Immunostimulatory Effects of Cytokine-Expressing Internal Ribosome Entry Site-Based RNA Adjuvants and Their Applications.

Developing new adjuvants that can effectively induce humoral and cellular immune responses while broadening the immune response is of great value. In this study, we aimed to develop single-stranded RNA adjuvants expressing (1) granulocyte monocyte colony-stimulating factor or (2) interleukin 18 based on the encephalomyocarditis virus internal ribosome entry site; we also tested their efficacy in combination with ovalbumin or inactivated influenza vaccines. Notably, cytokine-expressing RNA adjuvants increased the expression of antigen-presenting cell activation markers in mice. Specifically, when combined with ovalbumin, RNA adjuvants expressing granulocyte monocyte colony-stimulating factor increased CD4+ T-cell responses, while those expressing interleukin 18 increased CD8+ T-cell responses. Cytokine-expressing RNA adjuvants further increased the frequency of polyclonal T cells with the influenza vaccine and reduced the clinical illness scores and weight loss of mice after viral challenge. Collectively, our results suggest that cytokine-expressing RNA adjuvants can be applied to protein-based or inactivated vaccines to increase their efficacy.

Regulation of translation by ribosomal RNA pseudouridylation.

Pseudouridine is enriched in ribosomal, spliceosomal, transfer, and messenger RNA and thus integral to the central dogma. The chemical basis for how pseudouridine affects the molecular apparatus such as ribosome, however, remains elusive owing to the lack of structures without this natural modification. Here, we studied the translation of a hypopseudouridylated ribosome initiated by the internal ribosome entry site (IRES) elements. We analyzed eight cryo-electron microscopy structures of the ribosome bound with the Taura syndrome virus IRES in multiple functional states. We found widespread loss of pseudouridine-mediated interactions through water and long-range base pairings. In the presence of the translocase, eukaryotic elongation factor 2, and guanosine 5'-triphosphate hydrolysis, the hypopseudouridylated ribosome favors a rare unconducive conformation for decoding that is partially recouped in the ribosome population that remains modified at the P-site uridine. The structural principles learned establish the link between functional defects and modification loss and are likely applicable to other pseudouridine-associated processes.

A novel function for eukaryotic elongation factor 3: Inhibition of stop codon readthrough in yeast

Eukaryotic elongation factor 3 (eEF3) is one of the essential yeast ribosome-associated ATP-binding cassette type F (ABCF) ATPases. Previously, we found that eEF3 stimulates release of mRNA from puromycin-treated polysomes. In this study, we used a cell-free cricket paralysis virus (CrPV) internal ribosome entry site (IRES)-mediated firefly luciferase bicistronic mRNA translation system with yeast S30 extract. When eEF3 was partially removed from the crude extract, the product from the downstream ORF was increased by the readthrough of a UAA stop codon in the upstream ORF. eEF3 enhanced the release of luciferase from the polysome by eukaryotic release factor (eRF)1 and eRF3. These results suggest that eEF3 is a factor that assists eRFs in performing normal protein synthesis termination in yeast.

Elucidating the distinct contributions of miR-122 in the HCV life cycle reveals insights into virion assembly.

Efficient hepatitis C virus (HCV) RNA accumulation is dependent upon interactions with the human liver-specific microRNA, miR-122. MiR-122 has at least three roles in the HCV life cycle: it acts as an RNA chaperone, or 'riboswitch', allowing formation of the viral internal ribosomal entry site; it provides genome stability; and promotes viral translation. However, the relative contribution of each role in HCV RNA accumulation remains unclear. Herein, we used point mutations, mutant miRNAs, and HCV luciferase reporter RNAs to isolate each of the roles and evaluate their contribution to the overall impact of miR-122 in the HCV life cycle. Our results suggest that the riboswitch has a minimal contribution in isolation, while genome stability and translational promotion have similar contributions in the establishment phase of infection. However, in the maintenance phase, translational promotion becomes the dominant role. Additionally, we found that an alternative conformation of the 5' untranslated region, termed SLIIalt, is important for efficient virion assembly. Taken together, we have clarified the overall importance of each of the established roles of miR-122 in the HCV life cycle and provided insight into the regulation of the balance between viral RNAs in the translating/replicating pool and those engaged in virion assembly.

IRES-mediated translation in bacteria

Despite the similarity in fundamental goals of translation initiation between different domains of life, it is one of the most phylogenetically diverse steps of the central dogma of molecular biology. In a classical view, the translation signals for prokaryotes and eukaryotes are distinct from each other. This idea was challenged by the finding that the Internal Ribosome Entry Site (IRES) belonging to Plautia stali intestine virus (PSIV) could bypass the domain-specific boundaries and effectively initiate translation in E. coli. This finding led us to investigate whether the ability of PSIV IRES to initiate translation in E. coli is specific to this IRES and also to study features that allow this viral IRES to mediate prokaryotic translation initiation. We observed that certain IRESs may also possess the ability to initiate E. coli translation. Our results also indicated that the structural integrity of the PSIV IRES in translation in prokaryotes does not appear to be as critical as it is in eukaryotes. We also demonstrated that two regions of the PSIV IRES with complementarity to 16S ribosomal RNA are important for the ability of this IRES to initiate translation in E. coli.

DDX60 selectively reduces translation off viral type II internal ribosome entry sites

Co‐opting host cell protein synthesis is a hallmark of many virus infections. In response, certain host defense proteins limit mRNA translation globally, albeit at the cost of the host cell's own protein synthesis. Here, we describe an interferon‐stimulated helicase, DDX60, that decreases translation from viral internal ribosome entry sites (IRESs). DDX60 acts selectively on type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not by other IRES types or by 5′ cap. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not that of poliovirus or bovine enterovirus 1 (BEV‐1; type I IRES). Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit viral replication. Finally, DDX60 selectively modulates the amount of translating ribosomes on viral and in vitro transcribed type II IRES mRNAs, but not 5′ capped mRNA. Our study identifies a novel facet in the repertoire of interferon‐stimulated effector genes, the selective downregulation of translation from viral type II IRES elements.

Adjuvant effect of IRES-based single-stranded RNA on melanoma immunotherapy

BackgroundAdjuvant therapies such as radiation therapy, chemotherapy, and immunotherapy are usually given after cancer surgery to improve the survival of cancer patients. However, despite advances in several adjuvant therapies, they are still limited in the prevention of recurrences.MethodsWe evaluated the immunological effects of RNA-based adjuvants in a murine melanoma model. Single-stranded RNA (ssRNA) were constructed based on the cricket paralysis virus (CrPV) internal ribosome entry site (IRES). Populations of immune cells in bone marrow cells and lymph node cells following immunization with CrPVIRES-ssRNA were determined using flow cytometry. Activated cytokine levels were measured using ELISA and ELISpot. The tumor protection efficacy of CrPVIRES-ssRNA was analyzed based on any reduction in tumor size or weight, and overall survival.ResultsCrPVIRES-ssRNA treatment stimulated antigen-presenting cells in the drain lymph nodes associated with activated antigen-specific dendritic cells. Next, we evaluated the expression of CD40, CD86, and XCR1, showing that immunization with CrPVIRES-ssRNA enhanced antigen presentation by CD8a+ conventional dendritic cell 1 (cDC1), as well as activated antigen-specific CD8 T cells. In addition, CrPVIRES-ssRNA treatment markedly increased the frequency of antigen-specific CD8 T cells and interferon-gamma (IFN-γ) producing cells, which promoted immune responses and reduced tumor burden in melanoma-bearing mice.ConclusionsThis study provides evidence that the CrPVIRES-ssRNA adjuvant has potential for use in therapeutic cancer vaccines. Moreover, CrPVIRES-ssRNA possesses protective effects on various cancer cell models.

Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES.

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.

Pharmacophore-Based Discovery of Viral RNA Conformational Modulators.

New RNA-binding small-molecule scaffolds are needed to unleash the pharmacological potential of RNA targets. Here we have applied a pharmacophore-based virtual screening approach, seldom used in the RNA recognition field, to identify novel conformational inhibitors of the hepatitis C virus internal ribosome entry site. The conformational effect of the screening hits was assessed with a fluorescence resonance energy transfer assay, and the affinity, specificity, and binding site of the ligands were determined using a combination of fluorescence intensity and NMR spectroscopy experiments. The results indicate that this strategy can be successfully applied to discover RNA conformational inhibitors bearing substantially less positive charge than the reference ligands. This methodology can potentially be accommodated to other RNA motifs of pharmacological interest, facilitating the discovery of novel RNA-targeted molecules.

Multiple Cis-acting Polypyrimidine Tract Elements Regulate a Cooperative Mechanism for Triticum Mosaic Virus Internal Ribosomal Entry Site Activity.

Diverse elements within the 5′ untranslated region of an mRNA can influence the translation efficiency at the main AUG codon. We previously identified a core picornaviral like Y16X11-AUG motif with 16-nt polypyrimidine CU tract separated by an 11-nt spacer sequence from the 13th AUG codon, which is recognized as the preferred initiation site within the Triticum mosaic virus (TriMV) internal ribosome entry site (IRES) element. The motif is proposed to function as an internal ribosomal landing site at the designated start codon. Here, we exposed the cooperative role of multiple CU-rich segments flanking the TriMV YX-AUG motif to reach and drive internal initiation of translation at the preferred start site. We propose that these auxiliary domains may enhance the ribosome capacity and their delivery at proximity of the correct initiation site. These polypyrimidine tracts can be modulated with a cryptic AUG in a position-dependent manner to replace the native YX-AUG motif, and thus uncovering a new layer of control of start codon selection. In line with these observations, mass spectrometry analysis of proteins directly interacting with translationally impaired TriMV IRES mutants that bear these motifs indicated an enrichment in 40S and 60S ribosomal related proteins, revealing a new function of polypyrimidine tracts to regulate IRES-driven translation. Accessibility of these RNA regions for in trans interaction was validated by SHAPE analysis of the entire TriMV leader sequence and supported by the ability of anti-sense oligonucleotides designed to block the CU tracts accessibility to impair IRES activity. This is the first evidence that defines the core modular domains required for ribosomal recruitment and start codon selection in a complex, multi-AUG viral 5′ UTR for translation in plants.