T = 1 capsids, which is indifferent to sequences within the stem. The B3-containing virus-like particle has been crystallised and its structure was determined to 2.3 Å. A lower-resolution map encompassing density for the RNA has also been calculated. The presence of B3 results in increased ordering of the N-terminal helices located at the particle 3-fold axes, which extend by roughly one and a half turns to encompass residues 8–11, including R8 and K9. Under assembly conditions, STNV CP in the absence of RNA is monomeric and does not self-assemble. These facts suggest that a plausible model for assembly initiation is the specific RNA-induced stabilisation of a trimeric capsomere. The basic nature of the helical extension suggests that electrostatic repulsion between CPs prevents assembly in the absence of RNA and that this barrier is overcome by correct placement of appropriately orientated helical RNA stems. Such a mechanism would be consistent with the data shown here for assembly with longer RNA fragments, including an STNV genome. The results are discussed in light of a first stage of assembly involving compaction of the genomic RNA driven by multiple RNA packaging signal–CP interactions.We have examined the roles of RNA–coat protein (CP) interactions in the assembly of satellite tobacco necrosis virus (STNV). The viral genomic RNA encodes only the CP, which comprises a β-barrel domain connected to a positively charged N-terminal extension. In the previous crystal structures of this system, the first 11 residues of the protein are disordered. Using variants of an RNA aptamer sequence isolated against the CP, B3, we have studied the sequence specificity of RNA-induced assembly. B3 consists of a stem–loop presenting the tetra-loop sequence ACAA. There is a clear preference for RNAs encompassing this loop sequence, as measured by the yield of  ► Sequence specificity in satellite virus assembly. ► RNA-induced conformational change in a viral CP. ► RNA binding overcomes an electrostatic barrier to CP assembly. ► RNAs containing multiple preferred stem–loops assemble more efficiently. ► Evidence supports the existence of packaging signals in single-stranded RNA genomes. A great deal is known about the virion structure ,We have begun to study other examples of this class of virus to see which features of the assembly mechanism in the RNA phages are evolutionarily conserved and which are not.tructure  and it wEscherichiacoli. Expression results in assembly of T = 1 particles of the CP that are indistinguishable in the protein shell from the wild-type virion and that have been shown to encapsidate the mRNA.in vitro by alterations in calcium ion concentration and pH. This in vitro reassembly protocol has allowed us to show that STNV CP collapses the conformation of its cognate genomic RNA, implying its assembly in vivo has similar features to the mechanism described above for MS2.in vitro reassembly to probe the specificity of the CP interaction with genomic RNA and the roles of the CP arms in that process. We showed previously that CPs with N-terminal truncations of as few as 12 residues resulted in CPs that could not assemble.in vitro, we did not establish whether this effect was sequence/structure specific, or whether the multiple copies of this motif within genomes are functionally significant.To investigate the roles of the N-terminal arms in assembly, and the molecular details of virion self-assembly, we used a recombinant version of the STNV-1 CP expressed from a synthetic gene in To examine whether capsid assembly is sensitive to the preferred loop sequence motif derived by SELEX (AXXA), we prepared two 30-mer oligonucleotides encompassing variations on this motif . These cT = 1 VLPs occurred in this concentration range (T = 1 capsid in each reaction was used as a measure of assembly efficiency. This was estimated from the area of the peaks in the sedimentation coefficient c(S) versus (S) plots (The efficiency with which each of these fragments promotes reassembly of the recombinant STNV-1 CP was then assessed using a standardised reassembly protocol.on range . Higher S) plots . The disc(S) versus (S) plots. Under the same buffer conditions, the recombinant STNV VLP has a sedimentation coefficient of ~ 42 S. B3 is clearly more efficient at triggering capsid assembly than the oligonucleotide lacking the AXXA loop motif  and incomplete shells with the dimensions compatible with a T = 1 architecture (28 S)  and 60 copies of the RNA would likely exceed the capacity of the particle. This might explain the observation that B3 is less efficient at promoting assembly than the short RNAs, perhaps because its flanking sequences occlude stem–loop binding sites. The B3 reassembled VLP (B3 VLP), but not the B3 short or TR equivalents, formed crystals that diffracted to 2.3 Å resolution .T = 1 capsid.The structures of the CP shell and subunits are very similar to those of previously published structures. The CP has a backbone r.m.s.d. of 0.138 Å compared to the virion [Protein Data Bank (PDB) entry There is strong electron density for the B3 RNA that encompasses the smaller region of RNA density observed in the mRNA VLP. There is also significant additional density along the particle 2-fold axes adjacent to the tips of the N-terminal helices b. This min vitro can be triggered by a short RNA stem–loop, in vivo the virus packages a 1239-nt genome. We showed previously that this sequence for STNV-1 potentially contains up to 30 copies of stem–loops having the preferred (AXXA) motif, and there are similar numbers of these motifs in the other known viral strains.E. coli is only 590 nt long. It lacks eight copies of this preferred binding site with respect to the genome,Although capsid assembly T = 1 capsid formation are strikingly different for these two RNAs (T = 1 capsid (42 S) with additional peaks at 60 S and 30 S. In contrast, under the same conditions, STNV-C RNA forms a majority of T = 1 capsids with a slower sedimenting shoulder, which may represent incomplete particles. Only a small fraction of input material sediments faster than 42 S. These results suggest that assembly is cooperative in the presence of longer RNAs, since the CP concentration is sub-stoichiometric with respect to formation of a capsid around every RNA. They also suggest that assembly with the mRNA transcript is less faithful than with the genome. At higher concentrations, the genome also shows evidence of a faster sedimenting peak, which becomes more dominant at the highest CP concentration. The TEMs associated with these reactions show evidence of closely associated particles, suggestive of the formation of fused double capsids. Subsequent treatment with RNase confirms this, the double shells falling apart post-treatment, suggesting that they are due to multiple capsid assembly initiation events on the same RNA .The efficiency and fidelity of two RNAs . At the T = 1 capsid assembly than a mutated shorter sub-fragment. This makes sense if the multiple preferred binding sitesIt appears that a cognate genome is more faithful in et al., unpublished results). They are variations of the TR stem–loop oligonucleotides used here to trigger STNV assembly. The discrimination between genomes in assembly assays at single-molecule concentrations shown by their respective CPs re-emphasises the importance of both the existence of such sequences and their three-dimensional locations. This is especially true in light of the TR data described above. Many of the MS2 PSs contain a loop with an -AXXA- motif similar to TR, which triggers capsid assembly with STNV CP at the concentrations used here . We compared assembly efficiency and fidelity with iRNA (927 nt), which is ~ 25% shorter than the cognate STNV genome, and 5′ RNA (2469 nt), which is roughly twice as large as the natural genome. Both these RNAs encompass the TR site. Remarkably, both RNAs support assembly of VLPs, some of which sediment at 42 S, consistent with the appearance of T = 1 particles by TEM, but only at CP concentrations several times higher than those that trigger assembly with the mRNA and STNV-C RNAs. For the shorter iRNA, the highest CP concentration yields mostly T = 1 capsids  already identified in the mRNA VLP structure.In the presence of an RNA encompassing the B3 sequence, STNV CP assembles faithfully into a T = 1 shell? The answer may be provided by the structures of the B3 and mRNA VLPs. Positively charged side chains from one subunit  are positioned so that they contact the phosphodiester backbone of the base-paired section of the stem–loop around the 3-fold axis of the neighbouring trimer. Thus, the RNA stem–loops bridge between trimers, as Given the increased ordering of the protein N-termini clustered around 3-fold axes in the presence of preferred RNA fragment sequences, the simplest assumption is that the primary capsomere is a trimer. Previously, we analysed the state of aggregation of RNA-free STNV CP by svAUC. The CP is a monomer with only traces of two higher-order species that could be trimer or pentamer.in vitro. The assembly model for STNV described above is simply a variation on that theme. However, like the vast majority of assembly assays in vitro, the experiments reported here were carried out at relatively high concentrations of CP. In vivo, the CP concentration would steadily increase from zero during expression of viral genes. Under those circumstances, the protein would either begin to package genomic RNA immediately or remain as an unbound pool potentially interacting with other cellular RNAs and leading to inappropriate encapsidation.in vitro assays. The outcome of in vivo assembly reactions is usually a high yield of virions with the correct and complete protein architecture in which the cognate genome has been enclosed. How is such fidelity and efficiency achieved at low protein concentrations and in the presence of potential competitor RNAs?The dominant paradigm for the assembly of ssRNA viruses is based on the idea that charge neutralisation between the genome and positively charged domains or faces of the CP subunits drive assembly.in vitro assembly of both STNV and MS2 RNAs. At smFCS concentrations of CP (100 nM versus the 10 μM used here), altered behaviour is observed. Both STNV and MS2 genomic RNAs show evidence for conformational ensembles in equilibrium in the absence of RNA. Most of these are too large to fit into the confined space of the virion. CP binding brings about a rapid conformational collapse so that it is small enough to fit. This collapse is the result of multiple CP–RNA and CP–CP interactions. The complex post-collapse is a partially formed protein shell of the correct size and symmetry, which then recruits additional CPs to complete the capsid. Non-cognate and non-viral RNAs do not result in collapse and trigger assembly. However, in contrast to the cognate interactions, these are very inefficient and produce significant numbers of misassembled structures. These data are not easily reconciled with a purely electrostatic assembly mechanism driven by the CP. They are however consistent with the idea that each viral RNA contains dispersed PSs, B3-like or TR-like for STNVet al., unpublished results). Modelling the effects of such signals on assembly efficiency suggests that they are a more successful route towards high yields . PS affinity has a large electrostatic component rationalising previous reports of the importance of charge neutralisation.Recently, we introduced single-molecule fluorescence correlation spectroscopy (smFCS) to monitor the conformation of viral RNAs during in vitro reactions.in vitro assembly has been seen before in more complex plant viruses such as TCV and cowpea chlorotic mottle virus.The results here demonstrate, at higher CP concentrations, the effects of PS recognition and the cooperativity that result from their appropriate placement within a genomic RNA. The level of discrimination between cognate and non-cognate interaction is, however, reduced under these conditions, leading to inappropriate recognition of TR and TR-like sequences in the MS2 RNAs. At smFCS concentrations, the cognate discrimination is very clear. These results explain the heterologous encapsidation widely reported for in vivo for TCV consistent with this viewIt is interesting to speculate whether the N-terminal regions in other ssRNA virus CPs, which are involved in specifying the CP quasi-conformers as well as interacting with their genomes,et al.A260:280 ratios were determined by UV spectrophotometry.Recombinant STNV VLPs were purified according to Lane N-benzoyl-protected adenosine, N-dimethylformamidinyl-protected guanosine and N-acetyl-protected cytosine; no protective group is required for uracil. The 2′ hydroxyl group was protected by t-butyldimethylsilyl groups and the 3′ was protected by 2-cyanoethyl--phosphoramidite (Link Technologies). Ammonia-saturated methanol was used to remove protecting groups and to cleave RNA from CPG resin at room temperature for 24 h. Methanol was removed under vacuum and the pellet was resuspended in anhydrous dimethyl sulfoxide. One volume of TEA.3HF was added and incubated at room temperature to remove t-butyldimethylsilyl from the 2′ hydroxyl groups. Each RNA was butan-1-ol precipitated and resuspended in DEPC-treated water (Severn Biotech), purified by ion-exchange HPLC and analysed by negative-mode electrospray ionisation mass spectrometry.Aptamer B3 sequence (5′-CCU UUU CAA GAC AUG CAA CAA UGC ACA CAG-3′), Aptamer B3 4U sequence (5′-CCU UUU CAA GAC AUG CAU UUU UGC ACA CAG-3′), B3 short sequence (5′-CGU GCA ACA AUG CAC G-3′) and MS2 TR sequence (5′-ACA UGA GGA UUA CCC AUG U-3′) were synthesised using In vitro transcription from each template was performed using a MEGAScript in vitro transcription kit (Ambion) using the manufacturer's guidelines. All RNAs were purified by phenol:chloroform extraction and isopropanol precipitation. RNAs were analysed by 1% (w/w) agarose formaldehyde denaturing gel electrophoresis.Plasmids pETT22b-STNV2, pH 7.5) overnight at 4 °C using 0.1- to 0.5-mL Slide-A-Lyzers .All RNAs were prepared initially at a concentration of 20 μg/mL and STNV CP was either omitted or added at defined molar ratios for RNAs ≤ 30 nt long, or the RNA concentrations adjusted to allow comparison of assembly at constant phosphodiester concentration, all in 500 μL final volumes. Each reassembly reaction was extensively dialysed in reassembly buffer (50 mM Hepes and 3 mM CaClc(S) Lamm equation model.Each sample (0.32 mL) was placed in a 1.2-cm pathlength two-sector meniscus-matching epon centrepiece cell constructed with sapphire windows. These samples were centrifuged at 15,000 rpm in an An50-Ti rotor in an Optima XL-1 analytical ultracentrifuge at 20 °C. Changes in absorbance at 260 nm of the solute were detected by absorbance optics, with a total of 100 scans being taken over approximately 12 h. Buffer densities and viscosities were calculated by Sednterp version 1.09.et al.T = 1 VLPs would sediment at approximately 40 S, depending also on the size of RNA to be packaged. The sedimentation coefficient of the T = 1 mRNA VLP under these conditions was ~ 42 S. After continuous c(S) distribution analysis, peaks were integrated using size distribution of macromolecules as described by Schuck.T = 1 VLP.svAUC is used as a technique to determine the assembly efficiency of STNV VLPs. Previous work by Unge Each sample was added to a UV-irradiated Carbon/Formvar 300-mesh grid (Agar Scientific) and stained using 2% (w/v) uranyl acetate. Each reassembly reaction was analysed with either a Phillips CM-10 or a FEI Spirit G2 microscope at either 52,000 × or 49,000 × magnification, respectively.Recombinant STNV VLPs (~ 12 mg) were disassembled and STNV CP was purified by ion-exchange chromatography using the methodology described above. The total amount of CP was determined by UV spectrophotometry and sample quality was analysed by SDS-PAGE. Each RNA aptamer  was added at a ratio of 5:1  before being extensively dialysed in reassembly buffer. Reassembled VLPs were then concentrated to 500 μL by Amicon Ultra® spin concentrators (Millipore) and purified by size-exclusion chromatography.2. Drops were set up using 3 μL of VLP (7 mg mL− 1) and 3 μL of reservoir solution. Diamond-shaped crystals appeared after 4 weeks of incubation at 25 °C and measured about 0.1 μm in their longest dimension.The VLPs were crystallised using the conditions previously established for recombinant STNV.Crystals were prepared for data collection by cryoprotection with glycerol before flash cooling in liquid nitrogen. Addition of glycerol was carried out in a stepwise fashion as follows: soaking for 60 s in 20% (v/v) glycerol, 80% (v/v) reservoir solution followed by a 20-s soak in 25% (v/v) glycerol, 75% (v/v) reservoir solution and finally a 1-s soak in 30% (v/v) glycerol, 70% (v/v) reservoir solution.Diffraction data were collected at Diamond Light Source on beamline I02 at a wavelength of 0.9795 Å to a maximum resolution of 2.29 Å. High-quality low-resolution data were required to enable examination of the less ordered internal structure of the VLP. Three sweeps of data were therefore recorded from the same region of the crystal with varied detector and beamstop positions with resolution ranges of 217–7 Å, 116–3.8 Å and 30–2.29 Å. One hundred eighty degrees of data were recorded for each sweep with an oscillation angle of 1° for the very low resolution data and 0.5° for the medium- and high-resolution sweeps. All three sweeps of data were processed together using the 3dii option of the xia2 expert data reduction system .491VTZ and 3RQV), and this structure was used to provide initial phases for map calculation. Rigid-body refinement of the entire capsid was carried out using REFMAC5.The data were isomorphous with recombinant STNV VLPA-weighted 2m∣Fo∣ − D∣Fc∣ and m∣Fo∣ − D∣Fc∣ averaged maps were calculated using the Uppsala Software Suite.Sixty-fold σ4BCU.The coordinates and structure factors for the protein capsid in the presence of the B3 aptamer RNA have been deposited with the PDB with accession number The following are the supplementary materials related to this article.


The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and as catalyst. The central catalyst in an RNA world organism would have been a ribozyme that catalyzed RNA polymerization to facilitate self-replication. An RNA polymerase ribozyme was developed previously in the lab but it is not efficient enough for self-replication. The factor that limits its polymerization efficiency is its weak sequence-independent binding of the primer/template substrate. Here we tested whether RNA polymerization could be improved by a cationic arginine cofactor, to improve the interaction with the substrate. In an RNA world, amino acid-nucleic acid conjugates could have facilitated the emergence of the translation apparatus and the transition to an RNP world. We chose the amino acid arginine for our study because this is the amino acid most adept to interact with RNA. An arginine cofactor was positioned at ten different sites on the ribozyme, using conjugates of arginine with short DNA or RNA oligonucleotides. However, polymerization efficiency was not increased in any of the ten positions. In five of the ten positions the arginine reduced or modulated polymerization efficiency, which gives insight into the substrate-binding site on the ribozyme. These results suggest that the existing polymerase ribozyme is not well suited to using an arginine cofactor. According to the RNA world hypothesis, an early stage of life used RNA both as genome and as catalyst M in the millimolar range These polymerase ribozymes have a length in the range of 200 nucleotides. Therefore, self-replication would require the polymerization of about 200 ribozyme-encoding nucleotides. However, the best existing polymerase ribozymes favor variants of a single, short template sequence with the length of less than 20 nucleotides. By concatenating multiple copies of this sequence it was possible to extend a primer by 95 nucleotides This positive charge can be supplied by metal ions or by cationic organic molecules. The polymerase ribozyme was originally selected in the presence of 60 mM magnesium ions A of 12.5 In contrast to metal ions the polymerase ribozyme did not encounter cationic organic molecules during its history. Therefore, a potential benefit from those molecules would have gone undiscovered. Specifically, the amino acid arginine carries several advantages over other cationic cofactors. Most importantly, the guanidinium group does not establish a hydration shell in aqueous solution. This helps the binding of negatively charged RNAs because it avoids the enthalpic cost of displacing a hydration shell How could arginine cofactors compete with the high concentration of magnesium ions that are required by the polymerase ribozyme? In addition to the absence of a hydration shell our experiments carry two designs to help arginine compete with the magnesium ions. First, we decreased the free magnesium ion concentration from 184 mM to 64 mM, which facilitates near-optimal activity, and further down to 24 mM, which allows weak but quantifiable polymerization to occur In an RNA world, amino acid - nucleid acid conjugates or peptide - nucleic acid conjugates could have served in the roles of cofactors and could have established the first steps in a translation system In this study, arginine was used as a positively charged cofactor for the polymerase ribozyme. An arginine - nucleic acid conjugate was positioned at ten different positions on the ribozyme located near the substrate-binding site. We tested whether the positively charged arginine could be used by the ribozyme to increase polymerization efficiency. However, the arginine did not improve polymerization in any of these ten positions, suggesting that single arginines are not sufficient to improve the existing polymerase ribozyme. This also suggests that it may be harder than previously thought to take the first step in the development of the translation apparatus, via amino acid - nucleic acid conjugates.We used arginine-RNA and arginine-DNA conjugates to position the arginine cofactor on the ribozyme. Specific sequences for the nucleic acid handle of the conjugate made it possible to base pair the conjugate to different positions on the ribozyme . This stThe arginine-nucleic acid conjugates were synthesized by carbodiimide peptide coupling chemistry. Fmoc-protected arginine was activated as NHS ester and reacted with amino modified DNA or RNA. The nucleic acid sequences of these conjugates were chosen to pair to one of two target sites on the polymerase ribozyme, thereby forming a 5′-terminal duplex, or the P2 duplex. The choice of these target sites was based on their vicinity to the catalytic site  and becaTen polymerase ribozymes have been developed to date The first site for attaching the arginine conjugates is a duplex that extends the 5′-terminus of the polymerase ribozyme To measure the effect of each positioned arginine on ribozyme function we quantified the polymerization efficiency with and without the arginine modification. Additionally, we measured the influence of an amino group, which was used to couple the arginine with the nucleic acid handle. The polymerization efficiency was measured as the average number of nucleotides added to each primer molecule. This readout is sensitive enough to allow the detection of single hydrogen bonds between the ribozyme and the primer/template substrate When the 5′-duplex had a length of 7 base pairs, the DNA derivatives showed lower polymerization efficiency than the RNA derivatives . This caWhen the length of the 5′-duplex was 11 base pairs the arginine and amino modification showed an inhibitory effect on polymerization . While tWhen the 5′-duplex had a length of 15-base pairs the RNA/RNA duplex slightly decreased polymerization, in the absence of a P2 oligo .To explain the inhibitory effect of the 3′-terminal RNA 2′-hydroxyl group at the 5′-duplex we hypothesized that the distal terminus of the RNA/RNA duplex entered the active site and interfered with binding of the primer/template. To test whether the inhibitory effect of the 3′-terminal 2′-hydroxyl group could be due to insertion into the catalytic site we monitored whether the 5′-duplex could be used as a primer/template duplex and extended by the polymerase ribozyme. To obtain a templating sequence the 5′-terminus of the polymerase ribozyme was elongated by four nucleotides. Polymerization assays showed that the radiolabeled RNAs at the 5′-duplex were indeed extended by the polymerase ribozyme, with a strong dependence on the length of the 5′-duplex . The sam5′-GGCGCC-3′The second site on the ribozyme that was used for the attachment of conjugates is the P2 duplex, which was formed by the polymerase ribozyme base pairing to the RNA hexanucleotide An arginine or amino modification at the 5′-terminus of the P2 oligo resulted in an initial burst of polymerization but caused a stalling of polymerization after five or six nucleotides were added . This mi2+ (64 mM free Mg2+), which may be too high for arginine to compete with, to bind to phosphodiester oxygens. Therefore, we reduced the concentration of Mg2+ to 40 mM (24 mM free Mg2+), which is high enough to obtain quantifiable data from polymerization but perhaps low enough to see a positive effect of arginine cofactors 2+) but that single arginines could have a role in nucleic acid interactions in an RNA world, at these lower Mg2+ concentrations. However, even at this low concentration we did not detect increased polymerization efficiencies due to arginine . All chemicals were Molecular Biology grade or higher.Ribozymes were synthesized by in vitro transcription from PCR products, using bacteriophage T7 RNA polymerase as described 2CO3 for 2 hours at 10°C. Deprotected products were neutralized, ethanol precipitated, and purified by 7 M urea 20% PAGE. The overall yield was 5–10% for RNA-arginine conjugates and 10–20% for DNA-arginine conjugates, as calculated based on the nucleic acid. The identity of the DNA conjugates was confirmed by MALDI Mass Spectroscopy, using hydroxypicolinic acid and ammonium citrate as matrix and a DNA 12mer and 21mer as internal standard for calibration. Expected mass for our test amino modified DNA: 5330.6; found: 5330.7. Expected mass for the corresponding arginine modified DNA with Fmoc protection: 5709.0; found: 5708.7. Expected mass for deprotected arginine modified DNA: 5486.8. Found: 5486.9. Additionally, the identity of both DNA and RNA conjugates was confirmed by the migration pattern of 5′-radiolabeled samples in denaturing PAGE.Arginine conjugates were synthesized from amino-modified RNAs or DNAs via NHS-activated arginine. The amino modifications contained a tether to the nucleic acid by six methylene groups (C6-linker). In dry DMF, 143 mM a-amino-Fmoc-arginine were reacted with 143 mM N-hydroxy succinimide (NHS) and 143 mM N,N′-dicyclohexyl carbodiimide (DCC) for 1 hour at 50°C. After cooling to room temperature the reaction mixture was mixed with the 2.5-fold volume of an aqueous solution with 100 mM amino-modified nucleic acid and 200 mM MES/NaOH pH 6.5. After incubation for 2 hours at room temperature the reaction mixture was dried in vacuum, then deprotected in an excess of 100 mM NaOH for 1 hour at 40°C. RNA coupling products were deprotected in an excess of 50 mM NaOH and 50 mM Na2), or reactions annotated to contain 40 mM MgCl2. Reaction times were 24 hours for reactions with P2 oligo and 3 hours for reactions without P2 oligo if not indicated otherwise. Reaction times for reactions with 40 mM MgCl2 were 22 hours. The reason for the different incubation time is that in the absence of the P2 oligo polymerization is fast during the first hours and then stalls, whereas in the presence of the P2 oligo polymerization is slower during the first hours but extends further 2EDTA at pH 8.4) and a template-complementary RNA added in 20-fold excess over the template. The mixtures were heat denatured (2 min/80°C) and cooled to room temperature at 0.1°C/sec before loading and separating on 7 M urea 0.5× TBE 20% PAGE.Ribozyme reactions were performed as described Autoradiographs of the PAGE separations were recorded by a PMI phosphorimager (Bio-Rad) and quantitated using the software Quantity One. Shifts higher than 11 nt above the primer were counted as full-length extension. The values for “average nucleotides per primer” were obtained by multiplying the fraction of intensity for each band  with the number of added nucleotides corresponding to that band. For quantifying the effect of 2′-deoxy substitutions, the method was described previously


Penicillium species are among the most common fungi present in the environment and are usually considered non-pathogenic to humans. However, in immunocompromised hosts they can be virulent pathogens and can cause death. Penicillium digitatum is a plant pathogen that commonly causes a postharvest fungal disease of citrus called green mould; it very rarely causes systemic mycosis in humans. Here, we report a case of fatal pneumonia due to P. digitatum infection, as confirmed by repeated examination of cultured sputum.Aspergillus flavus and P. digitatum was confirmed in the patient’s serum and also later pleural fluid by using Ouchterlony double immunodiffusion testing with A. flavus and P. digitatum antigens. The patient was treated over a period of months with itraconazole, micafungin, voriconazole, amphotericin B, and antibacterials. However, the cavity enlarged, the pleural effusion increased, and the patient began producing purulent sputum. He died from progressive renal failure. From sputum culture only one fungus was isolated repeatedly on potato-dextrose agar in large quantities. This fungus was confirmed to be P. digitatum by molecular identification. Partial sequences of the beta-tubulin gene were determined by using the primers Bt2a and Bt2b for PCR amplification and sequencing and underwent a BLAST search at the National Centre for Biotechnology Information, these results confirmed that the isolated fungus was P. digitatum.A cavity was found in the left upper lung on routine chest X-ray in a 78-year-old undernourished male who had been diagnosed at age 66 with bronchial asthma and pulmonary emphysema. No increased sputum production was present. The presence of antigen-specific precipitating antibodies to P. digitatum. Our patient had pulmonary emphysema and was elderly, and undernourished. These factors might have facilitated the infection. In his case, antimycotics were ineffective in treating the lung involvement. Although human infection with P. digitatum is considered rare, it appears that this organism can be very virulent and resistant to antimycotics.To our knowledge, this is the first report of pulmonary infection with  Penicillium species are among the most common fungi in the environment and are usually considered non-pathogenic to humans[Penicillium digitatum is a plant pathogen that commonly causes a postharvest fungal disease of citrus called green mould[P. digitatum infection, as confirmed by repeated examination of cultured sputum.o humans. Howevero humans. Penicilen mould; it veryen mould. Here, wAspergillus antigen, negative; β-D glucan, negative), but antigen-specific precipitating antibodies to Aspergillus flavus and P. digitatum were confirmed in the patient’s serum and pleural fluid by Ouchterlony double immunodiffusion testing[A. flavus or P. digitatum and no bacteria or tubercle bacilli were detected in cultures of sputum or bronchial lavage fluids. We diagnosed the patient with lung aspergilloma and treated with itraconazole (100 mg/day) for 3 months. However, the cavity became larger and thicker-walled  revealed a thin-walled cavity about 4 cm across and containing a fungus ball in the left upper lobe (S1 + 2); a CT scan taken 2 years previously had revealed only a small cavity indicative of emphysematous change . We confirmed the presence of antigen-specific precipitating antibodies to P digitatum by using antigen derived from the patient’s sputum culture fluid or extracted directly from his sputum  to the patient’s sputum and crushed the mixture with a Mini-Beadbeater . It was then incubated with 0.125 mol of NHPenicillium digitatum is the most devastating pathogen of rotten citrus fruit and is responsible for 90% of production losses during post-harvest handling[Penicillium digitatum is widely distributed in soils throughout the world. People are commonly exposed to the spores of this airborne pathogen every day. Upon Ouchterlony double immunodiffusion testing, the sera of five of 770 patients with pulmonary lung disease were confirmed to have low titres of antigen-specific precipitating antibodies to P. digitatum against antigen . However, none of these patients had antigen-specific precipitating antibodies to P. digitatum in tests of antigen extracted from patient’ sputum culture fluid or sputum. In humans there is cross-reactivity to Aspergillus and Penicillium: most sera from patients with precipitins against Penicillium have precipitins against Aspergillus[P. digitatum and A. flavus, but we did not find any P. digitatum or A. flavus in sputum cultures or in bronchoalveolar lavage fluids. This led to a delay in diagnosis.handling. PenicilPenicillium species can cause opportunistic infections[Penicillium species infections have been treated successfully with itraconazole[Penicillium species have died despite treatment with ketoconazole[Penicillium species, are associated with much higher mortality rates in patients with nosocomial infections or infections complicating organ failure[A. flavus or Aspergillus fumigatus are lower than those against Penicillium spp.[fections. Patientconazole, amphoteconazole,9, or flconazole. Howeverconazole, amphoteconazole, or itraconazole. Pulmona failure. The minium spp..P. digitatum. Our patient had pulmonary emphysema and was elderly, and undernourished. He had no known history of exposure to citrus fruit pathogens, but he was likely to have been immunocompromised. These factors might have facilitated the infection. In his case, antimycotics were ineffective in treating the lung involvement. Although human infections with P. digitatum are considered rare, it appears that this organism can be very virulent and resistant to antimycotics.To our knowledge, this is the first report of pulmonary infection with Written informed consent was obtained from the kin of the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal form.The authors declare that they have no competing interests.A. flavus and P. digitatum in the patient’s serum. MW and YK confirmed the fungus as P. digitatum by molecular identification. KT and MT identified the fungus as P. digitatum, TT, HY, HM and KA contributed to discussions about the patient. All authors read and approved the final manuscript.CO examined the patient and contributed to manuscript preparation. NT examined the patient, took part in discussions about the patient, and was involved in manuscript preparation and editing. AS performed the tests for antigen-specific precipitating antibodies to The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/13/16/prepub


Bamboo mosaic virus (BaMV) and the Potato virus X (PVX) are members of the genus Potexvirus and have a single-stranded positive-sense RNA genome. The 3′-untranslated region (UTR) of the BaMV RNA genome was mapped structurally into ABC (a cloverleaf-like), D (a stem-loop), and E (pseudoknot) domains. The BaMV replicase complex that was isolated from the infected plants was able to recognize the 3′ UTR of PVX RNA to initiate minus-strand RNA synthesis in vitro.in vivo, we constructed chimera mutants using a BaMV backbone containing the PVX 3′ UTR, which was inserted in or used to replace the various domains in the 3′ UTR of BaMV. None of the mutants, except for the mutant with the PVX 3′ UTR inserted upstream of the BaMV 3′ UTR, exhibited a detectable accumulation of viral RNA in Nicotiana benthamiana plants. The in vitro BaMV RdRp replication assay demonstrated that the RNA products were generated by the short RNA transcripts, which were derived from the chimera mutants to various extents. Furthermore, the Vmax/KM of the BaMV 3′ UTR (rABCDE) was approximately three fold higher than rABCP, rP, and rDE in minus-strand RNA synthesis. These mutants failed to accumulate viral products in protoplasts and plants, but were adequately replicated in vitro.To investigate whether the 3′ UTR of PVX RNA is also compatible with BaMV replicase in vivo.Among the various studied BaMV/PVX chimera mutants, the BaMV-S/PABCDE that contained non-interrupted BaMV 3′ UTR was the only mutant that exhibited a wild-type level of viral product accumulation in protoplasts and plants. These results indicate that the continuity of the domains in the 3′ UTR of BaMV RNA was not interrupted and the domains were not replaced with the 3′ UTR of PVX RNA  Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome that is 6366 nt in length and contains a 5′ m7GpppG cap and a 3′ poly (A) tail  and andPotatVarious BaMV/PVX chimera mutants of the structural elements in the 3′ UTR of the BaMV and PVX were created to examine the relationship between structure and function based on a full-length infectious cDNA clone (pBS2-8) of BaMV strain S (BaMV-S) . The entNo BaMV-related RNA or protein products were detected in the protoplasts that were transfected with full-length RNA transcripts, BaMV-S/P, -/ABCP, -/ABCDP, -/PDE, -/PE, or -/ABCPE . However, the two detectable mutants were the insertion mutants BaMV-S/PABCDE (96%) and BaMV-S/ABCDEP (6%) at 48 h post inoculation Figure . Similarin vivo. This result was supported by in vitro translation assay that all constructs could translate the replicase at similar levels  for various time periods and then subjected to Northern blot analysis . The ress Figure B. Therefin vitro BaMV RNA replication. The T7 promoter-containing PCR products were prepared to allow the generation of short transcripts, which encompass the 3′ UTR of the individual mutant constructs in vitro. The rABCDE transcript, which contained the 3′ UTR of BaMV RNA and 40 additional adenylate residues at the 3′ end, was used as the positive control. The RNA accumulation of the rABCDE transcript in the in vitro replication assay was arbitrarily assigned as 100%. The rPABCDE construct with PVX 3′ UTR located upstream of the rABCDE exhibited 110% RNA accumulation. Conversely, the rP that represents the heterologous PVX 3′ UTR displayed an approximate RNA accumulation of 60%. The addition of the homologous BaMV ABC domains to the 5′ end of the PVX 3′ UTR (rABCP) resulted in an increase in the wild-type level , the addition of the BaMV ABC domain in rABCP (KM=295 nM) enhanced the interaction between the 3′ UTR of PVX and the BaMV replicase complex. The mutant rDE transcript exhibited a stronger affinity (KM=209 nM) but a slower initiation rate that cause a lower Vmax/KM than rABCDE in the interaction with the BaMV replicase complex.Transcripts of rABCDE, rABCP, rDE, and rP were studied to focus on the ABC and DE domains. A Michaelis-Menten hyperbolic graph, which was based on the determined initial velocity and substrate concentration Figure , was traly Table . The simin vitro replication assay  in the 3′ UTR. These abnormal chimeric 3′ UTRs may cause the failure in post-transcription RNA maturation (such as polyadenylation) and result in immature progeny RNAs.The 3′ UTR of BaMV RNA was proven to be essential for viral RNA replication in protoplasts -16. The  Figures  and 4. T pathway . The cat pathway ,24. AlteThe long-distance interaction between the 3′ and 5′ end of RNA plays a vital role in the synthesis of positive-strand RNA, because the 3′ end sequence of BMV , the satin vitro BaMV RNA replication assay may be attributed to the redundant potexviral-conserved hexamer motifs that are found in D domain of BaMV RNA and the 3′ UTR of PVX RNA. An alternative hypothesis is that the alteration of the distance between the ABC domain and the poly(A) region, which was demonstrated to be the initiation site of minus-strand RNA replication  methionine  in the reaction. Incubations were performed at 30°C for 1 h and terminated by the addition of sample buffer. The translation products were analyzed by 10% SDS-polyacrylamide gel electrophoresis. Gels were dried, and analyzed by a phosphorimager (Fujifilm BAS 2500).For RNA stability assay, RNAs were incubated with wheat germ extract supplemented with all amino acids. At various time points of post incubation, viral RNAs were isolated and analyzed by Northern blotting.EcoRI-T7-6228 (5′GCGAATTCTAATACGACTCACTATAGGGCGTTGCATGATCG3′), T7-PVX (5′TAATACGACTCACTATAGGGTAACTACGTCTACAT3′), and the down stream primers, PVX40T (5′T40ATTTATATTATTCATAC3′) and 40TGG (5′T40GG3′), were used to synthesize the PCR products. The pPVX/UTR, pBaMV40A, pBaMV-S/ABCP, -/ABCDP, -/PDE, -/PE, -/PABCDE, -/ABCPE, and -/ABCDEP were employed as the template to generate rABCP, rABCDP, rPDE, rPE, rPABCDE, rABCPE, and rABCDEP, respectively. The RNA transcripts were gel-purified and quantified by spectrophotometry.The RNA transcripts were generated directly from PCR-generated DNA templates using T7 RNA polymerase . The spe2 at 30°C for 30 min and the treatment was stopped by adding 25 mM EGTA. The 50-μl BaMV RNA replication reaction contained 25 μl of the RdRp preparation, 10 mM DTT, 3 mM MgCl2, 2 mM of each ATP, CTP, and GTP, 2 μM UTP, 2 μM (α-32P) UTP , 4.8 mg/ml bentonite, and 1 μg of RNA template. Reaction was incubated at 30°C for 1 hr and stopped by phenol/chloroform extraction, followed by ethanol precipitation. The pellet was dissolved in 5 μl water and treated with RNase A (32 μg/ml) and T1 (1.6 μg/ml) at 30°C for 30 min in RNase protection buffer  to collect only double-stranded RNA. After incubation at 37°C for 30 min with 66 mg/ml proteinase K [M and Vmax were calculated by the double-reciprocal Lineweaver-Burk plot.For exogenous RNA template activity assay, the detergent-solubilized RdRp preparation  was treaeinase K , RNA wasThe authors declare that they have no competing interests.in vitro BaMV replication assay, and protoplast and plant inoculation. JWL contributed to this work with BaMV replicase complex purification. YHH supplied the antibody and partially subsidized the study. CHT designed the experiments, analyzed data, and contributed to finalizing the manuscript. All authors read and approved the final manuscript.IHC and CHC performed most of the plasmids construction,


These results provide an alternative folding for this region and a foundation for designing experiments to probe its functional role in the influenza life cycle.Influenza A virus is a segmented single-stranded (−)RNA virus that causes substantial annual morbidity and mortality. The transcriptome of influenza A is predicted to have extensive RNA secondary structure. The smallest genome segment, segment 8, encodes two proteins, NS1 and NEP, via alternative splicing. A conserved RNA domain in the intron of segment 8 may be important for regulating production of NS1. Two different multi-branch loop structures have been proposed for this region. A combination of  Influenza A is a segmented, negative-sense RNA virus that causes substantial morbidity and mortality on an annual basis. It is estimated that approximately 25,000 deaths result from influenza infections each year in the United States alone The smallest segment of influenza A, segment 8 (NS1/NEP), codes for two proteins via alternative splicing  and is pKnowledge of RNA secondary structure informs interrogation of function by facilitating design and interpretation of mutational studies. Once function is established, potential therapeutics against RNA structure can be designed. For example, databases that correlate small molecule binding patterns to RNA structural motifs can facilitate rapid identification of lead therapeutic compounds for a given RNA structure in vivo and in vitroin vitro mapping experiments with small molecules  and isoenergetic microarrays were performed on the consensus sequence as determined from all available unique influenza A strains. Small molecules probe the structure of folded RNA by modifying the base or sugar of unpaired, or weakly paired, nucleotides. Universal isoenergetic microarrays contain 861 different modified pentamer and hexamer oligonucleotides, thus providing complementarity to most sequences. The oligonucleotides are designed to bind with roughly equivalent stability to unpaired regions of a target RNA in order to minimize the effect of sequence dependence on binding. Folded RNA is hybridized to the array and binding to a probe reveals unpaired, or weakly paired, regions of the molecule. This technique is helpful for the design of potential therapeutics To better define the folding of this region, 2, 300 mM KCl showed that probes bind strongly to sites 114, 115, and 116 in the hairpin loop of structure C, whereas these sites are involved in base pairing in structure B. Interpretation of probes complementary to sites 127 (strong binding), 128 and 91(medium binding) is more complicated because they have potential alternative binding sites. However, sites 127 and128 are likely accessible because this region also shows chemical mapping reactivity , but provides enough viral protein production to elicit an adapted immune response, then these strains may make ideal candidates for live attenuated vaccine development. Because conserved, structured RNA domains are likely fundamental to a wide variety of influenza strains, they could be universal mutation sites for the generation of attenuated viruses.in vivo, such as protein binding, can also elicit structural changes. The function of this domain is unclear, but its proximity to the 5′ splice site makes it a possible intronic splicing enhancer/inhibitor In conclusion, on the basis of experimental results, the 81–148 region of segment 8 folds into a hairpin structure. However, it should be noted that these results do not preclude the possibility that natural mutations at positions 108, 120, and 123 could favor a reorganization of nucleotides at the top of the structure as is seen in structure A or B, or that this region exists in a structural equilibrium with multiple conformations inside the cell. Other factors present An alignment was created for nucleotides 81 to 148 of the NS1 gene (numbering does not include the UTR) by downloading all available segment 8 sequences  from the NCBI Influenza Virus Resource GAATTCTAATACGACTCACTATAGGGTGATGCCCCAT TCCTTGATCGGCTTCGCCGAGATCAGAAGTCCCTAAGAGGAAGAGGCAGCACTCAAGCTT-3′5′-. For chemical mapping studies, an additional nine consensus nucleotides, 5′-TTGGTCTGG-3′, were added at the 3′ end of the consensus sequence, before the HindIII site, to serve as a primer binding site for reverse transcription. The DNA oligonucleotides were annealed and ligated into a digested pUC17 vector. The resulting plasmid was cloned into DH5-alpha cells [Invitrogen]. Plasmid was isolated from cloned cells using a Qiagen mini-prep kit and the sequence was confirmed by sequencing. PCR primers were designed to amplify only the T7 RNA polymerase promoter and the consensus sequence and the resulting product was used for in vitro RNA production. RNA was synthesized in vitro using a T7 RNA polymerase kit (Ambion). RNA products were purified on a denaturing 8% PAGE gel and passively eluted overnight in 0.3 M sodium acetate. After ethanol precipitation, the RNA was re-suspended in 0.5 X TE  buffer.DNA oligonucleotide templates were obtained from IDT containing a T7 RNA polymerase site, the consensus sequence for the 81 to 148 nt region, and EcoRI and HindIII restriction sites for cloning (underlined below). The sense DNA construct had the following sequence: 2 was added to each sample for a final concentration of 10 mM and the RNA was folded at 37°C for 10 min. As assayed by native gel electrophoresis, this folding protocol produced a single conformation, which runs identically to the single conformation generated with a slow-cooling method For all chemical mapping experiments, 10 µL samples were prepared as outlined below. All final concentrations listed are for the final 10 µL mapping samples. Ten pmol of RNA was combined with buffer , 100 mM KCl) with a pH of 7.5 (DMS) or 8.0 (NMIA and CMCT). Samples were heated to 95°C for 2 min and then placed on ice for 2 min. MgCl32P labeled RNA was folded as described above. Final folding buffer was 300 mM (or 1 M) KCl, 10 mM Tris-HCl, pH 7.0, and 10 mM MgCl2. Microarrays require higher monovalent ion concentrations than used in the chemical mapping experiments to achieve consistent binding Microarray experiments were carried out as previously described Figure S1Co-varation analysis of structural models from .  Base pair counts in the tables are based on an alignment of 1017 unique sequences. Tables A, B, and C correspond to their respective structures in (TIF)Click here for additional data file.Figure S2Denaturing 8% PAGE analysis for NMIA, DMS and CMCT mapping. Dideoxy ladders are shown in the first four lanes. NMIA, DMS, and CMCT lanes are marked as being a mock treated control or the amount of reagent used. Strong sites of modification are labeled to the right of the gel. These labels correspond to the site of modification, which is one nucleotide longer than the adjacent cDNA fragment.(TIF)Click here for additional data file.Figure S3Denaturing 8% PAGE analysis for DMS and CMCT mapping. Dideoxy ladders are shown in the first four lanes. DMS and CMCT lanes are marked as being a mock treated control or the amount of reagent used. Strong sites of modification are labeled to the right of the gel. These labels correspond to the site of modification, which is one nucleotide longer than the adjacent cDNA fragment.(TIF)Click here for additional data file.Figure S4Hybridization results for consensus sequence nucleotides 81–148 of influenza A NS1 mRNA. Bar graph represents relative probe binding intensities after hybridization in the following conditions: 300 mM KCl, 10 mM MgCl2, 10 mM Tris-HCl, pH 7.0 (Blue) and 1 M KCl, 10 mM MgCl2, 10 mM Tris-HCl, pH 7.0 (Red) at 4°C. The average intensities are plotted on the y-axis and the center of probe binding to the target RNA is shown on the x-axis.(TIF)Click here for additional data file.


Induction of type-I interferons (IFNs), IFN-α/β, is crucial to innate immunity against RNA virus infection. Cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors, including RIG-I and melanoma differentiation-associated gene 5 (MDA5), are critical pathogen sensors for activation of type-I IFN expression in response to RNA virus infection. MDA5 is required for type-I IFN expression in mouse models in response to infection by picornaviruses, such as encephalomyocarditis virus (EMCV) and coxsackievirus B3. Enterovirus 71 (EV71) belongs to picornaviridae and contains positive-stranded RNA genome that is linked with VPg protein at the 5′ end. Although a recent study showed that EV71 3C protease could suppress RIG-I-mediated IFN-β response, the cytoplasmic RIG-I-like receptor that is directly involved in the recognition of EV71 RNA remains unclear. Using EV71-derived RNA as an agonist, we demonstrate that MDA5 is involved in EV71 RNA-mediated IRF3 activation and IFN-β transcription. Our data also show that overexpression of the MDA5 protein reverses the suppression of IRF3 activation caused by EV71 infection. These results indicate that MDA5 is an important factor for EV71 RNA-activated type-I IFN expression. Furthermore, we also show that EV71 infection enhances MDA5 degradation and that the degradation could be inhibited by a broad spectrum caspase inhibitor. EV71 proteases also cleave certain cellular factors. For example, 2Apro cleaves the eukaryotic translation initiation factor 4G (eIF4G), blocking cap-dependent translation in a manner similar to that of the 2Apro of the poliovirus pro. The cleavage of CstF-64 may result in the inhibition of mRNA maturation in host cell Enterovirus 71 (EV71) is the causative agent of hand-foot-and-mouth disease which is mild illness in children, but occasionally EV71 infection can result in severe neurological complications, such as encephalitis, aseptic meningitis, and poliomyelitis-like paralysis Type-I IFNs, IFN-α/β, play important roles to initiate cellular antiviral response by activating the genes that establish the target cells to an antiviral state Several studies have shown that RIG-I binds to double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) bearing a 5′ triphosphate pro interacts with RIG-I, subsequently disrupting RIG-I-IPS-1 complex that is required for RIG-I-dependent type-I IFN response Although Type-I IFNs provide the first line of defense to limit virus replication, many viruses have evolved strategies to inhibit the IFN activation pathways. In picornaviruses, several mechanisms have been identified that suppress RLR signaling, including the degradation of the cytoplasmic RLR signal molecules, such as MDA5 and IPS-1 In our current study, we used RNAs derived from EV71-infected cells as agonists to investigate the role of RLRs in the EV71 RNA-mediated induction of type-I IFN response. Based on evidence of IRF3 activation and IFN-β mRNA synthesis, we demonstrate that overexpression of MDA5 or RIG-I protein could enhance the activation of IFN-β transcription in response to EV71 RNA. We also show that knockdown of MDA5, but not RIG-I, expression reduces EV71 RNA-mediated IRF3 activation and IFN-β mRNA production, and that exogenous MDA5 reverses the suppression of IRF3 activation and IFN-β expression caused by EV71 infection. These findings suggest that MDA5 plays a crucial role in innate response to EV71 infection. Furthermore, we show that EV71 infection induces the cleavage of MDA5, but not RIG-I, which may contribute partly to the suppression of type-I IFN production during EV71 infection.HeLa , Vero , and RD  cell lines were cultured in Dulbecco's modified Eagle medium  with 10% fetal bovine serum  and 1% penicillin-streptomycin-L-glutamate (PSG) mixture . The EV71 strains, TW2231/98 , BrCr , and MP4  were amplified in Vero cells that were cultured in DMEM containing 2% FBS. The amplified viruses were titrated by plaque formation assay with RD cells. For virus infection, cells were seeded and cultured overnight in 6-well plates. After the indicated treatments below, viruses were added to the treated cells at the indicated multiplicity of infection (MOI). After 1 h adsorption, the infected cells were maintained in DMEM with 2% FBS and 1% PSG. To block EV71 induced apoptosis, a broad spectrum caspase inhibitor, Q-VD-OPH  was added to cells during virus infection at the final concentration 20 nM. MTT   assay was performed to measure the cell viability after the caspase inhibitor treatment. In brief, after the treatment, the cells were washed with PBS, and then incubated with 1 mg/ml MTT in serum-free medium at 37°C for 3 h. At the end of MTT incubation, the converted dye was dissolved with 0.04N HCl in isopropanol. The absorbance of converted dye was calculated by the absorbance at wavelength 570 nm with subtraction at 630 nm.5′-GUUAUAGUUCUUGUCAAUA & 5′-UAUUGACAAGAACUAUAAC, final concentration: 30 nM; RIG-I siRNA sequence: 5′-GACUAGUAAUGCUGGUGUA & 5′-UACACCAGCAUUACUAGUC, final concentration: 50 nM) was transfected into HeLa or RD cells using the Lipofectamine 2000 transfection reagent  for the indicated durations.N-terminal FLAG-tagged MDA5 and RIG-I expression vectors were kindly provided by Dr. Michael Gale Jr. g for 20 min to remove the cells. After measuring the virus titer in the supernatant, viral RNA was extracted using the Trizol reagent. RT-PCR was performed to confirm the existence of EV71 genomic RNA using primers specific to sequences in the 3C region of the EV71 genome. The indicated amounts of EV71 RNA or cellular RNA were transfected into HeLa cells using the Lipofectamine 2000 transfection reagent. After the indicated times and treatments, the cell lysates and RNA were harvested for further analysis.Total RNA from infected Vero cells was isolated using the Trizol reagent . To isolate the EV71 genomic RNA, cultured medium from EV71-infected Vero cells was centrifuged at 2000× g for 5 min. Proteins in the lysates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to PVDF membranes, and probed with the anti-EV71 , anti-EV71 3C Cells were lysed with 100 mM Tris pH 7.5, 250 mM NaCl, 0.5% sodium deoxycholate, 0.5% NP-40, 1 mM PMSF, and phosphatase inhibitor  for the indicated durations. After vortexing and incubating on ice for 10 min, cell lysates were centrifuged at 10,000× 5′-TGCATCACGTCAATATGACC-3′; reverse 5′-CCTCATCACTAAATAAACAGC-3′), RIG-I gene coding region , IFN-β gene coding region  and primers complementary to the β-actin coding sequence . The amplified DNA products were analyzed by agarose gel electrophoresis. The TaqMan gene expression assay and the Applied Biosystems detector were used to quantify the relative amounts of IFN-β mRNA, as previously described −ΔΔCT method Cellular total RNA was isolated from treated HeLa and RD cells at the indicated time points using the Trizol reagent. Complementary DNA (cDNA) was generated from 2 µg of the RNA by reverse transcription with oligo(dT) primer. To detect the mRNA expression of MDA5, RIG-I, IFN-β and β-actin by regular RT-PCR, the cDNA that was described above was amplified using Taq DNA polymerase and a primer set complementary to the MDA5 gene coding region  positive control or the EV71-infected cellular RNA , lane 3.The total RNA from the EV71-infected cells consisted of genomic RNA, viral replication intermediate RNAs, and cellular RNAs. To rule out the involvement of exogenous cellular RNAs in the observed induction of the type-I IFN expression, EV71/MP4 viral RNA was extracted from virus particles in the supernatant of EV71-infected Vero cells to enrich the EV71 genomic RNA content and eliminate cellular RNA contaminants from the transfection mixture. The enriched EV71 viral RNA was verified by RT-PCR (data not shown), and transfected into HeLa cells. IRF3 phosphorylation and IFN-β mRNA expression were evaluated at 20 h post-transfection. As shown in Since the initiation of IFN-β signaling by viral RNAs in the cytoplasm requires the cytosolic receptors for the pathogen-associated molecules, such as the MDA5 and RIG-I proteins, we further investigated the roles of MDA5 and RIG-I in type-I IFN response to presence of EV71 viral RNA. Either MDA5 or RIG-I protein was overexpressed in HeLa cells by transfection with the FLAG-tagged MDA5 or RIG-I expression plasmids. At 24 h post-transfection, the enriched EV71 RNA was transfected into the cells. At 20 h after the EV71 RNA transfection, phosphorylated IRF3 and IFN-β mRNA expression were analyzed by immunoblotting and RT-PCR, respectively. The EV71 RNA alone was sufficient to stimulate IRF3 activation and IFN-β mRNA synthesis , lane 2,To verify the involvement of MDA5 and RIG-I in EV71 RNA-mediated type-I IFN response, the induced expression of endogenous MDA5 or RIG-I was knocked down using siRNA in HeLa cells. Initially, HeLa cells were transfected with the MDA5 siRNA, and incubated for 6 h. The EV71 viral RNA was transfected to the MDA5 knockdown cells. As shown in We have previously shown that MDA5 is involved in EV71 viral RNA-activated type-I IFN expression. However, IRF3 activation and IFN-β mRNA were not detected upon EV71 infection as shown in To examine the effect of RIG-I overexpression in EV71 infection, a plasmid expressing FLAG-tagged RIG-I was transfected to HeLa cells for 36 h, and then infected with EV71. At 9 h after EV71 infection, cell extracts were collected for immunoblotting analysis. As shown in In our evaluation of MDA5 overexpression, we found that EV71 infection resulted in the appearance of a shorter polypeptide (approximately 95 kDa) that was detected in immunoblots using the anti-MDA5 antibody . AlthougTo determine whether the observed EV71-induced MDA5 cleavage was strain-specific, the expression of MDA5 was induced in HeLa cells by transfection with poly(I:C), followed by infection with the TW2231/98 or the BrCr strain of the EV71 virus. We found that both the TW2231/98 and the BrCr strain also induced MDA5 cleavage . At 9 h post-infection, the integrity of the FLAG-tagged proteins was analyzed by immunoblotting with an anti-FLAG antibody. As shown in To determine whether MDA5 cleavage induced by EV71 affects the production of IFN-β mRNA, HeLa cells were transfected with a FLAG-MDA5 expressing plasmid for 14 h, and then infected with EV71 for 9 h in presence or absence of the caspase inhibitor, Q-VD-OPH. As shown in Although EV71 infection does not induce IRF3 activation, we showed that the presence of EV71 viral RNA in the cytoplasm of cells activates IRF3 and induces IFN-β gene expression. Based on these results, we further demonstrated that the overexpression of either MDA5 or RIG-I protein enhances the EV71 viral RNA-mediated induction of IFN-β expression. We also showed that the diminished MDA5, but not RIG-I, expression was correlated with the reduction of EV71 RNA-mediated IRF3 activation in gene knockdown experiments. Furthermore, the suppression of IRF3 activation by EV71 infection was strongly reversed by the pre-existing MDA5 protein. These data indicate that the MDA5 protein plays a crucial role in the activation of IRF3 and the induction of IFN-β transcription in response to EV71. Although the EV71 3C protein has been reported to down-regulate IFN-β transcription through interactions with host RIG-I protein or cleavage of the TRIF protein, an adaptor protein for TLR3 signaling −/− knockout mice, several studies have indicated that MDA5 signaling is essential for type-I IFN induction by picornaviruses, such as encephalomyocarditis virus (EMCV), Theiler murine encephalomyelitis virus (TMEV), poliovirus, and coxsackie B virus In the cytoplasm of cells, the RNA helicases, RIG-I and MDA5, play a key role in the recognition of PAMPs that are generated following RNA virus infection. RIG-I has been implicated in the recognition of RNA with free 5′-triphosphate ends or intermediate length of synthetic poly(I:C), −/− and RIG-I−/−, are warranted.In our study, the EV71 viral RNA, which was extracted from the supernatant of EV71-infected Vero cells, was directly delivered to host cells by RNA transfection. Using this procedure, in contrast to natural virus infection, IRF3 phosphorylation and IFN-β expression were activated. Unlike poly(I:C) treatment, this approach provides a system to investigate the regulation of type-I IFN expression in response to the agonist from EV71. Nevertheless, though the purified EV71 viral RNA was shown to primarily consist of positive-stranded EV71 genomic RNA, we cannot exclude absolutely the presence of replication intermediate dsRNA, or cellular RNA contaminants in the RNA preparation. Because we assayed IRF3 activation and IFN-β mRNA expression before large amount of viral protein was expressed, the triggering signal for the response could be the result of the originally transfected viral RNA. However, we do not rule out the possibility that the replication intermediate dsRNA from the second cycle of replication has been generated and involved in the activation of IRF3. To further investigate whether EV71 viral genomic RNA could be the PAMP that is responsible for the MDA5-mediated innate response, in vitro-transcribed viral RNA, that was treated with alkaline phosphatase to remove 5′ phosphates, was used as an agonist for stimulating the expression of IFN-β mRNA . AlthougIn addition to RIG-I and MDA5, LGP2 is an inducible cytosolic RIG-I like receptor that also contains the DExD/H RNA helicase and the C-terminal repressor domains. Because LGP2 does not contain CARD domains and cannot therefore transduce the activation of downstream molecules through CARD-CARD interactions, LGP2 may function as a negative regulator of type-I IFN activity pro. Through this mechanism, EV71 may strongly suppress protein synthesis to diminish the host immune response. We found that EV71 infection suppresses total IRF3 expression at the late stage of infection. Under these conditions, poly(I:C) barely stimulated IRF3 activation and IFN-β expression in EV71 infected cells  Cell extracts were analyzed for MDA5 protein expression by immunoblotting using an anti-MDA5 antibody. (B) Total RNA was isolated from the transfected RD cells, and relative amount of the IFN-β mRNA was measured using real-time RT-PCR.(TIFF)Click here for additional data file.Figure S2RIG-I siRNA was sufficient to decrease in vitro-transcribed RNA-induced IFN-β expression. An empty pcDNA3 plasmid was digested with EcoRI. RNA was transcribed from the linearized plasmid by MEGAscript in vitro transcription kit  with T7 polymerase. The synthesized RNA was purified by RNeasy mini kit  (A) HeLa cells were transfected with scrambled siRNA or siRNA against RIG-I for 24 h, followed by transfection with 3 µg of the in vitro-transcribed RNA for 20 h. Cell extracts and total RNA were collected from the transfected HeLa cells. The expression of RIG-I, MDA5, phosphorylated IRF3, total IRF3, and β-actin was detected by immunoblotting. (B) Relative amount of IFN-β mRNA in RIG-I knockdown cells was measured by real-time RT-PCR.(TIF)Click here for additional data file.Figure S3Parallel comparison for MDA5 and RIG-I on IFN-β gene activation upon EV71 infection. HeLa cells were transfected with an empty plasmid or a plasmid expressing the FLAG-MDA5 or FLAG-RIG-I protein for 38 h. The transfected cells were subsequently infected with the MP4 strain of the EV71 virus at 2 MOI. At 9 h post-infection, cell extracts were analyzed by immunoblotting using anti-FLAG M2, anti-MDA5, anti-RIG-I, anti-3C, and anti-β-actin antibodies. (B) Real-time RT-PCR was performed to measure the relative amount of IFN-β mRNA expression.(TIFF)Click here for additional data file.Figure S4EV71 induces the cleavage of endogenous MDA5 protein. HeLa cells were transfected with 1 µg of EV71 RNA. At 14 h post-transfection, the cells were infected with the MP4 strain of the EV7 virus at 2 MOI. At 3 and 6 h post-infection, cell extracts and total RNA were collected for analyzing. (A) Cell extracts were analyzed by immunoblotting using anti-MDA5, anti-EV71, anti-β-actin, and anti-PARP antibodies. (B) Real-time RT-PCR was performed to measure the relative amount of IFN-β mRNA expression.(TIF)Click here for additional data file.Figure S5Overexpressed MDA5, but not RIG-I, is degraded during EV71 infection. (A) HeLa cells were transfected with an empty plasmid or a plasmid expressing the FLAG-MDA5 or FLAG-RIG-I proteins for 24 h. The transfected cells were subsequently infected with the MP4 strain of the EV71 virus at 2 MOI. At 9 h post-infection, cell extracts were analyzed by immunoblotting using anti-FLAG M2, anti-3C, and anti-β-actin antibodies. (B) The extracts from the FLAG-MDA5 transfected cells were also analyzed by immunoblotting using an anti-MDA5 antibody. The asterisk indicates the putative cleavage product of the MDA5 protein.(TIF)Click here for additional data file.Figure S6Examining the effect of caspase-dependent MDA5 cleavage on IFN-β mRNA production upon EV71 infection. HeLa cells were transfected with a plasmid expressing the FLAG-MDA5 for 22 h The transfected cells were subsequently infected with EV71/MP4 strain in presence or absence of a broad spectrum caspase inhibitor, Q-VD-OPH, at final concentration of 20 nM. Cell extracts and total RNA were collected at 12 h post-infection. The cell extracts were assayed by immunoblotting using anti-MDA5, anti-PARP, anti-V71 3C, and anti-β-actin antibodies (A). Relative amount of IFN-β mRNA in the cells was measured by real-time RT-PCR (B).(TIF)Click here for additional data file.Figure S7In vitro-transcribed EV71 viral RNA stimulates IFN-β mRNA production. A plasmid which contains full-length cDNA of EV71/4643 strain in pCR-XL-TOPO vector (kindly provided by Dr. Jen-Ren Wang) was digested with MluI. EV71 viral RNA was transcribed from the linearized plasmid by MEGAscript in vitro transcription kit  with T7 polymerase. The synthesized RNA was purified by RNeasy mini kit  and treated with FastAP alkaline phosphtase  for 30 min at 37°C to remove 5′-triphosphates. After inactivating the enzyme activity at 75°C for 5 min, the dephosphorylated RNA was extracted by phenol-chloroform and precipitated by isopropanol. For the preparation of control RNA, a pcDNA3 empty plasmid was linearized by NotI and performed the in vitro transcription and RNA purification, as described above. HeLa cells were transfected with 2 µg of control or dephosphorylated in vitro-transcribed EV71 RNA. At 24 h post-transfection, total RNA was isolated and analyzed by real-time PCR for measuring relative amount of IFN-β mRNA expression. IVT, in vitro transcription.(TIF)Click here for additional data file.Figure S8Total IRF3 expression is reduced during EV71 infection. HeLa cells were infected with the MP4 strain of the EV71 virus. At 4 h post-infection, the cells were transfected with 2 µg of poly(I:C) or transfection reagent alone. At 16 h post-transfection, cell extracts were analyzed by immunoblotting using anti-phospho-IRF3, anti-total IRF3, anti-EV71 3C, and anti-β-actin antibodies.(TIF)Click here for additional data file.


RNA editing by adenosine deaminases that act on RNA converts adenosines to inosines in coding and non-coding regions of mRNAs. Inosines are interpreted as guanosines and hence, this type of editing can change codons, alter splice patterns, or influence the fate of an RNA. A to I editing is most abundant in the central nervous system (CNS). Here, targets for this type of nucleotide modification frequently encode receptors and channels. In many cases, the editing-induced amino acid exchanges alter the properties of the receptors and channels. Consistently, changes in editing patterns are frequently found associated with diseases of the CNS. In this review we describe the mechanisms of RNA editing and focus on target mRNAs of editing that are functionally relevant to normal and aberrant CNS activity. RNA editing is a site specific, post transcriptional modification of RNA. Two types of RNA editing can be distinguished. On the one hand, insertion-deletion type editing inserts or removes single or multiple nucleotides from an RNA molecule  seemingly only affects metazoan nuclear encoded RNAs. Deamination of adenosines leads to the formation of inosines which are recognized as guanosines by most cellular machineries , Ca2+(cac), and glutamate-gated Cl− channels  are found to undergo ADAR-mediated RNA editing Bass, . A totalAMPA GluA2 subunit mRNA was the first target discovered. It is edited mainly at two coding sites leading to a glutamine to arginine and arginine to glycine conversion  exit efficiency, whereas unedited GluA2Q isoforms readily tetramerize and are transported to the synaptic membrane  and hotspot 2 (or+262/263/264 site), respectively  to methionine (AUI) codon change  gene is intronless and undergoes A to I RNA editing leading to an isoleucine to valine exchange  has been shown to induce epileptic seizures. RNA editing makes the channel insensitive to 4-AP by disrupting the interaction between the pore lining and the channel blocker  are classified into two types: Low voltage activated (LVA) and High voltage activated (HVA) channels  binds to the IQ domain located at the C-terminus of the pore forming α1 subunit. The formation of a Ca2c) is found widely distributed in the CNS. 5-HT2cR belongs to the G-protein-coupled receptor superfamily that stimulates phospholipase C (PLC) activity . Both these proteins co-localize on the apical membrane of choroid plexus cells  independent of G-protein coupling. Expression of the unedited INI isoform increases ERK1/2 phosphorylation in transfected HEK293 cells while expression of the edited VGV form decreases phosphorylation. However, this activation is significantly reduced upon β-arrestin depletion  and Filamin B (FLNb) are amongst a group of newly identified mammalian editing targets  is critical for this interaction. Again, FLNa repeats 21–24 are involved in this interaction. Coexpression of filamin in heterologous cells enhances the whole cell current density by ∼2.7-fold most likely by properly positioning functional KDrosophila melanogaster  belong to a conserved protein family that were the first proteins identified responsible for familial Alzheimer disease  in a yeast two hybrid screen. The region of interaction between CyFIP2 and FMRP overlaps with the FMRP dimerization site . Hu proteins preferentially bind to AU rich RNA elements (ARE) where they can act as RNA stabilizers and regulators of polyadenylation and translation  binds to YCAY enriched regions located upstream of repressed exons and downstream of activated exons . Glioblastomamultiforme (GBM) is a grade IV tumor with a survival rate of less than 18 months in children and adults  is characterized by slow degeneration of upper and lower motor neurons with a consequent loss of voluntary movement . Reduced editing at this site may limit cell death progression by suppressing postsynaptic excitation. Thus, editing might influence recovery after SCI (Barbon et al., Epilepsy is a common neurological disorder characterized by seizures caused by neuronal hyperexcitability (Bozzi et al., v1.1 have also been linked to seizures (Vollmar et al., v1.1 channels lies in the S6 segment. This is the target site of many drugs blocking the channel (Decher et al., Increased editing at the R/G site of the GluA2 transcripts and also of K2C pre-mRNA have been linked to different psychiatric disorders such as schizophrenia, depression, and bipolar disorder (Table Changes in the editing pattern of 5-HTer Table . Editinger Table . Howeverer Table . Previouer Table . Analysier Table . Dereguler Table . Howeverer Table .Current studies on RNA editing have clearly shown that adenosine deamination is most abundant in the CNS where it plays a major role in the diversification of the transcriptome. Three major processes seem to be primarily affected by A to I editing: first, many receptors and channels are modulated in their primary response and sensitivity to stimuli. Second, in many cases receptor assembly and retention in the ER seems to be affected by RNA editing. Finally, cytoskeletal components required for both outgrowth of neurons but also to the structuring of the cortical cytoskeleton and the anchoring of receptors is affected by RNA editing. It is one of the challenges to understand how these three processes are interconnected possibly being regulated through neuronal activity that may feed back on the process of RNA editing itself.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.