Replication of alphaviruses requires a pseudoknot that involves the poly(A) tail.

Alphaviruses, such as the Sindbis virus and the Chikungunya virus, are RNA viruses with a positive sense single-stranded RNA genome that infect various vertebrates, including humans. A conserved sequence element (CSE) of ∼19 nt in the 3' noncoding region is important for replication. Despite extensive mutational analysis of the CSE, no comprehensive model of this element exists to date. Here, it is shown that the CSE can form an RNA pseudoknot with part of the poly(A) tail and is similar to the human telomerase pseudoknot with which it shares 17 nt. Mutants that alter the stability of the pseudoknot were investigated in the context of a replicon of the Sindbis virus and by native gel electrophoresis. These studies reveal that the pseudoknot is required for virus replication and is stabilized by UAU base triples. The new model is discussed in relation to previous data on Sindbis virus mutants and revertants lacking (part of) the CSE.

Investigating the correlation between Xrn1-resistant RNAs and frameshifter pseudoknots.

Xrn1-resistant RNA structures are multifunctional elements employed by an increasing number of RNA viruses. One of such elements is the coremin motif, discovered in plant virus RNAs, of which the structure has been hypothesized to form a yet unelucidated pseudoknot. Recently, the coremin motif was shown to be capable of stalling not only Xrn1, but scanning ribosomes as well. Following that observation, in this study we demonstrate that the coremin motif can promote -1 ribosomal frameshifting, similar to better-characterized viral frameshifting pseudoknots. Since this function was lost in concert with substitutions that were known to disturb Xrn1-resistance, we developed a frameshifting screen for finding novel Xrn1-resistant RNAs by randomizing parts of the coremin motif. This yielded new insights into the coremin motif structure, as Xrn1-resistant variations were identified that more clearly indicate a pseudoknot interaction. In addition, we show that the Xrn1-resistant RNA of Zika virus promotes frameshifting as well, while known -1 programmed ribosomal frameshifting pseudoknots do not stall Xrn1, suggesting that promoting frameshifting is a universal characteristic of Xrn1-resistant RNAs, but that Xrn1-resistance requires more than just a frameshifting pseudoknot.

A database of flavivirus RNA structures with a search algorithm for pseudoknots and triple base interactions.

MOTIVATION: The Flavivirus genus includes several important pathogens, such as Zika, dengue and yellow fever virus. Flavivirus RNA genomes contain a number of functionally important structures in their 3' untranslated regions (3'UTRs). Due to the diversity of sequences and topologies of these structures, their identification is often difficult. In contrast, predictions of such structures are important for understanding of flavivirus replication cycles and development of antiviral strategies. RESULTS: We have developed an algorithm for structured pattern search in RNA sequences, including secondary structures, pseudoknots and triple base interactions. Using the data on known conserved flavivirus 3'UTR structures, we constructed structural descriptors which covered the diversity of patterns in these motifs. The descriptors and the search algorithm were used for the construction of a database of flavivirus 3'UTR structures. Validating this approach, we identified a number of domains matching a general pattern of exoribonuclease Xrn1-resistant RNAs in the growing group of insect-specific flaviviruses. AVAILABILITY AND IMPLEMENTATION: The Leiden Flavivirus RNA Structure Database is available at https://rna.liacs.nl. The search algorithm is available at https://github.com/LeidenRNA/SRHS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

All genera of Flaviviridae host a conserved Xrn1-resistant RNA motif.

After infection by flaviviruses like Zika and West Nile virus, eukaryotic hosts employ the well-conserved endoribonuclease Xrn1 to degrade the viral genomic RNA. Within the 3' untranslated regions, this enzyme encounters intricate Xrn1-resistant structures. This results in the accumulation of subgenomic flaviviral RNAs, an event that improves viral growth and aggravates viral pathogenicity. Xrn1-resistant RNAs have been established throughout the flaviviral genus, but not yet throughout the entire Flaviviridae family. In this work, we use previously determined characteristics of these structures to identify homologous sequences in many members of the genera pegivirus, hepacivirus and pestivirus. We used structural alignment and mutational analyses to establish that these sequences indeed represent Xrn1-resistant RNA and that they employ the general features of the flaviviral xrRNAs, consisting of a double pseudoknot formed by five base-paired regions stitched together by a crucial triple base interaction. Furthermore, we demonstrate that the pestivirus Bungowannah virus produces subgenomic RNA in vivo. Altogether, these results indicate that viruses make use of a universal Xrn1-resistant RNA throughout the Flaviviridae family.

Structural features of an Xrn1-resistant plant virus RNA.

Xrn1 is a major 5'-3' exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3' untranslated region of several beny- and cucumovirus RNAs harbors a so-called 'coremin' motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence to deduce putative structural features responsible for Xrn1 stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5' proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3' proximal hairpin loop sequence is variable. The sequence of the 10-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae, Potyviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn1-stalling motifs are more widespread among RNA viruses than previously realized.

G-quadruplexes within prion mRNA: the missing link in prion disease?

Cellular ribonucleic acid (RNA) plays a crucial role in the initial conversion of cellular prion protein PrP(C) to infectious PrP(Sc) or scrapie. The nature of this RNA remains elusive. Previously, RNA aptamers against PrP(C) have been isolated and found to form G-quadruplexes (G4s). PrP(C) binding to G4 RNAs destabilizes its structure and is thought to trigger its conversion to PrP(Sc). Here it is shown that PrP messenger RNA (mRNA) itself contains several G4 motifs, located in the octarepeat region. Investigation of the RNA structure in one of these repeats by circular dichroism, nuclear magnetic resonance and ultraviolet melting studies shows evidence of G4 formation. In vitro translation of full-length PrP mRNA, naturally harboring five consecutive G4 motifs, was specifically affected by G4-binding ligands, lending support to G4 formation in PrP mRNA. A possible role of PrP binding to its own mRNA and the role of anti-prion drugs, many of which are G4-binding ligands, in prion disease are discussed.

Stimulation of ribosomal frameshifting by RNA G-quadruplex structures.

Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads, so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the DNA level; however, exploration of the biological roles of G4s at the RNA level is just emerging. Here we show that G4 RNA when introduced within coding regions are capable of stimulating -1 ribosomal frameshifting (-1 FS) in vitro and in cultured cells. Systematic manipulation of the loop length between each G-tract revealed that the -1 FS efficiency positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in higher -1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon readthrough as well. These results suggest a potentially novel translational gene regulation mechanism mediated by G4 RNA.

Structural and thermodynamic signatures that define pseudotriloop RNA hairpins.

Pseudotriloop (PTL) structures in RNAs have been recognized as essential elements in RNA folding and recognition of proteins. PTL structures are derived from hexaloops by formation of a cross-loop base pair leaving a triloop and 3' bulged out residue. Despite their common presence and functional importance, insufficient structural and thermodynamic data are available that can be used to predict formation of PTLs from sequence alone. Using NMR spectroscopy and UV-melting data we established factors that contribute to the formation and stability of PTL structures derived from hepatitis B virus and human foamy virus. The NMR data show that, besides the cross-loop base pair, also a 3' pyrimidine bulge and a G-C loop-closing base pair are primary determinants of PTL formation. By changing the G-C closing base pair into C-G, the PTL switches into a hexaloop. Comparison of these rules with regular triloop hairpins and PTLs from other sources is discussed as well as the conservation of a PTL in human foamy virus and other spumaretroviruses.

Tospovirus ambisense genomic RNA segments use almost complete repertoire of stable tetraloops in the intergenic region.

The intergenic regions of the ambisense RNA segments of viruses from the Tospovirus genus form large extended RNA structures that regulate virus replication. Using comparative structure analysis, we show the presence of conserved alternative conformations at the apical parts of these structures. In one conformation, a branched Y-shape, the 5'-proximal hairpin arms are mostly capped by exceptionally stable tetraloop motifs. The tetraloop hairpins are folded in both virus and virus-complementary sense RNAs, and different tetraloops can functionally replace each other. Folding simulations show that the branched Y-shape structures can undergo a conformational transition to alternative extended rod-like conformations. Functional importance of both alternatives is supported by nucleotide covariations. The balanced equilibrium between alternative structures is evidenced by native gel electrophoresis of mutant RNA transcripts with shifted equilibria. The tetraloops play a role in the stability and dynamics of structures but may also be recognized by proteins involved in translation and/or replication.

The subgenomic promoter of brome mosaic virus folds into a stem-loop structure capped by a pseudo-triloop that is structurally similar to the triloop of the genomic promoter.

In brome mosaic virus, both the replication of the genomic (+)-RNA strands and the transcription of the subgenomic RNA are carried out by the viral replicase. The production of (-)-RNA strands is dependent on the formation of an AUA triloop in the stem-loop C (SLC) hairpin in the 3'-untranslated region of the (+)-RNA strands. Two alternate hypotheses have been put forward for the mechanism of subgenomic RNA transcription. One posits that transcription commences by recognition of at least four key nucleotides in the subgenomic promoter by the replicase. The other posits that subgenomic transcription starts by binding of the replicase to a hairpin formed by the subgenomic promoter that resembles the minus strand promoter hairpin SLC. In this study, we have determined the three-dimensional structure of the subgenomic promoter hairpin using NMR spectroscopy. The data show that the hairpin is stable at 30°C and that it forms a pseudo-triloop structure with a transloop base pair and a nucleotide completely excluded from the helix. The transloop base pair is capped by an AUA triloop that possesses an extremely well packed structure very similar to that of the AUA triloop of SLC, including the formation of a so-called clamped-adenine motif. The similarities of the NMR structures of the hairpins required for genomic RNA and subgenomic RNA synthesis show that the replicase recognizes structure rather than sequence-specific motifs in both promoters.

A recombinant RNA bacteriophage system to identify functionally important nucleotides in a self-cleaving ribozyme.

BACKGROUND: RNA bacteriophages like Qbeta and MS2 are well known for their high mutation rate, short infection cycle and strong selection against foreign inserts. The hammerhead ribozyme (HHRz) is a small self-cleaving RNA molecule whose active residues have previously been identified by mutational analysis of each individual base. Here the functionally important bases of HHRz were determined in a single screening experiment by inserting the HHRz into the genome of MS2. FINDINGS: The minimal HHRz of satellite Tobacco ringspot virus was cloned into the genome of RNA bacteriophage MS2. Sequence analysis of the surviving phages revealed that the majority had acquired single base-substitutions that apparently inactivated the HHRz. The positions of these substitutions exactly matched that of the previously determined core residues of the HHRz. CONCLUSIONS: Natural selection against a ribozyme in the genome of MS2 can be used to quickly identify nucleotides required for self-cleavage.

RNA structural constraints in the evolution of the influenza A virus genome NP segment.

Conserved RNA secondary structures were predicted in the nucleoprotein (NP) segment of the influenza A virus genome using comparative sequence and structure analysis. A number of structural elements exhibiting nucleotide covariations were identified over the whole segment length, including protein-coding regions. Calculations of mutual information values at the paired nucleotide positions demonstrate that these structures impose considerable constraints on the virus genome evolution. Functional importance of a pseudoknot structure, predicted in the NP packaging signal region, was confirmed by plaque assays of the mutant viruses with disrupted structure and those with restored folding using compensatory substitutions. Possible functions of the conserved RNA folding patterns in the influenza A virus genome are discussed.

Megalencephalic leukoencephalopathy with subcortical cysts: a variant update and review of the literature.

The leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterized by infantile-onset macrocephaly and chronic edema of the brain white matter. With delayed onset, patients typically experience motor problems, epilepsy and slow cognitive decline. No treatment is available. Classic MLC is caused by bi-allelic recessive pathogenic variants in MLC1 or GLIALCAM (also called HEPACAM). Heterozygous dominant pathogenic variants in GLIALCAM lead to remitting MLC, where patients show a similar phenotype in early life, followed by normalization of white matter edema and no clinical regression. Rare patients with heterozygous dominant variants in GPRC5B and classic MLC were recently described. In addition, two siblings with bi-allelic recessive variants in AQP4 and remitting MLC have been identified. The last systematic overview of variants linked to MLC dates back to 2006. We provide an updated overview of published and novel variants. We report on genetic variants from 508 patients with MLC as confirmed by MRI diagnosis (258 from our database and 250 extracted from 64 published reports). We describe 151 unique MLC1 variants, 29 GLIALCAM variants, 2 GPRC5B variants and 1 AQP4 variant observed in these MLC patients. We include experiments confirming pathogenicity for some variants, discuss particularly notable variants, and provide an overview of recent scientific and clinical insight in the pathophysiology of MLC.

Stem-loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting.

-1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base-base and base-sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in -1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.

Xrn1-resistant RNA motifs are disseminated throughout the RNA virome and are able to block scanning ribosomes.

RNAs that are able to prevent degradation by the 5'-3' exoribonuclease Xrn1 have emerged as crucial structures during infection by an increasing number of RNA viruses. Several plant viruses employ the so-called coremin motif, an Xrn1-resistant RNA that is usually located in 3' untranslated regions. Investigation of its structural and sequence requirements has led to its identification in plant virus families beyond those in which the coremin motif was initially discovered. In this study, we identified coremin-like motifs that deviate from the original in the number of nucleotides present in the loop region of the 5' proximal hairpin. They are present in a number of viral families that previously did not have an Xrn1-resistant RNA identified yet, including the double-stranded RNA virus families Hypoviridae and Chrysoviridae. Through systematic mutational analysis, we demonstrated that a coremin motif carrying a 6-nucleotide loop in the 5' proximal hairpin generally requires a YGNNAD consensus for stalling Xrn1, similar to the previously determined YGAD consensus required for Xrn1 resistance of the original coremin motif. Furthermore, we determined the minimal requirements for the 3' proximal hairpin. Since some putative coremin motifs were found in intergenic regions or coding sequences, we demonstrated their capacity for inhibiting translation through an in vitro ribosomal scanning inhibition assay. Consequently, this study provides a further expansion on the number of viral families with known Xrn1-resistant elements, while adding a novel, potentially regulatory function for this structure.

Single-walled carbon nanotubes as scaffolds to concentrate DNA for the study of DNA-protein interactions.

Genomic DNA in bacteria exists in a condensed state, which exhibits different biochemical and biophysical properties from a dilute solution. DNA was concentrated on streptavidin-covered single-walled carbon nanotubes (Strep-SWNTs) through biotin-streptavidin interactions. We reasoned that confining DNA within a defined space through mechanical constraints, rather than by manipulating buffer conditions, would more closely resemble physiological conditions. By ensuring a high streptavidin loading on SWNTs of about 1 streptavidin tetramer per 4 nm of SWNT, we were able to achieve dense DNA binding. DNA is bound to Strep-SWNTs at a tunable density and up to as high as 0.5 mg mL(-1) in solution and 29 mg mL(-1) on a 2D surface. This platform allows us to observe the aggregation behavior of DNA at high concentrations and the counteracting effects of HU protein (a histone-like protein from Escherichia coli strain U93) on the DNA aggregates. This provides an in vitro model for studying DNA-DNA and DNA-protein interactions at a high DNA concentration.

Stimulation of ribosomal frameshifting by antisense LNA.

Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12-18 nt. Antisense oligonucleotides bearing locked nucleic acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.

Functional analysis of the SRV-1 RNA frameshifting pseudoknot.

Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109-1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for -1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in -1 frameshifting.

Role of an RNA pseudoknot involving the polyA tail in replication of Pepino mosaic potexvirus and related plant viruses.

Pepino mosaic virus (PepMV) is a potexvirus of the family Alphaflexiviridae within the order of Tymovirales that threatens tomato production worldwide. PepMV possesses a positive-strand RNA genome with a 5'-methylguanosine cap and a 3'-polyA tail. Previously, using partially-purified viral RNA polymerase important secondary structures within the 3'-untranslated region (UTR) of PepMV RNA were identified. Here we show that an RNA pseudoknot can be formed in the 3'-UTR that includes part of the polyA tail. Using protoplasts, we demonstrate that the pseudoknot is required for replication of PepMV RNA. Mutational analysis and native gel electrophoresis further show that the pseudoknot is stabilized by UAU base triples, as is the human telomerase RNA pseudoknot. The presence of a pseudoknot in several other members of the Alpha- and Betaflexiviridae is supported by covariance analysis and native gel electrophoresis of other potexvirus, capillovirus and trichovirus RNAs. The ubiquitous presence of the pseudoknot in viruses of the Betaflexiviridae, suggests that the pseudoknot is a typical trait of the Betaflexiviridae that may have been adopted by many potexviruses during evolution.

In vitro and in vivo studies of the RNA conformational switch in Alfalfa mosaic virus.

The 3' termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3' untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3' UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3' UTR of AMV RNAs.