The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells.DOI:http://dx.doi.org/10.7554/eLife.01535.001 A virus is basically molecules of DNA or RNA inside a protein shell, and in order to reproduce, it must infect a living cell and take control of it. However, the attacked cell will fight back and try to eliminate the invader. Activation of this so-called innate immune response requires the host cells to recognize that they have been infected, which they do by detecting the tell-tale molecules that indicate the presence of the virus.When an RNA virus infects a cell, the tell-tale molecules are often atypical RNA molecules carried by the virus or produced as the virus replicates. Recognition of this ‘foreign’ material by receptor proteins inside the cell triggers the production of molecules called interferons, which activate the innate defence systems that eliminate the virus.Different receptor proteins recognize different RNA viruses. For example, a receptor called MDA5 is known to respond to the picornaviruses, some of which can cause inflammation of the brain and heart muscle. However, the identities of the specific RNA molecules that are recognized by the MDA5 receptor have not been known. Deddouche et al. have now identified one such RNA molecule with the help of a second receptor protein, called LGP2.The LGP2 receptor is not able to give the signal to produce interferons, so it is thought to work by binding to the RNA molecule to form a complex that is then relayed to MDA5 to give this signal. By isolating the complexes of LGP2 receptor from picornavirus-infected cells and sequencing the associated RNA, it was possible to identify the mystery RNA trigger. Deddouche et al. then tested picornaviruses in which this piece of RNA had been deleted from the genome, and found that the mutant viruses triggered a much weaker interferon response. By providing insight into the ways that some viruses are detected by the innate immune system, this research may inform future work on the development of new treatments to control viral infection.DOI:http://dx.doi.org/10.7554/eLife.01535.002 Viral infection in mammals triggers a rapid innate immune response involving the production of antiviral proteins and proinflammatory mediators, prominent among which are the type I interferons  . IFNs arMDA5 and RIG-I are activated by different RNA viruses . RIG-I iCardiovirus genus of picornaviruses. Picornaviruses are single-stranded positive-strand (sense) RNA viruses that replicate in infected cells via a negative-strand (antisense) intermediate. We purify RNA directly from complexes obtained by immunoprecipitation of LGP2 and show that this method enriches for MDA5 stimulatory RNA corresponding to a portion of the EMCV antisense RNA. Deletion of the region encoding this antisense RNA generates viruses that produce less stimulatory RNA and are less potent at inducing IFN in infected cells or mice. Conversely, in vitro synthesis of the same sequence generates an MDA5 agonistic RNA. Thus, a discrete region of the EMCV negative-strand RNA acts as a physiologically-relevant MDA5 agonist in infected cells.Immunoprecipitation of RIG-I from cells infected with influenza A virus (IAV) or Sendai virus has allowed the identification of physiological RIG-I agonists . In thisIfih1 and Dhx58 encoding MDA5 and LGP2, respectively. We infected Ifih1−/− (MDA5-deficient), Ifih1−/+ (MDA5-sufficient), Dhx58−/− (LGP2-deficient) or Dhx58+/+ (LGP2-sufficient) MEFs and assessed the induction of IFN-β and the interferon-stimulated protein IFIT-1. The upregulation of Ifit1 or Ifnb1 mRNA was greatly impaired in MDA5- or LGP2-deficient MEFs infected with EMCV  LGP2 from EMCV-infected HeLa cells transiently expressing a FLAG-tagged LGP2 protein , extractHaving established a method to purify IFN stimulatory RNA from LGP2/RNA complexes isolated from EMCV-infected HeLa cells, we subjected it to deep sequencing analysis. We pooled RNA extracted from multiple independent control or LGP2 IPs and carried out Illumina sequencing in duplicate . Approximately, 30 million reads of 60 nts in length were obtained from each sequencing sample and were mapped to the human and the EMCV genomes. The total number of reads mapping to EMCV represented around 30% in LGP2 IP samples vs only 4% and 6% in ctrl IP and input, respectively , which iTo validate these findings, we used strand-specific RT-PCR with primers for the L antisense or, as a control, the VP1 antisense regions  RNA sequence was sufficient to trigger an MDA5-dependent IFN response. We generated, by in vitro transcription, RNAs corresponding either to the AS or the sense strand of the EMCV L region. After CIP treatment to remove any RIG-I-stimulatory activity linked to the presence of the 5′ triphosphates, we tested the stimulatory potential of these RNAs in MEFs deficient for the se assay . The RNAse assay . As a cose assay , which sse assay . We concse assay  can trigac and EMCV ΔLzn) or complete (EMCVΔL) deletions of that region  EMCV  EMCV . More imWT) EMCV . These rac, ΔLzn or ΔL viruses was due to the absence of L region RNA rather than absence of L protein function, we additionally used an EMCV ZnC19AC22A strain. This virus carries two mutations in the zinc domain of the L protein, which inactivate function , which produce vast amounts of IFNs in response to virus infection   . EMCV ΔLA levels . SimilarFMDV was comparable to that induced by EMCVΔL . FMDV belongs to a different picornavirus genus and the FMDV L region sequence and FMDV leader protein share little similarity with those of EMCV. Notably, the induction of both Ifit1 and Ifnb1 in response to EMCV Ly EMCVΔL  and reply EMCVΔL . HoweverC19AC22A strains and the outputs of innate immune stimulation were measured. Higher levels of IFN-α were found in the serum of mice 24 hr after infection with EMCV ZnC19AC22A when compared to EMCVΔL and WT  were infected with EMCV WT, EMCVΔL and EMCV ZnL and WT . In addiL and WT . MeasureL and WT . We concSeveral types of RNA can trigger MDA5-dependent responses in experimental settings  but it rThe finding that pulldown of LGP2 allows isolation of MDA5 stimulatory RNA is in agreement with the fact that both RLRs are required for IFN production upon EMCV infection  and withDeep sequencing of LGP2-associated stimulatory RNA was revealing. We were not able to identify specific enrichment for any human sequences (data not shown), a finding that does not support a dominant role for RNAseL cleaved self RNA in MDA5 activation . This isAnother intriguing question is how a fragment of L AS RNA is generated upon viral infection. We have not been able to find a report describing the generation of small antisense RNA fragments during EMCV replication. However, Northern blots of RNA from EMCV-infected cells screened with an L AS sequence probe reveal many fragments smaller than the EMCV genome ranging between 200 and 2000 nucleotides (data not shown). Moreover, a previous study has reported the generation of a short subgenomic RNA fragment derived from the degradation of the non-polyadenylated genome of flaviviruses by the exonuclease XRN1 . It is pPicornavirus replication involves a stage where the positive-stranded genome anneals with a newly-formed negative strand in a full-length double-stranded RNA structure known as the replicative form (RF). Negative-strand RNA from the RF then serves as the template to produce new progeny viral genomes through a replicative intermediate (RI) consisting of many positively-stranded progeny RNA genomes in the process of being synthesised and still partially hybridised to the full-length antisense RNA template. A recent study showed that negative-strand RNA synthesis is absolutely required for IFN induction during infection of mengovirus, a strain of EMCV . Our resOur investigation has resulted in the identification of a specific region of the EMCV negative-strand RNA as a determinant of innate immunity to the virus. To our knowledge, this is the first study to identify an MDA5 agonist bound to LGP2 in infected cells. Picornaviruses constitute a large family of viruses that encompasses many human and animal pathogens including some of economical or medical importance including Poliovirus, Coxsackievirus, Rhinovirus, and EMCV . The L rThe human LGP2 sequences were amplified from cDNA derived from IFN-A/D -treated HEK293 cells using a forward primer containing the 3xFLAG epitope sequence with the following oligos . PCR products were then introduced into the pcDNA3.1 TOPO plasmid  to generate the 3xFLAG LGP2 expression constructs. For production of the recombinant LGP2 protein, the 3xFLAG-human LGP2 sequence was amplified using the forward primer 5′-actcgagttatggactacaaagaccatgacgg-3′ and the reverse primer 5′-ttgcggccgctcagtccagggagaggtccga-3′ and cloned into the XhoI and NotI sites of the pBacPAK-His3-GST plasmid. Recombinant 3xFLAG LGP2 was expressed as a GST-tagged protein in SF9 insect cells using a baculovirus expression system and purified on a single step by affinity chromatography using Glutathione Sepharose matrix . The 3xFLAG LGP2 was eluted by GST tag cleavage using in-house 3C enzymatic digestion. A final polishing step was then performed on a superdex 200 10/300 GL column . Protein purity was then verified on an acrylamide gel and the protein yield was quantified using a Nanodrop apparatus .The M2 (Sigma) and IgG1 isotype match control  antibodies were used for immunoprecipitation. M2 antibody was used at 1/5000 dilution for Western blot. Anti-mouse HRP antibody  was used at 1/10,000 dilution for Western blot. IFN A/D (PBL Assay Science) was used at 100 units/ml for 24 hr to pre-treat cells. Ribavirin powder (Sigma) was reconstituted in DMSO and used at 4 mM final concentration.Ifih1−/−, Ifih1+/−, Ddx58−/−, Ddx58+/+, Ddx58−/+, Dhx58−/− and Dhx58+/+, MEFs were generated as previously described  Cell services. HEK293 selected for responsiveness to RLR agonists were previously described . WT, Ifiescribed , immortaescribed . Brieflyc19AC22A, EMCV ΔL and EMCV LFMDV (mengovirus strain), were produced as described  were in vitro transcribed to generate full length EMCV RNA using T7 Megascript kit (Ambion) as per manufacturer’s instructions. Products were then digested with DNase I and purified with phenol:chloroform:isoamylalcohol (25:24:1), followed by chloroform extraction and ethanol precipitation. RNAs were transfected into HeLa cells to generate viruses, which were subsequently amplified on BHK21 cell monolayers until cytopathic effects were observed. Cell lysates were then freeze-thawed three times, cleared and centrifuged for 2 hr at 22,000 rpm  at 4°C on 20% sucrose cushion to purify viral particles. The pelleted viral particles were resuspended in 10 mM Tris pH7, 2 mM MgCl2 containing buffer and viral content quantified by plaque assay on Vero cells.Influenza A virus (IAV) (PR8 strain) and EMCV were as previously described . EMCV Znescribed . Other E6 pfu of the indicated virus in 200 μl of PBS. Control mice were injected with 200 μl of PBS. Serum and organs were collected from culled animals 24 hr after injection. All animal experiments were performed in accordance with national and institutional guidelines for animal care and were approved by the London Research Institute Animal Ethics Committee and by the UK Home Office (project licence PPL 80/2309).IFNAR1 KO mice were obtained from Michel Aguet (University of Lausanne) and backcrossed 14 times to C57BL/6J. The mice were bred at CRUK or at St Mary’s Hospital (kind gift from Cecilia Johansson) in specific-pathogen free conditions. For infection, 10- to 12-week-old C57BL/6-IFNAR1 KO mice were injected intraperitoneally with 10The IVT RNA used as a positive control for IFN induction was the IVT neomycin sequence previously described . The in Viral genomic RNAs were extracted from purified viral suspension using Trizol LS reagent (Life Technologies) as per manufacturer’s recommendations. For RNA extracted from infected HeLa cells, HeLa cells were infected with EMCV or IAV at an MOI of 1 for 16 hr before total RNA was extracted using Trizol. For separation of RNA into ss and dsRNA fractions, ssRNAs were first precipitated in presence of 2M LiCl. Double-stranded RNAs were then ethanol precipitated from the supernatant in presence of 0.7M LiCl. RNA pellets were washed with 70% ethanol, dried, and resuspended in RNase free water.Calf intestinal phosphatase , Terminator , RNaseT1 (Ambion Life Technologies), Polyphosphatase (Epicenter Biotechnologies), RNase A  and RNase III (Ambion Life Technologies) were used as per manufacturer’s recommendations. A control reaction omitting the enzyme was carried out in parallel. RNA was recovered by extraction with phenol:chloroform:isoamylalcohol (25:24:1), followed by chloroform extraction and precipitation with ethanol and sodium acetate in the presence of glycogen (Ambion). All RNAs were quantified using a Nanodrop apparatus.Ifnb1, Ifit1, IFNB1, gapdh, GAPDH, VP1 and L genes, RNA was extracted from 1 × 105 MEFs, BM-DCs or HeLa cells using the RNeasy kit  or from infected mouse organs using Trizol.For quantitative RT-PCR analysis of To generate the NS L AS Influenza segment, we amplified the L EMCV fragment amplified using the forward primer 5′-aaaccatggatggccacaaccatggaac-3′ and the reverse primer 5′-aaaccatggctgtaactcgaaaacgactt-3′ and introduced it into NcoI site of the pPolI_NS plasmid encoding the NS segment sequence of Influenza WSN strain (gift from Ervin Fodor). vRNP were then reconstituted in HEK 293T cells as previously described in , mouse Ifnb1 ID Mm00439546_s1, mouse gapdh ID 4308313, human IFNB1 ID Hs02621180_s1, human gapdh ID 402869. To detect viral RNA, Express Syber GreenER reagent (Life Technologies) was used in combination with the following primers: fw 5′-gcgcactctctcacttttga-3′ and rv 5′-tcgaaaacgacttccatgtct-3′ for detection of the L region or fw 5′-cctcttctccccctttgtgt-3′ and rv 5′-caggtccggcactataaacc-3′ for detection of the VP1 region. Data were normalized to levels of gapdh.RNA was treated with DNase I (Qiagen) prior to reverse transcription using superscript II reverse transcription reagents (Life Technologies) according to the manufacturer’s instructions. PCR was performed with TaqMan Universal PCR master Mix (Applied Biosystem Life Technologies) and the following taqman reagent assays: mouse Ifit1 ID Mm00515153_m1For strand-specific detection of viral RNA, RNA was first reverse transcribed using Superscript II (Life Technologies) in the presence of primers specific for L antisense (5′- ggccgtcatggtggcgaataagcgcactctctcacttttga-3′) or VP1 antisense (5′- ggccgtcatggtggcgaataacaggtccggcactataaacc-3′). cDNAs were then subjected to Exonuclease I (New England Biolabs) treatment and purified using Qiaquick PCR purification kit (Qiagen). In the next step, quantitative RT-PCR was performed in presence of Express Syber GreenER (Invitrogen) using the following primers: common forward primer 5′-aataaatcataaggccgtcatggtggcgaataa-3′ in combination with either L reverse primer 5′-aataaatcataatcgaaaacgacttccatgtct-3′ or 1D reverse primer 5′- aataaatcataacctcttctccccctttgtgt-3′ to detect L or 1D region respectively. RNA levels were calculated by comparison with a dilution curve of cDNA from EMCV-infected cells.5 of HEK293 cells or IFN-A/D treated MEFs were transfected with 200 ng of p125Luc  and 50 ng pRL-TK  using Lipofectamine 2000 (Life Technologies) as per manufacturer’s instructions. Cells were incubated for 6–8 hr and were then transfected with water (mock control) or with test or control RNAs using lipofectamine. Luciferase activity was analysed in cell lysates 12 to 16 hr later using the Dual luciferase reporter Assay system (Promega). In all cases, firefly luciferase values were divided by Renilla luciferase values to normalise for transfection efficiency. All data are shown as fold increase relative to reporter cells transfected with water alone.IFN-β luciferase reporter assay: 2.5 × 105 cell/well. The cells were then transfected with test or control RNAs using lipofectamine. After overnight culture, mIFN-α was measured in cell supernatants by ELISA as described previously (MEF assay: MEFs were pre-treated with 100 units/ml of IFN-A/D (PBL interferon) for 24 hr and plated into 24-well plates at 0.5 × 10eviously  or MEF R5 cell/well. The cells were then transfected with the indicated amounts of test or control RNAs using lipofectamine. After overnight culture, HeLa RNA was extracted for RT-PCR analysis of IFNB1 induction.HeLa assay: HeLa cells were pre-treated with 100 units/ml of IFN-A/D (PBL Assay Science) for 24 hr and plated into 24-well plates at 0.5 × 102, 200 mM NaCl, 0.5% NP40, 1X protease inhibitor cocktail , 0.5 U/ml RNasin [Promega]). Lysate was incubated for 30 min on ice, cleared by centrifugation for 10 min and the supernatant was collected. At this stage a small fraction of the input was collected for protein and RNA extraction. 5 μg of M2-anti-FLAG (Sigma) or IgG1 isotype control antibody was added to 500 μl of lysate and incubated on a rotating shaker for 1 hr at 4°C. 300 μl of washed Gamma Bind Plus Sepharose Beads  were then added to the mixture for another 1 hr. The beads were then precipitated by centrifugation and washed four times with 1 ml of lysis buffer. The beads were then split into two samples for protein or RNA extraction. Proteins were extracted from Protein/RNA complexes by boiling the beads for 5 min in SDS sample buffer for Western blot analysis. RNAs were purified from the beads by Phenol/chlorophorm extraction followed by ethanol precipitation. RNA was then quantified using a Nanodrop apparatus and the same amount of RNA from each sample was tested for IFN stimulatory activity as indicated.Around 10–15 million HeLa cells were transfected with 30 µg of 3xFLAG LGP2 IP plasmids using lipofectamine reagent following the manufacturer instructions. The cells were incubated for 16 hr, lipofectamine was washed away and the cells were incubated in fresh medium for 6–8 hr. HeLa cells expressing the 3xFLAG constructs were then infected with EMCV at an MOI of 1 for 16 hr. The cells were subsequently washed and lysed in lysis buffer  in lysis buffer for 1 hr at 4°C on a rotating shaker. The rest of the IP was performed the same way as for the LGP2 IP from infected cells.Input material pooled from five replicate cultures was subjected to immunoprecipitation with anti-FLAG or control IgG1 antibody (see above). RNA extracted from 10 independent IPs was pooled and ribosomal RNA was removed using Ribo-Zero rRNA Removal Kit (Human.Mouse.Rat)  according to the manufacturer’s protocol. After ribosomal RNA removal, the RNA was tested for its ability to activate the IFN-β promoter luciferase reporter. The RNA was then prepared for Illumina sequencing using an optimised version of the original Directional mRNA-Seq Library Prep protocol (Pre-Release Protocol Rev.A). The original protocol was optimised from 1 μg of total RNA input to a total input of up to 173 ng. Reagents supplied by Illumina included 10× v1.5 sRNA 3′ Adaptor; SRA 5′ Adaptor; SRA RT primer; PCR Primer GX1/GX2. The remaining reagents recommended on the protocol were outsourced from alternative vendors. To analyse both coding and non-coding RNA regions, the poly(A) purification step was omitted. After fragmentation of the total RNA (optimised incubation of 2 min at 80°C), the RNA was checked for efficient fragmentation sizing, ribosomal RNA contamination and RIN using the Agilent 2100 Bioanalyzer QC Pico RNA chip. After library preparation, the PCR reaction was optimised by replacing the recommended PCR polymerase with Kapa Hifi DNA Polymerase  in addition to optimising the number of PCR cycles from 12 to 18 cycles using the recommended PCR cycling conditions. The final library preparation was then size selected at 150 bp–450 bp using a 2% Agarose E-gel (Invitrogen) to remove unwanted regions in excess of 500 bp which was detected using the Agilent 2100 Bioanalyzer QC DNA 1000 chip. Next generation sequencing and library preparation was performed in the Advanced Sequencing Facility (ASF) at the London Research Institute on the Genome Analyzer IIx (GAIIx) with a single-end 72 bp sequencing run alongside a PhiX control of 1–5% in every lane of the flowcell. Sequencing typically generated ∼30 million 60 bp single-end reads.Alignment to the EMCV strain pEC9 genome (genbank accession ID DQ288856) was performed using Bowtie  was used for statistical analysis of data.An unpaired two-tailed Student’s  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  As it stands now, the definition of this ‘L’ RNA ligand is still ‘fuzzy’. In particular, the authors stated that the L fragments generated by in vitro transcription or chemical synthesis did not stimulate the MDA5/LGP2 pathway. Thus, it is not clear what makes the L antisense RNA fragment a unique ligand for LGP2. At the minimum, the authors should perform deletion and/or point mutagenesis of the L RNA sequence to determine if there are specific sequence(s) in this region that renders it a good LGP2 ligand. The EMCV mutant Zn2) Of note, the study does not identify the EMCV RNA structure that activates MDA5 but the sequence that preferentially binds to LGP2. The authors should therefore amend the claim that it identities a natural MDA5 agonist, or that the L-region RNA itself constitutes an MDA5 agonist. Given that L-region RNA on its own fails to activate MDA5, one must assume that the antisense L-region RNA enriched by LGP2 is merely contained in the same RNA strand as the actual MDA5 agonist, which also remains unidentified. Does introduction of the antisense EMCV L-region into previously inactive RNA transcripts render them MDA5 agonists?3) How long is the antisense L RNA that is proposed to be the LGP2 ligand? Is it a uniform size or heterogeneous? Is it possible that the RNA ligand is actually longer than those detected by RNA-seq because the parts of RNA not bound (protected) by LGP2 are degraded by RNases? This could be one of the reasons that the in vitro transcribed antisense L RNA has no activity.4) While a lot of evidence is presented that suggests that LGP2 delivers the agonist to MDA5, no direct evidence is presented. Especially considering the fact that pure L region antisense fragment does not recapitulate the observed phenotype, more evidence is required to corroborate the claim that LGP2 serves as a means to preselect agonists for MDA5 activation in the context of EMCV infection. Is MDA5 associated with the RNA in the LGP2 pulldowns? Do MDA5 and LGP2 interact after ligand capture? Does this interaction induce the formation of MDA5 filaments or IFN expression?5) 6) In cells infected by EMCV virus lacking the L region, can the dsRNA and ssRNA be isolated from the gels  and show 1) As it stands now, the definition of this ‘L’ RNA ligand is still ‘fuzzy’. In particular, the authors stated that the L fragments generated by in vitro transcription or chemical synthesis did not stimulate the MDA5/LGP2 pathway. Thus, it is not clear what makes the L antisense RNA fragment a unique ligand for LGP2. At the minimum, the authors should perform deletion and/or point mutagenesis of the L RNA sequence to determine if there are specific sequence(s) in this region that renders it a good LGP2 ligand. The EMCV mutant ZnC19AC22Acan be a good starting point for mutagenesis because this virus can induce IFN-β quite well. It is formally possible that any RNA sequence, when placed in this region, could be a ligand for LGP2. It is important to have a better characterization of the antisense L RNA fragment, especially given that this RNA could not be detected by Northern blotting and that currently there is no explanation for how this RNA is generated in the EMCV infected cells.We have now been able to generate an L region antisense RNA by in vitro transcription that possesses MDA5-stimulatory activity new . In contC19AC22A EMCV would provide useful information. However, this is not a trivial exercise as it involves the rescue of hundreds of viruses. As it is, we have found it quite problematic to rescue and grow the mutant virus, which grows poorly. Nevertheless, we do show that 3 different EMCV mutants that carry either full or partial deletion of the L acidic region or the L zinc domain all display impaired ability of the viral RNA to interact with LGP2 .Notably, this recombinant EMCV behaves much like the EMCVΔL virus .2) Of note, the study does not identify the EMCV RNA structure that activates MDA5 but the sequence that preferentially binds to LGP2. The authors should therefore amend the claim that it identities a natural MDA5 agonist, or that the L-region RNA itself constitutes an MDA5 agonist. Given that L-region RNA on its own fails to activate MDA5, one must assume that the antisense L-region RNA enriched by LGP2 is merely contained in the same RNA strand as the actual MDA5 agonist, which also remains unidentified. Does introduction of the antisense EMCV L-region into previously inactive RNA transcripts render them MDA5 agonists?We thank the reviewers for these thoughtful comments. We feel that the claim that we have identified an MDA5 agonist is now warranted by the new data in As suggested by the reviewers, we also have tried to introduce the EMCV L antisense sequence into a heterologous RNA. For that experiment, we chose to introduce it into the influenza virus NS segment, a known RIG-I agonist. As shown in the new 3) How long is the antisense L RNA that is proposed to be the LGP2 ligand? Is it a uniform size or heterogeneous? Is it possible that the RNA ligand is actually longer than those detected by RNA-seq because the parts of RNA not bound (protected) by LGP2 are degraded by RNases? This could be one of the reasons that the in vitro transcribed antisense L RNA has no activity.We can indeed not exclude that partial degradation of the RNA does take place and that LGP2 protects a fragment of 171 nucleotides as revealed by our sequencing data. However, the fact that we can now synthesise this fragment and reveal stimulatory activity confirms that it is sufficiently long to activate MDA5. It is remains possible that the LGP2 agonist is actually smaller than 171nts. The full characterisation of this RNA will require further work, which we feel is beyond the scope of the present report.4) While a lot of evidence is presented that suggests that LGP2 delivers the agonist to MDA5, no direct evidence is presented. Especially considering the fact that pure L region antisense fragment does not recapitulate the observed phenotype, more evidence is required to corroborate the claim that LGP2 serves as a means to preselect agonists for MDA5 activation in the context of EMCV infection. Is MDA5 associated with the RNA in the LGP2 pulldowns? Do MDA5 and LGP2 interact after ligand capture? Does this interaction induce the formation of MDA5 filaments or IFN expression?To address the reviewers’ comments, we have tried to co-immunoprecipitate MDA5 and LGP2. To do this, we have coexpressed FLAG-tagged LGP2 together with HA-tagged MDA5 in HeLa cells but, oddly, we lose MDA5 detection when the cells are infected with EMCV (data not shown). We do not know the reason for this phenomenon but attribute it to the capacity of MDA5 to form insoluble filaments upon activation that are lost from the lysate during preclearing. We have attempted to circumvent the issue by using lysate from uninfected cells that has been spiked with RNA from EMCV infected Vero cells prior to MDA5 or LGP2 immunoprecipitation but this has failed to reveal any co-precipitation (data not shown). Because these experiments are negative they remain inconclusive and, therefore, we have not included them in the paper. Thus, we do not at present have proof that LGP2/MDA5/RNA form a tripartite complex and, consequently, have amended the term “complex” throughout the text. It is in fact possible that LGP2 acts upstream of MDA5 to remodel agonistic RNA or that its interaction with MDA5 is very transient and unlikely to be captured by biochemical means. We now refer to these possibilities in the revised Discussion. We thank the reviewers for allowing us to clarify the issue.5): There is still a substantial level of IFN-β induction by the EMCV virus lacking the L RNA. Is this L-independent activity dependent on RIG-I or MDA5? If it is MDA5 or LGP2 dependent, it suggests that there are other MDA5 ligands in the virus infected cells. These could conceivably be dsRNA as previously thought as MDA5 ligands. Indeed, the authors showed that dsRNA isolated from EMCV infected cells induced IFN-β .The notion that there are additional MDA5 triggers besides the L region antisense RNA is a possibility that we had considered in the original Discussion. We make this point clearer with textual revisions. As regards the question of whether the activity remaining in the EMCV delta L virus is MDA5 dependent, we now state in the revised Discussion that this point is unclear and have added a new figure to support that statement new .6) In cells infected by EMCV virus lacking the L region, can the dsRNA and ssRNA be isolated from the gels  and shown to stimulate IFN-β? This could help clarify where the antisense L RNA comes from and whether it has an indirect effect on the generation of dsRNA.We have isolated the dsRNA and ssRNA fractions from cells infected with EMCV virus lacking the L region as requested (data not shown). We have refrained from including the results in the revised manuscript as we are not entirely sure what they mean, especially in the face of the new data on generation of the MDA5 stimulatory activity in vitro.


During HIV-1 maturation, the step driven by its protease (PR) controling RNA condensation remains poorly documented.in vitro demonstration that HIV-1 RNA behaves within the HIV-1 particle as an up-regulator of PR. The resulting fast processing of RNA-bound nucleocapsid protein (NC) both in N- and C-termini from its Gag precursor clearly leads to RNA condensation. The critical step consists of the secondary cleavage releasing the Gag C-terminal p6 domain from the NCp15 intermediate. Remarkably, such processing is optimal in more physiological conditions than classically used for in vitro HIV-1 PR assay, thus allowing a useful protection of the crucial NC zinc fingers. The related mechanism implies PR sequestration by clusters of NCp15 assembled along the RNA chains, highlighting a fast condensation of RNA:NC ribonucleoproteic complexes as an opportune step within the overall process of maturation, prior to the conical capsid reassembly.Our methodology, mainly inspired by B. M. Alberts’ concept of macromolecular machines, combines biochemical and biophysical approaches with purified components. They provide a clear These data support a new biological paradigm of a protease dramatically controlled by a RNA molecule to optimize its action changing the targeted nucleoprotein architecture from an assembly mode to a functional one.


Bioinformatics and in vivo analyses show that Dis3p has a conserved and functional mitochondrial-targeting signal (MTS). A clean and marker-less chromosomal deletion of the Dis3p MTS results in a defect in the decay of intron and mirror RNAs, thus revealing a role for Dis3p in mitochondrial RNA decay.Mitochondria are essential organelles that harbor a reduced genome, and expression of that genome requires regulated metabolism of its transcriptome by nuclear-encoded proteins. Despite extensive investigation, a comprehensive map of the yeast mitochondrial transcriptome has not been developed and all of the RNA-metabolizing proteins have not been identified, both of which are prerequisites to elucidating the basic RNA biology of mitochondria. Here, we present a mitochondrial transcriptome map of the yeast S288C reference strain. Using RNAseq and bioinformatics, we show the expression level of all transcripts, revise all promoter, origin of replication, and tRNA annotations, and demonstrate for the first time the existence of alternative splicing, mirror RNAs, and a novel RNA processing site in yeast mitochondria. The transcriptome map has revealed new aspects of mitochondrial RNA biology and we expect it will serve as a valuable resource. As a complement to the map, we present our compilation of all known yeast nuclear-encoded ribonucleases (RNases), and a screen of this dataset for those that are imported into mitochondria. We sought to identify RNases that are refractory to recovery in traditional mitochondrial screens due to an essential function or eclipsed accumulation in another cellular compartment. Using this  Saccharomyces cerevisiae, by the group of Ephrussi and Slonimski is a haploid derivative of S288C[Yeast wild-type strain, BY4741  in a 14 mL round-bottom culture tube, followed by overnight incubation at 30°C and 245 rpm shaking. Over-night cultures were used to inoculate the experimental culture (1 L of YPD in a 4 L Belco baffle-bottom flask) to an OD600 of 0.5, followed by incubation at 30°C and 245 rpm for two days. dis3Δmts). Highly purified yeast mitochondria were isolated from ~10 g wet-weight of each culture according to published protocols[Four independent yeast cultures were grown for RNAseq analysis  steps were performed by the genomic core facility at the Huntsman Cancer Institute including validation of RNA quality, quantity and purity; library preparation using the Illumina TruSeq Directional RNA protocol with fragmentation to reduce insert size to 30-300 bases, no DSN treatment, no polyA selection, no ribosomal or tRNA subtraction, and no size selection; and 101 cycle directional paired-end sequencing on an Illumina HiSeq2000 platform. Resulting sequence data was processed using Illumina's CASAVA software, version 1.7.0. to retain only high-quality reads, which were mapped with Bowtie using the Galaxy main instance-28. Avad5’-3’ direction and a dataset in the 3’-5’ direction, for a total of 8 datasets. Only the 5’-3’ datasets were used for this study to avoid the possibility of double-counting the expression of bases from inserts shorter than 202 bp. The 8 datasets have been deposited at the NCBI short read archive with the following accession numbers . WT experiment-GCDA5 , WT experiment-GCDA8 , mutant experiment-N29dis3A5 , mutant experiment-N29dis3A8 .Due to paired-end sequencing, two RNAseq datasets result from each RNA sample, a dataset of reads in the TM III reverse transcriptase (Invitrogen) and the recommended protocol were used to synthesize cDNA from 50 ng of mitochondrial RNA from yeast strain BY4741, except the RNA was incubated with primers and dNTPs at 70°C for 5 min. Five units of RNase H (New England Biolabs) were used to digest the RNA complementary to the cDNA. The cDNA was then diluted 1:5 and 2 μL was used as a template for PCR. Immolase hot start DNA polymerase (Bioline) was used to amplify the spliced product with the following reaction conditions: 1x immobuffer, 3 mM MgCl2, 0.3 mM dNTP, 1 μM of forward and reverse primer, and 2.5 units of polymerase in a 50 μL reaction volume. The reaction was denatured at 95°C for 7 min, followed by 35 cycles of 95°C for 30s / 50°C for 30s / 72°C for 30s, and then 4°C hold. Reaction products were separated on a 1% agarose, 1X TAE horizontal gel, detected using ethidium bromide staining, and visualized with GeneSnap software (Syngene). A single 444 bp band was seen, which is the sized predicted for amplification of alternative-spliced aI5β. The PCR product was purified using the Qiaquick PCR purification kit (Qiagen), and then sequenced in both directions using the forward and reverse PCR primers, which resulted in 2-fold coverage over the splice junction. Gene-specific primers used to generate cDNA, amplify the alternative-spliced product, and sequence the resulting PCR product are described in Table S2 in Full-length Dis3p and Arg8p were modeled using the I-TASSER web accessible program,31. Predhttp://rzlab.ucr.edu/scripts/wheel/wheel.cgi. Dis3p has a 17 amino acid amphipathic alpha helix starting with lysine 11 (K11). Arg8p has a 15 amino acid amphipathic alpha helix starting with leucine 6 (L6). Hydrophilic residues are circles, hydrophobic residues are diamonds, potentially negatively charged are triangles, and potentially positively charged are pentagons. The most hydrophobic residue is green, and the amount of green is decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are coded red with pure red being the most hydrophilic (uncharged) residue, and the amount of red decreasing proportionally to the hydrophilicity. The potentially charged residues are light blue.The N-terminus of Dis3p and Arg8p were assayed for the ability to form an amphipathic alpha helix using the Helical Wheel Projections program Version: Id: wheel.pl,v 1.4 2009-10-20 21:23:36 don Exp, created by Don Armstrong and Raphael Zidovetzki at the University of California Irvine. Web address: ARG8 gene was cloned from yeast strain BY4741 into the low-copy pRS415 yeast shuttle vector[ARG8-ARG8. We also engineered a plasmid that contained no MTS coding sequence (pMTS∆-ARG8), or contained the MTS from DIS3 in place of the native MTS (pMTSDIS3-ARG8). Plasmids were transformed into an ARG8-deletion strain (arg8-∆0) that is isogenic to BY4741. Growth on arginine-free medium, which was scored after 3 days at 30°C, requires localization of Arg8p to the mitochondrial matrix. Plasmids and primers used in this experiment are described in Table S3 in The le vector. To faciS2 cell culturing, immunofluorescence, and fractionation were performed as described. PrimersS. cerevisiae Dis3p/Rrp44p was obtained from SGD and used as a blastp query . The COBALT program was used to perform a multiple sequence alignment of the first 50 sequences.The complete 1001 amino acid sequence of CUP1 promoter between the DIS3 promoter and ORF. This homologous recombination event simultaneously deleted the MTS (codons 2-29) and maintained expression of DIS3. The CUP1-containing cassette was then removed by a second homologous recombination event, resulting in a clean and marker-less deletion of the MTS to make strain dis3Δmts. Primers used for this experiment are described in Table S5 in A novel homologous recombination strategy was used to insert the dis3Δmts RNA samples) for a total of 12 experimental reactions plus a no-RT control for each individual RNA analyzed. In each case the resulting Ct values of the 6 WT reactions were compared to the 6 dis3Δmts reactions P-values were calculated using the Student’s t-test function built into Microsoft Excel .Gene-specific primers used to generate cDNA and amplify the target are described in Table S6 in To obtain sufficient quantities of total mitochondrial RNA for RNAseq analysis, we grew two independent cultures of yeast BY4741, a derivative of S288C, for two days in rich media. Within the fist day the cells completed the diauxic shift from fermentative growth on dextrose to aerobic growth on ethanol. During fermentative growth the cells divide rapidly and provide the abundant number of fresh cells required for mitochondrial isolation. Aerobic growth during the second day results in slow growing but mitochondrial-active cells. Highly purified mitochondrial RNA from each culture was subjected one time to Illumina Hiseq2000 deep sequencing, which resulted in approximately 9 million mapped reads for each RNA sample .To create a reference for mapping RNAseq data, we built a model of the S288C mitochondrial genome using DNA sequence information from the Saccharomyces Genome Database , the public repository for this organism . We annoATP9 gene is followed by tRNA-Ser and then VAR1 on the same transcript, and the order of abundance is also 5’ to 3’. These results are similar to the results obtained from sequencing the human mitochondrial transcriptome in that co-transcribed RNAs have differential abundance, which points to the importance of attenuated transcription between genes or post-transcriptional RNA processing/decay to provide differential gene expression[Mapping the RNAseq reads to our mitochondrial genomic annotation demonstrated clear differential expression of the 35 genes required for oxidative phosphorylation, the group introns, and the HEG-related ORFs . Notablyxpression,41.Sequencing the mitochondrial transcriptome allowed us to determine for the first time the exact 3’ end of all 24 tRNAs. This is made possible by the presence of the non-encoded 3’ terminal CCA that is added post-transcriptionally to all tRNAs. Detection of the CCA addition site also allowed nucleotide-level resolution of the 5’ end of all tRNAs based on tRNA-structure prediction . It is nTo annotate the 5’ end of primary transcripts we analyzed the mitochondrial genomic sequence for the presence of the presumed 9-base yeast mitochondrial promoter, referred to as nonanucleotide,45. We fThe yeast mitochondrial RNA polymerase is of phage T7 origin, but theori) have been proposed, although not all of the ori are active, and those that are active are strain dependent[ori are immediately downstream of the 20-base promoter and conform to a 5-part consensus sequence [ATP8 and ATP9 both have non-canonical dodecamer sequences, suggesting an alternative mechanism of mRNA cleavage ,54. The cleavage . In thisochondria. We haveodecamer . Thus, n BI3 ORF . Heptakaidecamer cleavage sites are particularly easy to identify in our RNAseq data because numerous reads end precisely at the 5’ cleavage nucleotide, and numerous other reads begin precisely at the 3’ cleavage nucleotide. The heptakaidecamer is found 18 times in the S288C mitochondrial genome, but in our RNAseq dataset we only find reads associated with the RNA ends of four of them . 15S rRNA / Trp transcript, and a fourth cleavage site within the RPM1 transcript , RNA 3’ ends, or active We precisely mapped the 3’ terminal nucleotide of all 24 mitochondrial-encoded tRNA due to the presence of non-encoded CCA trinucleotide. Simultaneously identifying the boundaries and measuring the quantities of all tRNA should provide a valuable tool to investigate tRNA metabolic pathways. Mitochondrial tRNA is processed by endonucleolytic cleavage at the 5’ and 3’ end by RNase P and RNase Z respectively, but most molecular components of the processing, modifying, surveillance, stability, and decay complexes have not been identified.Yeast mitochondrial RNA abundance has been measured numerous times, but with various strains, techniques and standards, and only on a limited number of RNAs. We chooVAR1 has the lowest RNA abundance of any mitochondrial gene  in the cytoplasm of ular RNAs,91,92. Donuclease,94 and honuclease,95-97. Donuclease-101. in vitro in the absence of any other proteins[Our bioinformatic analysis of all yeast RNases identified Dis3p as a candidate for mitochondrial localization. Although numerous proteomic studies show Dis3p in mitochondria, the same studies do not find other components of the exosome within the mitochondria-65. Ther proteins,97,104. dis3∆mts strain shows significantly more bI2, 3, and 4 intron RNA than in the wild type. Due to greater variability in the results for bI1, the difference did not reach our stringent threshold of significance. However, RT-qPCR confirmed the RNAseq results and showed a significant hyper-accumulation of bI1 intron RNA. Hyper-accumulation of intron RNA can be due to enhanced transcription of the primary transcript, which could be detected as an accumulation in the amount of the first exon. Accumulation of intron RNA could also result from a defect in splicing, as un-spliced RNA is often more stable than freed intron RNA, and this can be scored as a decrease in the accumulation of ligated exons[dis3∆mts is likely due to a defect in RNA decay, which is consistent with Dis3p endo- and exo-ribonuclease activity. However, this study has not addressed if Dis3p interacts directly with any of these RNAs, or if ribonuclease activity is required for these effects.RNAseq analysis of the ted exons. We founsuv3-1 allele specifically affects the splicing of these two introns[suv3-1 background, which causes a defect in recycling of Mrs1p and hence a block in splicing[ We could find nothing in common among the four affected introns that would suggest a reason for their hyper-accumulation in the mutant, while bI5 and all other introns are largely unaffected. A somewhat similar phenomenon was found for the aI5β and bI3 introns, in that the o introns, due to o introns. These to introns. It was o introns, which io introns,108. It  splicing. Thus thSUV3 or DSS1 transcripts, thereby depleting mitochondrial Suv3p or Dss1p. However, we consider this unlikely because the most dramatic defect of SUV3 or DSS1 perturbation is massive accumulation of the omega intron RNA[dis3∆mts strain Click here for additional data file.


Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important “classical” anthelmintics like levamisole and pyrantel, as well as “novel” anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite  Xenopus oocyte expression system to investigate the diversity of anthelmintic receptors in the parasitic nematode Oesophagostomum dentatum. We demonstrate effects of the ‘novel’ anthelmintics tribendimidine and derquantel on these reconstituted receptors, revealing, for the first time, the subunits that make up a tribendimidine- and derquantel-sensitive receptor from a parasitic nematode. We show that the receptor structure and pharmacology can be plastic, and depends on subunit composition and stoichiometry. The factors that affect the diversity of the receptor may contribute to anthelmintic resistance. Our results demonstrate the presence of acetylcholine receptor subtypes that may serve as anthelmintic targets and suggest that pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.Parasitic nematode infections of humans and animals are world-wide. In humans, they cause disease and perpetuate a cycle of poverty. In animals, the parasites cause welfare problems and production loss. In developing countries, the debilitating effect of nematode parasites in school children limits their education, and in adults reduces productivity. These two factors, along with the production loss associated with nematode infections of animals that affects human nutrition, sustain poverty. Treatment and prophylaxis of these parasites requires the use of anthelmintic drugs. Here, we employ the  Ascaris, hookworms, Strongyloides stercolaris, trematodes and tapeworms C. elegans suggest that tribendimidine acts as a cholinergic agonist like levamisole and pyrantel B-subtype nAChR from Ascaris suumHuman nematode parasite infections are of public health concern in many developing countries where over a billion people are infected C. elegans, possesses two muscle nAChR types: one sensitive to levamisole and one sensitive to nicotine Ascaris suum, there are three pharmacologically separate nAChR types on the body muscle: the N-type that is preferentially activated by nicotine; the L-type that is preferentially activated by levamisole and; the B-type that is preferentially activated by bephenium Oesophagostomum dentatum there are four muscle nAChRs which are defined by their single-channel conductances: G 25 pS, G 35 pS, G 40 pS and G 45 pS. The G 35 pS receptor type of O. dentatum is reduced in a levamisole-resistant isolate O. dentatum has a similar channel conductance to the L-type from the distantly related species, A. suumThe model nematode, O. dentatum isolates are still unknown because the O. dentatum genes encoding muscle nAChR subunits have not yet been identified. In order to determine a mechanistic explanation for the variability and pharmacological diversity of the muscle nAChR types in parasitic nematodes like Oesophagostomum dentatum, we took advantage of the Xenopus oocyte expression system However, the precise molecular mechanisms of levamisole and pyrantel resistance occurring in Ode-unc-38, Ode-unc-29 Ode-unc-63 and Ode-acr-8, which are homologues of the C. elegans levamisole muscle receptor genes Xenopus laevis oocytes to recapitulate the levamisole-sensitive AChRs of O. dentatum. In a previous study on H. contortus which is closely related to O. dentatum, we have reported that co-expression of four AChR subunits  in Xenopus oocytes resulted in the robust expression of a levamisole sensitive nAChR Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr8) and express them in Xenopus oocytes. Of particular note is the evidence that these receptor subtypes are pharmacological targets of the new anthelmintic tribendimidine. Moreover, we demonstrate that the nAChR reconstituted with all four subunits is more sensitive to levamisole than pyrantel and has a single channel conductance of 35 pS, corresponding to the L-type previously observed in vivo in O. dentatum and A. suum. Another receptor type, made of 3 subunits, was more sensitive to pyrantel and the new anthelmintic derquantel had a selective effect against receptors activated by pyrantel rather than levamisole, providing the first insight of its molecular target composition in any parasitic nematode. These results provide a basis for understanding the greater diversity of the nAChR repertoire from parasitic nematodes and for deciphering the physiological role of nAChR subtypes targeted by distinct cholinergic anthelmintics. This study will facilitate more rational use of cholinergic agonists/antagonists for the sustainable control of parasitic nematodes impacting both human and animal health.In this study our first objective was to clone the four putative subunit genes O. dentatum, C. elegans and H. contortus in clade V of the nematode phylum, we used a candidate gene approach to identify full-length subunit cDNAs from O. dentatum of unc-63, unc-29 and acr-8 homologues. The previously available O. dentatum unc-38-like full-length cDNA sequence (Accession number GU256648) allowed the rapid cloning of the Ode-unc-38 subunit cDNA. The UNC-38 and UNC-63 subunits being closely related in the UNC-38 “core” group unc-63 and unc-38 cDNA sequences from different nematode species allowed the design of degenerate primers that were able to specifically amplify the unc-63 homologue from O. dentatum. To identify the unc-29 homologue from O. dentatum, degenerate primers from the H. contortus and C. elegans unc-29 sequences were used to amplify a partial 288 bp cDNA sequence, providing specific information to get the full-length cDNA by RACE-PCR standard procedures. To identify the acr-8 homologue from O. dentatum, a partial cDNA of 861 bp was cloned using primers from H. contortus acr-8, allowing subsequent identification of the full-length cDNA encoding Ode-ACR-8 subunit. The four O. dentatum subunit transcripts were trans-spliced at their 5′end with the splice leader 1 (SL1). The predicted nAChR proteins displayed typical features of LGIC subunits including a secretion signal peptide, a cys-loop domain consisting of 2 cysteines separated by 13 amino acid residues and four transmembrane domains (TM1-4)  of: Ode-unc-29, Ode-unc-38, Ode-unc-63 and Ode-acr-8.Our investigation of Ode-unc29∶Ode-unc-63 cRNA, regularly produced currents of >50 nA in response to 100 µM acetylcholine or cholinergic anthelmintics, indicating that the two subunits could form functional receptors , or the Pyr-nAChR.More than 75% of injected oocytes responded to acetylcholine. For our standard test concentrations, except for 100 µM acetylcholine and 100 µM levamisole, we used 30 µM drug concentrations of different important anthelmintics to limit effects of open-channel block. Interestingly, the novel anthelmintic tribendimidine elicited currents when perfused on oocytes expressing Ode-UNC-29 and Ode-UNC-63. The potency series of the agonists (anthelmintics) based on the normalized currents, Ode(29–63). The 50EC values for pyrantel and tribendimidine were less than the 50EC for acetylcholine but the maxI values were larger for pyrantel and tribendimidine  receptor. When we injected a 1∶5 ratio of Ode-unc-29∶Ode-unc-63 cRNA with the same 1∶1∶1 ratio of ancillary proteins, the mean amplitudes of the pyrantel and tribendimidine currents were significantly increased . The large increase in the size of the currents with increased Ode-unc-63 cRNA suggests that increased UNC-63 favors expression of receptors with a stoichiometry of (UNC-63)3∶ (UNC-29)2Ode-29∶Ode-63 cRNA, the normalized currents of 30 µM nicotine and 100 µM levamisole became significantly bigger than 100 µM acetylcholine (2∶ (UNC-29)3. The increase in currents with the 1∶5 and 5∶1 Ode-29∶Ode-63 cRNA was not due to variability in currents with different batches of oocytes because we observed the same effects when we injected 1∶1, 1∶5 and 5∶1 Ode-29∶Ode-63 cRNA in the same batch of oocytes.We were interested to see if there was evidence of pharmacological changes associated with variation in stoichiometry of the lcholine  suggestiunc-38 and injected Ode-unc-29∶Ode-unc-63∶Ode-unc-38 cRNAs in the ratio 1∶1∶1, along with the three H. contortus ancillary factors. Oocytes injected with this mix responded with larger currents of around 250 nA to 100 µM acetylcholine and to all the other agonists tested except bephenium and thenium (Ode(29–63–38), or the Pyr/Tbd-nAChR. The potency series of the agonists based on the normalized currents was 30 µM pyrantel ≈30 µM tribendimidine >100 µM acetylcholine >30 µM nicotine ≈100 µM levamisole (50EC for pyrantel was less than the 50EC for tribendimidine and acetylcholine (Ode(29–63) receptor.We subsequently added  thenium . Pyrantevamisole  C. The dlcholine . The Hillcholine , showingOde-acr-8 and injected Ode-unc-29∶Ode-unc-63∶Ode-acr-8 cRNA in the ratio 1∶1∶1 along with the three H. contortus ancillary factors into the oocytes, we observed even bigger receptor responses to 100 µM acetylcholine. We also observed responses to all the agonists we tested, including bephenium and thenium , or the ACh-nAChR.When we added  thenium , which sOde(29–63–8) receptor, the potency series of the agonists (anthelmintics) based on the normalized currents, was 100 µM acetylcholine >30 µM tribendimidine ≈100 µM levamisole ≈30 µM pyrantel >30 µM nicotine ≈30 µM thenium ≈30 µM bephenium  was bigger than maxI for tribendimidine (75.0±6.2%) and levamisole (73.0±2.4%).For the ephenium . Here, tephenium  show thaOde(29–63–8) receptor to pyrantel suggest the presence of open-channel block which is more pronounced with Ode(29–63–8) than with the other types. Firstly, the maintained application of 30 µM pyrantel produced a response that declined rapidly despite the concentration being maintained, and secondly, on wash-out of pyrantel the response ‘rebounded’ or increased temporally before declining. Pronounced open-channel block with pyrantel has been observed at the single-channel level Two characteristic features of the response of the Ode-unc-29∶Ode-unc-63∶Ode-acr-8∶Ode-unc-38 cRNA in the ratio 1∶1∶1∶1 along with the three H. contortus ancillary factors. These subunits reconstituted a receptor type on which 100 µM levamisole produced the biggest responses of >1.5 µA, sometimes reaching >4.5 µA, Ode(29–63–8–38) or the Lev-nAChR. The potency series of the agonists based on the normalized currents, was 100 µM levamisole >100 µM acetylcholine >30 µM tribendimidine >30 µM thenium ≈30 µM bephenium ≈30 µM pyrantel >30 µM nicotine (50EC for tribendimidine was less than the 50EC for levamisole and acetylcholine (Ode(29–63–8–38), was most sensitive to levamisole. It is clear from the observations above that each of the four subunits: Ode-UNC-29, Ode-UNC-63 Ode-ACR-8, and Ode-UNC-38 may combine and be adjacent to different subunits as they form different pentameric nAChRs.Finally, we injected all our subunits in a mix of nicotine . Here, tlcholine . The doslcholine . We haveOde (29-63-38-8) and Ode (39-63-38), Ode(29–63–8–38) and pyrantel is more potent on Ode(39-63-38). 50EC dose-ratio of 1.7 . When we tested the antagonist effects of derquantel on Ode(29–63–8–38) using pyrantel as the agonist, we saw mixed non-competitive and competitive antagonism: there was a right-shift in the 50EC and a reduction to 41% of the maximum response by 0.1 µM derquantel, Ode(39-63-38-8) receptors that are preferentially activated by levamisole and which are competitively antagonized by derquantel and; a smaller number of receptors like Ode(39-63-38) that are preferentially activated by pyrantel and antagonized non-competitively by derquantel. To test this further we examined the antagonist effects of derquantel on pyrantel and levamisole activation of Ode (39-63-38). Derquantel antagonized both agonists non-competitively, Ode(39-63-38-8) receptors can give rise to expression of a smaller number of other receptors including Ode(39-63-38). We comment further on these observations in our discussion. Nonetheless, the competitive and non-competitive antagonism of derquantel when levamisole is used on the two different receptor subtypes, We tested the antagonist effects of derquantel on the levamisole and pyrantel concentration-response plots of 50EC concentrations of acetylcholine for the different expressed receptors varied (Ode(29–63–38), Ode(29–63–8) and Ode(29–63–8–38), receptors which produced large enough currents to determine reversal potentials.The s varied , suggestOde(29–63–8–38), and less in the other two receptor types. The potentiating effect of calcium on the acetylcholine currents indicates the presence of positive allosteric binding sites on one or more subunits of the pentameric nAChR Ode(29–63–8)  was similar, 1.3 mV, also giving a permeability ratio Ca/PNaP of 0.4 (Ode(29–63–8–38), the shift was 16.0 mV  receptor, was therefore much more permeable to extracellular calcium and positively allosterically We observed two different effects of increasing calcium from 1 mM to 10 mM on the acetylcholine currents. One effect was potentiation of the current amplitudes, an effect that was voltage-dependent . The vol29–63–8) . Using t29–63–8)  Legend, a of 0.4 . For Ode 16.0 mV , correspOesophagostomum dentatum body muscle have demonstrated the presence of different nAChR types with conductances of 25, 35, 40 and 45 pS, showing the presence of four or more receptor types. We have shown in experiments here, that combinations of four nAChR subunits from this parasite can produce four pharmacologically different receptor types. We found that the expressed Ode(29–63–38–8) Lev-nAChR type was most sensitive to levamisole and had a high permeability to calcium and hypothesized that this type may be the G35 type. Accordingly we investigated the single-channel properties of this type under patch-clamp using 10 µM levamisole as the agonist. In control oocytes injected with only the ancillary proteins, we recorded no nAChR-like channel currents in 4 oocytes. We found however, that in patches from oocytes expressing the Ode(29–63–8–38) type, that more than 90% of patches contained active, recognizable nAChR channels with one main conductance level. + .Single-channel studies in τ, at the different potentials and observed values of 2.4±0.4 ms at +100 mV and 2.9±0.5 ms at −100 mV  receptor. We found that the requirement for all the ancillary proteins was not essential and that each protein, when absent reduced currents produced by the expressed receptors . We have not tested the possibility that post-translational modification may serve as an additional mechanism to produce different receptor types.We have shown previously in the parasitic nematodes, Ode(29–63–38–8), Ode(29–63–8) and Ode(29–63–38) produce the largest current responses to acetylcholine. On the basis of the larger size of the currents responses to acetylcholine and the smaller currents of Ode(29–63) mixture, we speculate that the other three receptor types have more important physiological roles in the worm. Ode(29–63–38) was the most sensitive type to acetylcholine with an 50EC of 3.5±0.2 µM but it had a lower calcium permeability, Ca/PNaP of 0.5, than Ode(29–63–38–8) that had an acetylcholine 50EC of 4.2±0.2 µM. However, Ode(29–63–38–8) had greater responses to levamisole and a calcium permeability, Ca/PNaP of 10.4. Thus the addition of ACR-8 is responsible for increasing the calcium permeability and the response to levamisole. Since the Ode(29–63–38–8) receptor produced the biggest current to levamisole and had a single-channel conductance of 35.1±2.4 pS, close to the in vivo G 35 pS type that is reduced with levamisole resistance, we suggest that Ode(29–63–38–8) subunit combinations give rise to the in vivo G 35 pS channel in O. dentatum. There may also be pH regulatory effects on channel activity of Ode(29–63–38–8). The presence of histidine (pKa 6.0) in the ACR-8 .Ode(29–63–38–8) produces the biggest response to levamisole, while Ode(29–63–38) produces the biggest response to pyrantel. If these two types of receptors exist together in the nematode parasite, our observations suggest that combinations of levamisole and pyrantel will produce a bigger therapeutic response than either of the two anthelmintics given alone and that combination therapy may be advantageous. However, very few reports of combining levamisole and pyrantel for treatment are available. In one report T. muris infected mice, combination of levamisole and pyrantel treatment produced antagonism but these authors did not determine the 50EC dose for pyrantel pamoate and used a very high dose of 300 mg/Kg dose, above the normal therapeutic dose. At this level we anticipate the presence of open-channel block limiting the effects of the therapeutic combination The different nAChR types have different sensitivities to cholinergic anthelmintics: for example Ode(29–63–38–8), the Lev-nAChR, and on Ode(29-63-38), the Pyr/Trbd-nAChR. Interestingly, the mode of action of derquantel varied (competitive or non-competitive) with the receptor subtypes when activated by the same agonist, levamisole. The difference in the mode of action will relate to the preferred non-equivalent binding sites of derquantel, of levamisole and of pyrantel on the receptor subtypes. The competitive antagonism of levamisole suggests a common binding site for derquantel on the Ode(29–63–38–8) receptor; the non-competitive antagonism of pyrantel and levamisole by derquantel on Ode(29–63–38) suggests negative allosteric modulation and different non-equivalent binding sites for derquantel and the agonists, levamisole and derquantel, on this receptor. Interestingly the loss of the ACR-8 subunit, which reduces the levamisole sensitivity of the Ode(29-63-38) receptor, changes the antagonism of levamisole from competitive to non-competitive. Thus ACR-8 may contribute to a high affinity binding site of levamisole and a competitive site of derquantel. When ACR-8 was not present derquantel behaved non-competitively. We have interpreted the mixed non-competitive competitive antagonism of pyrantel by derquantel on Ode(29–63–38–8) to be due to a mixed expression of receptor subtypes with mostly the levamisole sensitive Ode(29–63–38–8) receptors being present and a smaller proportion of other receptors like Ode(29-63-38) being present. This is certainly possible because, as we have shown, the subunits can and do combine in different ways to form 4 types of receptor. Although less likely, it may be that: 1) expression of Ode(29-63-38-8) produces only one receptor type, 2) levamisole and pyrantel bind to separate sites on this receptor, 3) derquantel acts competitively, binding at the levamisole site but; 4) derquantel acts as a negative allosteric modulator when pyrantel binds to its receptor. Further study is required to elucidate details of binding sites on the different receptor types. Despite these possibilities we have seen potent antagonist effects of derquantel on both the Ode(29–63–38-8) and the Ode(29–63–38) subtypes suggesting that derquantel could still be active against levamisole resistance associated with reduced ACR-8 expression.We tested the effects of derquantel on the Ode(29–63–38), Ode(29–63–8) and Ode(29–63–38–8) types. Thus tribendimidine could remain an effective anthelmintic if levamisole resistance in parasites were associated with null mutants or decreased expression of acr-8 and/or unc-38.We were also interested in the effects of the novel cholinergic anthelmintics tribendimidine C. elegans.The different functional properties of the nAChR types indicate that each type may have different physiological roles to play in the parasite and the proportion of the different types present at the membrane surface is likely to be regulated. This regulation could allow changes in calcium permeability, desensitization, receptor location, receptor number and anthelmintic sensitivity. The control is expected to include dynamic physiological processes and developmental process that take place over different time scales. An adaptable and diverse receptor population may allow changes, even during exposure to cholinergic anthelmintics. We know little about these processes, their time-scales and how they may be involved in anthelmintic resistance in parasitic nematodes but some information is available from C. elegans there are a number of genes involved in processing and assembly of the subunits of AChR; specific examples include Cel-ric-3, Cel-unc-74 and Cel-unc-50Cel-unc-74 encodes a thioredoxin-related protein required for the expression of levamisole AChR subunits Cel-unc-50 encodes a transmembrane protein in the Golgi apparatus Cel-unc-50 mutants, levamisole nAChR subunits are directed to lysosomes for degradation. We found that acetylcholine currents and pharmacological profile of the receptors were sensitive to the presence of the accessory proteins, RIC-3, UNC-74 & UNC-50, suggesting the possibility that the expression level of these proteins could contribute to anthelmintic resistance in parasitic nematodes.In O. dentatum receptor subunits can be used to reconstitute different functional levamisole receptor types and that levamisole is a full agonist in one of the main types. All of the four main O. dentatum receptor types responded to nicotine, which supports our use of the term nAChR. In contrast, in the expressed levamisole-sensitive AChRs of C. elegans, levamisole is a partial agonist, requires five subunits (Cel-UNC-38:Cel-UNC-29:Cel-UNC-63:Cel-LEV-1:Cel-LEV-8) and does not respond to nicotine. The C. elegans nicotine-sensitive muscle receptor is a separate homopentamer of ACR-16 O. dentatum and the expressed AChR of H. contortusXenopus oocytes expressing H. contortus subunits. Furthermore, we saw differences and similarities between the expressed O. dentatum receptors and the expressed A. suum receptors A. suum AChR, such that varying the amounts of the subunit cRNAs injected changed the pharmacology of the expressed receptors. This is similar to the Ode(29–63), in that only two subunits were used to reconstitute a Pyr-nAChR and varying the amount of injected subunit cRNAs affected the pharmacology of the receptor. Whereas no ancillary factors were used to reconstitute the A. suum nAChRs, we observed that the three ancillary factors were required to reconstitute the O. dentatum receptors. Another observation is that the amounts of cRNA we injected to reconstitute the O. dentatum receptors were far less than what was injected to reconstitute the A. suum receptors. These demonstrate differences between the expressed receptors of nematodes in the same clade  and/or in different clades (O. dentatum and A. suum). In addition we have also demonstrated tribendimidine's effect on all four O. dentatum receptor types and in two of the receptor types, the 50EC values show tribendimidine was more potent than the other agonists across the types. We found that pyrantel was the most potent agonist of two types of O. dentatum receptors but it is only most potent in one of the types of H. contortus. The real and significant differences between the effects of pharmacological agents on the model nematode C. elegans and on the parasitic nematode O. dentatum, both Clade V nematodes, shows that species variation requires that effects of anthelmintics on the relevant nematode parasite are tested as a vital reality check.We show here that four All animal care and experimental procedures in this study were in strict accordance with guidelines of good animal practice defined by the Center France-Limousin ethical committee (France). The vertebrate animals (pig) studies were performed under the specific national (French) guidelines set out in the Charte Nationle portant sur l'ethique de l'experimentation animal of the Ministere De Lensiengnement Superieur et de la Recherch and Ministere De L'Agriculture et de la Peche experimental agreement 6623 approved by the Veterinary Services (Direction des Services Vétérinaires) of Indre et Loire (France).O. dentatum as previously described These studies were carried out on the levamisole-sensitive (SENS) isolate of O. dentatum SENS isolate were used for total RNA preparation using TRIzol (Invitrogen) according to the manufacturer's instructions. First-strand cDNA synthesis on resuspended and DNase-treated total RNA was carried out with the oligo (dT) RACER primer and superscript III reverse transcriptase  as previously described O. dentatum unc-29, unc-63 and acr-8 homologues, 5′- and 3′- rapid amplification of complementary ends (RACE) polymerase chain reactions (PCR) were performed using the splice leader sequence primer (SL1) with internal reverse primers and internal forward specific primers with reverse transcription 3′-site adapter primers, respectively. PCR products were cloned into the pGEM-T vector (Promega). Positive identifications were made by BLAST analysis against all Haemonchus contortus entries in the H. contortus information resource data base. Primers designed on unc-29 sequence from H. contortus and C. elegans  allowed PCR amplification of a 288 bp fragment corresponding to partial O. dentatum unc-29 homologue. New gene specific primer sets were designed on this sequence for the 5′- and 3′-RACE-PCR providing the full-length cDNA sequence of Ode-unc-29. To identify the unc-63 homologue from O. dentatum, degenerate primers were designed based on the Ode-unc-38 mRNA sequence (accession number GU256648) and the unc-63 and unc-38 sequences of cDNAs available from multiple nematode species . An 861 bp partial coding sequence of Ode-acr-8 cDNA was amplified using a sense primer (Hco-acr-8-FT1 TATGGTTAGAGATGCAATGGTT) and an antisense primer (Hco-acr-8-RE8 GTGTTTCGATGAAGACAGCTT) from H. contortus Hco-acr-8. The product of this reaction was cloned and sequenced, and the data were used to design primers to get the 5′ and the 3′ ends of the target transcript. To amplify the full-length coding sequence of Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, we used the Phusion DNA polymerase (Finnzymes) and gene specific primer pairs containing HindIII or XhoI and ApaI restriction enzyme sites , purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel), ligated into the respective corresponding sites of the pTB-207 expression vector with T4 DNA Ligase (New England Biolabs), and resulting constructs were transformed into E. coli DG1 cells (Eurogentec) in vitro cRNA synthesis from linearized plasmid DNA templates. The cRNAs were precipitated by lithium chloride, resuspended in RNAse-free water and stored at −80°C.Ten adult males from C. elegans: UNC-29 NM_059998, UNC-38 NM_059071, UNC-63 NM_059132, ACR-8 JF416644; H. contortus: Hco-unc-29.1 GU060980, Hco-unc-38 GU060984, Hco-unc-63a GU060985, Hco-acr-8 EU006785, Hco-unc-50 HQ116822, Hco-unc-74 HQ116821, Hco-ric-3.1 HQ116823; O. dentatum: Ode-unc-29 JX429919, Ode-unc-38 JX429920, Ode-unc-63 HQ162136, Ode-acr-8 JX429921.Database searches, prediction of conserved motifs and phylogenetic analyses for the cDNAs were carried out as already described Xenopus laevis ovaries were obtained from NASCO  and defolliculated using 1–2 mg/ml collagenase type II and Ca2+-free OR2 . Alternatively, defolliculated oocytes were purchased from Ecocyte Bioscience . Oocytes  were microinjected with ∼36 nL of a cRNA injection mix containing 50 ng/µL of each subunit cRNA, as described in H. contortus ancillary factors ric-3, unc-50 and unc-74 were added to each mix. Microinjected oocytes were incubated at 19°C for 2–5 days. The oocytes were incubated in 200 µL of 100 µM BAPTA-AM for ∼3 hours prior to recordings, unless stated otherwise. In control experiments, we recorded from un-injected oocytes. Recording and incubation solutions used are reported in 2+  to reduce K+ currents and lower opening of Ca2+-activated Cl− channels. Patch electrodes were pulled from thin-walled capillary glass tubing , coated close to the tip with sylgard and fire-polished. The electrodes were filled with a high pipette solution {composition in mM: CsCl 35, CsAc 105, CaCl2 1, HEPES 10} containing 10 µM levamisole. Axopatch 200B amplifier  was used to amplify currents which were sampled at 25 kHz with Digidata 1320A (Axon Instruments) and filtered at 1.5 kHz (3-pole Bessel). The linear least squares regression was used to estimate the conductances of the channels since the slopes were linear and did not show rectification.Oocyte-attached (outside-out) patch-clamp procedure was used to obtain all recordings at room temperature (20°C–25°C). Oocytes were prepared for the recordings by removal of the vitelline membrane with forceps after placing in hypertonic solution, as already described BpK values, the graphs were fitted with the Gaddum/Schild 5EC0 shift and the bottom of the curves were set to zero.Acquired data were analyzed with Clampfit 9.2  and Graphpad Prism 5.0 software . The peak of currents in BAPTA-soaked oocytes was measured. The response to 100 µM ACh was normalized to 100% and the responses to the other agonists normalized to that of ACh. For all dose-response relationships, the mean ± s.e of the responses is plotted. Dose-response data points were fitted with the Hill equation as described previously Figure S1O. dentatum nAChR subunits with the H. contortus and C. elegans homologues. The sequences were aligned with the MUSCLE algorithm A–D. Amino acid alignments of the four (TIF)Click here for additional data file.Figure S2Oesophagostomum dentatum , Caenorhabditis elegans (Cel), Haemonchus contortus (Hco) and Teladorsagia circumcincta (Tci). Numbers at each branch indicate percentage boostrap values corresponding to 1000 replicates. The scale bar represents substitutions per site. The C. elegans acr-16 nAChR subunit was used as an outgroup. Distance analyses were performed on full-length cDNA sequences. Multiple alignment was performed using Muscle program with standard parameters Distance tree showing relationships of nicotinic acetylcholine receptors (nAChR) subunit sequences in (TIF)Click here for additional data file.Figure S3Ode(29–63–38–8) or Lev-nAChR subtype. This receptor subtype was used to test the effect of sequentially removing the ancillary proteins on the reconstitution. All responses were normalized to control 100 µM ACh (unfilled bar) currents in oocytes expressing this receptor subtype with all the ancillary proteins (A). (B) Effect of removing unc-74 from the mix on the Ode(29–63–38–8)/Lev-nAChR subtype. Note the relative change in ACh and Lev responses. (C) Effect of removing ric-3 on the Ode(29–63–38–8) : Lev-nAChR subtype. The ACh current responses were reduced to <50% of the control. (D) Effect of removing unc-50 on the Ode(29–63–38–8) or Lev-nAChR subtype. Note the dramatic decrease in currents elicited by all agonists.Effect of the ancillary proteins on receptor reconstitution. (A) Bar chart (mean ± se) of agonist-elicited currents in the (TIF)Click here for additional data file.Results S1Supplementary results.(DOCX)Click here for additional data file.Text S1Ca/PNaP ratio. All assumptions for the permeability calculation are noted. For these set of experiments, we increased external Ca2+ in the recording solution from 1 mM to 10 mM without changing the concentration of the other ions. We used the GHK equation to calculate the permeability ratio, Ca/PNaP. Due to BAPTA treatment before recordings, we assumed the internal [Ca2+] to be negligible; we also assumed permeability to Na and K, NaP and KP, are equal. Ionic activities were used for the calculations (activity coefficients: 0.56 for Ca2+ and 0.72 for Na+ and K+). Below is the GHK equation used to calculate the calcium permeability ratio, with the activity coefficients for Na+, K+ and Ca2+ inserted: rev = RT/F In{(PNa*0.72[Na]o+PK*0.72[K]o+4P′*0.56[Ca]o)/{PNa*0.72[Na]i+PK*0.72[K]i)}E. Where R is universal gas constant (8.314 JK−1mol−1); F is Faraday's constant (96485 Cmol−1); T is temperature ; and Ca/PNa{1/(1+eFErev/RT)}P′ = P. A table of the primers (Table S1) used for the PCR and cloning of the four O. dentatum AChR subunits is also included here in Supplementary information indicating how the calcium permeability measurements were conducted and the equation used for calculating the (PDF)Click here for additional data file.