The impact of RNA structure on picornavirus IRES activity

Internal ribosome entry site (IRES) elements consist of cis-acting regions that recruit the translation machinery to an internal position in the mRNA. The biological relevance of RNA structure-mediated mechanisms involved in internal ribosome recruitment is now emerging from the structural and functional analysis of viral IRES elements. However, because IRES elements found in genetically distant mRNAs seem to be organized in different RNA structures, the definition of the structural requirements for IRES activity is challenging and demands multidisciplinary approaches. This review discusses the latest reports that establish a relationship between RNA structure and IRES function in picornavirus genomes, the first RNAs described to contain these specialized regulatory elements.     

A conserved RNA structure within the HCV IRES eIF3-binding site

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is recognized specifically by the small ribosomal subunit and eukaryotic initiation factor 3 (eIF3) before viral translation initiation. Using extensive mutagenesis and structure probing analysis, we show that the eIF3-binding domain of the HCV IRES contains an internal loop structure (loop IIIb) and an adjacent mismatched helix that are important for IRES-dependent initiation of translation. NMR studies reveal a unique three-dimensional structure for this internal loop that is conserved between viral isolates of varying primary sequence in this region. These data indicate that internal loop IIIb may be an attractive target for structure-based design of new antiviral agents.     

NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element

The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.     

Structural features of the Seneca Valley virus internal ribosome entry site (IRES) element: a picornavirus with a pestivirus-like IRES

The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the Flaviviridae. The SVV IRES has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates that the SVV IRES is more closely related to the CSFV IRES, including the presence of a bipartite IIId domain. Mutagenesis of the SVV IRES, coupled to functional assays, support the core elements of the IRES structure model, but surprisingly, deletion of the conserved IIId(2) domain had no effect on IRES activity, including 40S and eIF3 binding. This is the first example of a picornavirus IRES that is most closely related to the CSFV IRES and suggests the possibility of multiple, independent recombination events between the genomes of the Picornaviridae and Flaviviridae to give rise to similar IRES elements.     

Utilization of a mammalian cell-based RNA binding assay to characterize the RNA binding properties of picornavirus 3C proteinases.

Using an assay capable of detecting sequence-specific RNA/protein interactions in mammalian cells, we demonstrate that the poliovirus and rhinovirus 3C proteinases are able to bind structured target RNA sequences derived from their respective 5' noncoding regions in vivo. Specific RNA binding by poliovirus 3C was found to be dependent on the integrity of stem-loop d of the RNA cloverleaf structure located at the 5' end of poliovirus genomic RNA. In contrast, mutation of stem-loop b did not prevent this in vivo interaction. However, mutation of stem-loop b, which serves as the RNA binding site for a cellular co-factor important for efficient poliovirus replication, did significantly attenuate the efficiency of 3C RNA binding in vivo and 3CD RNA binding in vitro. This in vivo protein:RNA binding assay was also used to identify several residues in 3C that are critical for RNA binding, but dispensable for 3C proteinase activity. The mammalian cell-based RNA binding assay described in this study may have considerable potential utility in the future detection or analysis of in vivo RNA/protein interactions unrelated to the 3C/RNA interaction described here.     

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.     

NMR analysis of the Mg2+-binding properties of aequorin, a Ca2+-binding photoprotein

Aequorin, which is a calcium-sensitive photoprotein and a member of the EF-hand superfamily, binds to Mg2+ under physiological conditions, which modulates its light emission. The Mg2+ binding site and its stabilizing influence were examined by NMR spectroscopy. The binding of Mg2+ to aequorin prevented the molecule from aggregating and stabilized it in the monomeric form. To determine the structural differences between Mg2+-bound and free aequorin, we have performed backbone NMR assignments of aequorin in the Mg2+-free state. Mg2+ binding induces conformational changes that are localized in the EF-hand loops. The chemical shift difference data indicated that there are two Mg2+-binding sites, EF-hands I and III. The Mg2+ titration experiment revealed that EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and that only EF-hand III seems to be occupied by Mg2+ under physiological conditions.     

Translational efficiency of BVDV IRES and EMCV IRES for T7 RNA polymerase driven cytoplasmic expression in mammalian cell lines

Mammalian T7 polymerase-based cytoplasmic expression systems are common tool for molecular studies. The majority of these systems include the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). To carry out a cap-independent translation process, this type of IRES might require the expression of an extensive array of host factors, what is a disadvantage. Other IRESes might be less dependent on the host cell factors, but their biology is characterized to a lesser degree. Here, we compare the translational efficiencies of bovine viral diarrhea virus (BVDV) IRES with that of ECMV. Both IRESes were tested in reporter vectors containing the T7 promoter, an IRES of choice and the coding sequence of the enhanced green fluorescent protein (EGFP). To provide for the expression of T7 RNA polymerase, the corresponding gene was isolated from Escherichia coli and inserted into pCDNA3.1-Hygro(+). After co-transfection of the T7 RNA polymerase encoding vector with either of the two IRES-containing reporter vectors into T7 baby hamster kidney (T7-BHK), human embryonic kidney (HEK) 293T, chinese hamster ovary (CHO) and HeLa cells, the translational efficiency of the reporter construct was studied by fluorescence microscopy and flow cytometry. In T7-BHK, HEK 293T and HeLa cells the translational efficiency of BVDV IRES was two to three times higher than that of EMCV IRES. In CHO cells, BVDV IRES and EMCV IRES were equally efficient. An analysis of the secondary structure of respective mRNAs showed that their ΔG values were–544.00 and–469.40 kcal/mol for EMCV IRES and BVDV IRES harboring molecules, respectively. As EMCV IRES-containing mRNA is more stable, it is evident that other, still unidentified factors should be held responsible for the enhanced translational efficiency of BDVD IRES. Taken together, our results indicate the potential of BVDV IRES as a replacement for EMCV IRES, which is now commonly used for T7 polymerase driven cytoplasmic expression of genes of interest or virus cDNA rescue experiments.     

Structure of a conserved retroviral RNA packaging element by NMR spectroscopy and cryo-electron tomography

The 5′-untranslated regions of all gammaretroviruses contain a conserved “double-hairpin motif” (Ψ^CD) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming “kissing” interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([Ψ^CD]₂, 132 nt, 42.8 kDa) using a ²H-edited NMR-spectroscopy-based approach. This approach enabled the detection of ¹H-¹H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional ¹H-¹H correlated and ¹H-¹³C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D′ and SLC′ to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C′ to SL-D′) that gives rise to an elongated overall shape (ca 95 Å × 45 Å ×  25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [Ψ^CD]₂ simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [Ψ^CD]₂ functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.     

Unusual loop-sequence flexibility of the proximal RNA replication element in EMCV

Picornaviruses contain stable RNA structures at the 5' and 3' ends of the RNA genome, OriL and OriR involved in viral RNA replication. The OriL RNA element found at the 5' end of the enterovirus genome folds into a cloverleaf-like configuration. In vivo SELEX experiments revealed that functioning of the poliovirus cloverleaf depends on a specific structure in this RNA element. Little is known about the OriL of cardioviruses. Here, we investigated structural aspects and requirements of the apical loop of proximal stem-loop SL-A of mengovirus, a strain of EMCV. Using NMR spectroscopy, we showed that the mengovirus SL-A apical loop consists of an octaloop. In vivo SELEX experiments demonstrated that a large number of random sequences are tolerated in the apical octaloop that support virus replication. Mutants in which the SL-A loop size and the length of the upper part of the stem were varied showed that both stem-length and stability of the octaloop are important determinants for viral RNA replication and virus reproduction. Together, these data show that stem-loop A plays an important role in virus replication. The high degree of sequence flexibility and the lack of selective pressure on the octaloop argue against a role in sequence specific RNA-protein or RNA-RNA interactions in which octaloop nucleotides are involved.     

Measurement of the specific and non-specific binding energies of Mg2+ to RNA

Determining the non-specific and specific electrostatic contributions of magnesium binding to RNA is a challenging problem. We introduce a single-molecule method based on measuring the folding energy of a native RNA in magnesium and at its equivalent sodium concentration. The latter is defined so that the folding energy in sodium equals the non-specific electrostatic contribution in magnesium. The sodium equivalent can be estimated according to the empirical 100/1 rule (1 M NaCl is equivalent to 10 mM MgCl₂), which is a good approximation for most RNAs. The method is applied to an RNA three-way junction (3WJ) that contains specific Mg²⁺ binding sites and misfolds into a double hairpin structure without binding sites. We mechanically pull the RNA with optical tweezers and use fluctuation theorems to determine the folding energies of the native and misfolded structures in magnesium (10 mM MgCl₂) and at the equivalent sodium condition (1 M NaCl). While the free energies of the misfolded structure are equal in magnesium and sodium, they are not for the native structure, the difference being due to the specific binding energy of magnesium to the 3WJ, which equals $\text{Δ}G?$ 10 kcal/mol. Besides stabilizing the 3WJ, Mg²⁺ also kinetically rescues it from the misfolded structure over timescales of tens of seconds in a force-dependent manner. The method should generally be applicable to determine the specific binding energies of divalent cations to other tertiary RNAs.     

The mechanism of translation initiation on Aichivirus RNA mediated by a novel type of picornavirus IRES

Picornavirus mRNAs contain IRESs that sustain their translation during infection, when host protein synthesis is shut off. The major classes of picornavirus IRESs (Types 1 and 2) have distinct structures and sequences, but initiation on both is determined by their specific interaction with eIF4G. We report here that Aichivirus (AV), a member of the Kobuvirus genus of Picornaviridae, contains an IRES that differs structurally from Type 1 and Type 2 IRESs. Its function similarly involves interaction with eIF4G, but its eIF4G-interacting domain is structurally distinct, although it contains an apical eIF4G-interacting motif similar to that in Type 2 IRESs. Like Type 1 and Type 2 IRESs, AV IRES function is enhanced by pyrimidine tract-binding protein (PTB), but the pattern of PTB's interaction with each of these IRESs is distinct. Unlike all known IRESs, the AV IRES is absolutely dependent on DHX29, a requirement imposed by sequestration of its initiation codon in a stable hairpin.     

The structure of a rigorously conserved RNA element within the SARS virus genome

We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-Å resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3′ end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90° kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold.     

NMR analysis of bovine tRNATrp: conformation dependence of Mg2+ binding

NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).     

Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements

The translation of picornavirus genomic RNAs occurs by a cap-independent mechanism that requires the formation of specific ribonucleoprotein complexes involving host cell factors and highly structured regions of picornavirus 5' noncoding regions known as internal ribosome entry sites (IRES). Although a number of cellular proteins have been shown to be involved in picornavirus RNA translation, the precise role of these factors in picornavirus internal ribosome entry is not understood. In this report, we provide evidence for the existence of distinct mechanisms for the internal initiation of translation between type I and type II picornavirus IRES elements. In vitro translation reactions were conducted in HeLa cell cytoplasmic translation extracts that were depleted of the cellular protein, poly(rC) binding protein 2 (PCBP2). Upon depletion of PCBP2, these extracts possessed a significantly diminished capacity to translate reporter RNAs containing the type I IRES elements of poliovirus, coxsackievirus, or human rhinovirus linked to luciferase; however, the addition of recombinant PCBP2 could reconstitute translation. Furthermore, RNA electrophoretic mobility-shift analysis demonstrated specific interactions between PCBP2 and both type I and type II picornavirus IRES elements; however, the translation of reporter RNAs containing the type II IRES elements of encephalomyocarditis virus and foot-and-mouth disease virus was not PCBP2 dependent. These data demonstrate that PCBP2 is essential for the internal initiation of translation on picornavirus type I IRES elements but is dispensable for translation directed by the structurally distinct type II elements.     

Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods

RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na(+)]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg(2+) salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs.     

Analysis of the EIAV Rev-responsive element (RRE) reveals a conserved RNA motif required for high affinity Rev binding in both HIV-1 and EIAV

A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.