Fusarium oxysporum. The virus, termed Hadaka virus 1 (HadV1), has 11 (+)RNA genomic segments, the largest number in known (+)RNA viruses. Nevertheless, HadV1 lacked a typical structural protein of polymycoviruses and was not pelleted by standard ultracentrifugation, implying an unusual capsidless nature of HadV1. This study reveals a potential novel lifestyle of multisegmented RNA viruses.Fungi collectively host various RNA viruses. Examples include encapsidated double-stranded RNA (dsRNA) viruses with diverse numbers of genomic segments (from 1 to 12) and capsidless viruses with nonsegmented (+)RNA genomes. Recently, viruses with unusual intermediate features of an infectious entity between encapsidated dsRNA viruses and capsidless (+)RNA viruses were found. They are called polymycoviruses, which typically have four to eight dsRNA genomic segments associated with one of the virus-encoded proteins and are phylogenetically distantly related to animal (+)RNA caliciviruses. Here, we identified a novel virus phylogenetically related to polymycoviruses, from the phytopathogenic fungus  Fusarium oxysporum, was characterized. The genomic RNA of HadV1 consisted of an 11-segmented positive-sense RNA with highly conserved terminal nucleotide sequences. HadV1 shared the three conserved segments with known polymycoviruses but lacked the PASrp-encoding segment. Unlike the known polymycoviruses and encapsidated viruses, HadV1 was not pelleted by conventional ultracentrifugation, possibly due to the lack of PASrp. This result implied that HadV1 exists only as a soluble form with naked RNA. Nevertheless, the 11 genomic segments of HadV1 have been stably maintained through host subculturing and conidiation. Taken together, the results of this study revealed a virus with a potential novel virus lifestyle, carrying many genomic segments without typical capsids or PASrp-associated forms.The search for viruses infecting fungi, or mycoviruses, has extended our knowledge about the diversity of RNA viruses, as exemplified by the discovery of polymycoviruses, a phylogenetic group of multisegmented RNA viruses with unusual forms. The genomic RNAs of known polymycoviruses, which show a phylogenetic affinity for animal positive-sense single-stranded RNA [(+)RNA] viruses such as caliciviruses, are comprised of four conserved segments with an additional zero to four segments. The double-stranded form of polymycovirus genomic RNA is assumed to be associated with a virally encoded protein  in either of two manners: a capsidless colloidal form or a filamentous encapsidated form. Detailed molecular characterizations of polymycoviruses, however, have been conducted for only a few strains. Here, a novel polymyco-related virus named Hadaka virus 1 (HadV1), from the phytopathogenic fungus  Note that large-scale evolutionary analysis of RNA viruses has still been controversial because only limited RdRP motifs are conserved  for their replication, into three groups based on their genome types: double-stranded RNA (dsRNA), positive-sense single-stranded RNA [(+)RNA], and negative-sense single-stranded RNA [(−)RNA] , 3. Wolfonserved , 6. HowePolymycovirus” in the family “Polymycoviridae” in the realm Riboviria, according to the taxonomy proposal submitted to the ICTV . These 10 candidates were isolated from different fungal species but share four conserved genomic RNA segments, termed dsRNA1, dsRNA2, dsRNA3, and dsRNA4, that putatively encode an RdRP, a protein with unknown function (containing a transmembrane domain and a zinc-finger-like motif), a methyltransferase (MTR), and a proline-alanine-serine-rich protein (PASrp), respectively (17–17–17–Members of the kingdom Fungi host diverse RNA viruses (RNA mycoviruses) . Fungal 10–ectively , 17–22. ly 17–17–. RdRPs oly 17–17–, 24. How17–17–17–, 22, whi17–17–17–.Polymycoviridae, has been reported to be associated with PASrp in a colloidal form  region and the elongation factor 1 alpha gene , three of the NGS contigs showed low-identity (approximately 23 to 33% identity at the amino acid sequence level) but significant BLASTX hits to each dsRNA1-, dsRNA2-, and dsRNA3-encoded protein of the known polymycoviruses  and uninfected (VF) fungal isolates . Download FIG S2, JPG file, 0.6 MB.Comparison between the putative proteins encoded on the HadV1 genome and the putative polymycovirus PASrps. (A) Comparison of the HadV1 genomic segments with the PASrp-encoding segments of 10 polymycoviruses. RdRP, MTR, and ZF denote RNA-dependent RNA polymerase, a putative methyltransferase, and a zinc-finger-like protein, respectively. The black boxes indicate hypothetical ORFs. The values shown on the right indicate the estimated molecular mass  of each hypothetical protein. (B) Amino acid compositions of the putative proteins. GenBank accession numbers for the amino acid sequences of polymycovirus PASrps are listed in Copyright © 2020 Sato et al.2020Sato et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Because PASrp is a hallmark of polymycoviruses, further attempts were made to search HadV1 proteins for PASrp-like proteins. Some genomic segments of HadV1, such as S4 and S5, have coding capacities similar to those of the PASrp-encoding segments of polymycoviruses . The pro10.1128/mBio.00450-20.3FIG S3FIG S3, JPG file, 1.4 MB.Multiple-sequence alignment of the deduced amino acid sequences of the HadV1 hypothetical proteins  and the polymycovirus PASrps using PSI-Coffee. The black arrow indicates scores of consistency within the alignment. Download Copyright © 2020 Sato et al.2020Sato et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the http://www.rib.okayama-u.ac.jp/pmi/Supplemental%20Material.html). AfuTmV1 shared BLASTP-detectable RdRP sequences with only some vesiviruses in the family Caliciviridae in addition to polymycoviruses (see Table S1 at the URL mentioned above). These results suggested that HadV1 and polymycoviruses have very distant phylogenetic relationships to known viruses except for vesiviruses.Based on a BLASTP search, the RdRP sequences of HadV1 and the proposed polymycoviruses, except for AfuTmV1, showed similarity exclusively to RdRPs of polymycoviruses and related viruses listed in Caliciviridae and Astroviridae rather than with dsRNA viruses in the families Partitiviridae and Amalgaviridae . The multiple-sequence alignment showed that HadV1 RdRP as well as RdRPs of polymycoviruses possessed several domains conserved in these picornavirus-like supergroup members RNA viruses in the families aviridae . Thus, waviridae  or membe members . A phylo members . However members . As prev members , HadV1 a members .10.1128/mBio.00450-20.4FIG S4http://www.rib.okayama-u.ac.jp/pmi/Supplemental%20Material.html. The alignment was visualized with MEGA X. Download FIG S4, JPG file, 2.0 MB.Part of the multiple-sequence alignment of the RdRP sequences of HadV1 and polymycoviruses. The filled and unfilled orange circles indicate published and unpublished polymycoviruses, respectively. Viruses that are closely related to polymycoviruses but not assigned to any taxa  are indiCopyright © 2020 Sato et al.2020Sato et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00450-20.5FIG S5FIG S5, JPG file, 0.3 MB.Maximum likelihood phylogenetic tree based on RdRP amino acid sequences. This tree shows all viruses hidden in Copyright © 2020 Sato et al.2020Sato et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Taken together, HadV1 as well as phylogenetically closely related polymycoviruses seem to show a higher phylogenetic affinity for (+)RNA viruses than for dsRNA viruses.The 11 dsRNA segments were maintained during repeated subculture of single HadV1-infected isolates. We tested by one-step RT-PCR how frequently HadV1 was vertically transmitted via asexual spores  present F. oxysporum by HadV1 on a nutrient-rich medium (The preparation of isogenic HadV1(−) and HadV1(+) fungal subisolates allowed us to investigate HadV1 effects on the host fungus. Colony size and morphology were indistinguishable between the HadV1(−) and HadV1(+) subisolates, suggesting asymptomatic infection of h medium .10.1128/mBio.00450-20.6FIG S6F. oxysporum isolate 7n. (A) Microscopic observation of conidia harvested from isolate 7n. (B) Detection of HadV1 RNA1 (RdRP-encoding segment) in 7n single conidial subisolates by direct colony one-step RT-PCR. Conidia taken from younger (2- to 4-day-old) (top) or older (10- to 12-day-old) (bottom) mycelia were tested. The ef1F. oxysporum was employed as a control for RT-PCR. (C) Frequency of HadV1 vertical transmission through conidiation. Single conidial subisolates were prepared from young (2- to 4-day-old) and old (10- to 12-day-old) colonies and tested for the presence of virus. HadV1(−) and HadV1(+) indicate the absence and presence of virus, respectively. The assay was repeated twice (Rep1 and Rep2). (D and E) Electrophoretic gel profile of dsRNA fractions from 7n single conidial subisolates. HadV1(−) (7n-cfn) and HadV1(+) (7n-cvn) subisolates were obtained from old (D) or young (E) colonies. All the conidial subisolates here are different from the subisolates used in n and 7n-cvn in panel E were used in n” represents a different independent subisolate. Download FIG S6, JPG file, 0.5 MB.All-or-none transmission of viral dsRNA segments to single conidial subisolates of Copyright © 2020 Sato et al.2020Sato et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00450-20.7FIG S7F. oxysporum isolate 7n. (A) Colony morphology of representative HadV1(+) and HadV1(−) cultures on PDA medium. The conidial subisolates described in the legend of t test  using R software. Note that mycelial growth was slightly faster than that in FIG S7, JPG file, 0.5 MB.Comparison of mycelial growth between HadV1-free (−) and HadV1-infected (+) single conidial subisolates of Copyright © 2020 Sato et al.2020Sato et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the g for 1.5 h) of resuspended particles on a 20% sucrose cushion (Alphachrysovirus), harbored in an isolate (A-60) of F. oxysporum. Chrysoviruses have multipartite dsRNA genomes enclosed in icosahedral capsids  were detected in the pellet but not in the supernatant . HoweverNext, we examined where the dsRNA segments were fractionated after Ucfg. For HadV1, the resuspended PEG precipitant (PEG-ppt) and the supernatant after PEG-NaCl-mediated precipitation (PEG-sup) were separately ultracentrifuged , which sg for 1 to 2 h). Thus, we next examined whether presumed HadV1 particles were pelleted by Ucfg of mycelial extracts that were obtained without organic solvent extraction , from Cryphonectria parasitica was also pelleted . The experimental procedure is described in the legend of Copyright © 2020 Sato et al.2020Sato et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the These combined results showed that HadV1 has an extremely low sedimentation coefficient compared to the previously characterized polymycoviruses, the membrane vesicle-associated capsidless hypoviruses, and the encapsidated viruses, suggesting a unique capsidless nature of HadV1.ef1α and benA), which could be observed in the RNase A-untreated samples, were undetectable in all the RNase-treated samples RNA, 3 segments of which showed significant similarity to the conserved segments  of polymycoviruses (in vitro (F. oxysporum with HadV1-derived dsRNA were unsuccessful (data not shown). Thus, the infectivity of purified viral dsRNA, which was reported previously for two polymycoviruses  and endornaviruses (family Endornaviridae) had historically been classified as dsRNA viruses by the ICTV until recently because their dsRNA molecules as a replicative form accumulate in large amounts in infected host cells and are readily detectable. However, the ICTV has reclassified them as (+)RNA viruses according to RdRP- and/or RNA helicase-based phylogeny . Note that the phylogenetic positions of members of branch II proposed by Wolf et al.   –41 and c–viridae) . Some Haviridae) . There aHypoviridae, Narnaviridae, and Endornaviridae, have nonsegmented (+)RNA genomes  and ScV23S, lacks antiviral RNA silencing  medium . The PDA cultures were placed on a laboratory bench near windows at room temperature (23°C to 27°C). For subculture, a 1-mm3 fresh mycelial plug was transferred to new PDA in flat petri dishes (9 cm in diameter) every 7 to 10 days. For a long-term stock, mycelial plugs were stored in 10% (vol/vol) glycerol at −80°C.The F. oxysporum isolate 7n was collected from a tomato in a vegetable market in Pakistan. Isolate 7n was identified as F. oxysporum by sequencing of PCR products of the ITS amplified with primers ITS1 and ITS4 . Virus-free isogenic subisolates of isolate 7n were obtained by single-conidium isolation as described below.The and ITS4 , the IGSand ITS4 , and ef1and EF-2   , was used as a control host of an encapsidated virus. In addition, a Japanese strain (9859-1) of F. oxysporum f. sp. lycopersici race 2, a generous gift from Masashi Matsusaki of the Aichi Agricultural Research Center, was used in several experiments as a virus-free control  . As a virus-free isogenic control for A-58, one of its conidial subisolates (A-58-cf1) was used. A standard strain (EP155) of C. parasitica infected with the exemplar strain of hypovirus, CHV1, was also used  of  control . A Pakislso used .dsRNA was extracted from mycelia cultured on PDA-cellophane for 2 days by cellulose column chromatography as previously described . The dsR+ membrane  in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). After overnight blotting, dsRNA on the membrane was denatured as described previously by Sun et al. . The plausible ORFs were predicted using Open Reading Frame Finder (https://www.ncbi.nlm.nih.gov/orffinder/) provided by the NCBI.The complete genomic sequence of HadV1 was determined by Sanger sequencing of clones from a random cDNA library and by 3′ RLM-RACE. The random cDNA library was prepared by a PCR-based method  with dsRCaliciviridae, 6 members of the family Astroviridae, and 12 members of the family Partitiviridae. The full names of the viruses and GenBank accession numbers of their RdRP sequences are shown in http://www.rib.okayama-u.ac.jp/pmi/Supplemental%20Material.html, except for PjPmV1 (see above). Some amino acid sequences were trimmed before multiple-sequence alignment, according to the experimentally determined polyprotein cleavage site or putatively annotated RdRP domain. The sequences were aligned by using PSI-Coffee  , 13 members of the family . 11.00) . The obt. 11.00) . The phy. 11.00) .F. oxysporum isolate 7n were suspended in sterilized water and spread onto PDA medium to obtain single conidial subisolates. Whether 7n single conidial subisolates were HadV1 positive or negative was determined by mycelial direct one-step RT-PCR  mixed with the respective specific primers (see Table S3 at the URL mentioned above) and 0.5 μl of the 10-fold-diluted cDNA products in a total reaction mixture volume of 10 μl. The PCR cycle conditions were according to the manufacturer’s standard 3-step program.The presence of a full set of HadV1 genomic segments in conidial HadV1(+) subisolates was confirmed by RT-PCR . Total s2) using a mortar and pestle. The frozen mycelial powder was homogenized in 4 volumes (vol/wt) of 0.1 M sodium phosphate (pH 7.0) and 0.004 volumes (vol/wt) of 2-mercaptoethanol. The homogenate was mixed with a one-quarter volume of CCl4 and immediately centrifuged at 2,000 × g for 20 min at 4°C. This CCl4 extraction step was repeated once. The supernatant was mixed with 8% (wt/vol) PEG  and 1% (wt/vol) NaCl and stirred for 2 h on ice. The solution was centrifuged at 15,000 × g for 20 min at 4°C. The resultant pellets were suspended in 0.05 M phosphate buffer (pH 7.0). The suspension was again centrifuged at 8,000 × g for 10 min at 4°C to remove undissolved PEG. The supernatant was overlaid onto a 20% (wt/vol) sucrose cushion in a 5:2 volume ratio and ultracentrifuged at 100,000 × g for 1.5 h at 4°C with an Optima L-100K ultracentrifuge . After ultracentrifugation, the resultant pellet was dissolved in 0.05 M sodium phosphate (pH 7.0). The dsRNA contained in each fraction, equivalent to the content in 250 mg of mycelia, was purified by PCIA and CIA extraction followed by cellulose column chromatography.Crude virus particle fractions were obtained by a conventional method as previously described  27, 28,–32. Fung–g) for 20 min at 4°C to remove cell debris. The supernatant was optionally subjected to CCl4 extraction (g for 1.5 h at 4°C with an Optima L-100K ultracentrifuge. The resultant pellet was suspended in 0.05 M sodium phosphate (pH 7.0). The dsRNA in each fraction was purified as mentioned above. The ssRNA in each fraction was enriched by PCIA and CIA extraction followed by 2 M lithium chloride (LiCl) precipitation.For milder purification , the frotraction , bottom.2. The mycelial powder was homogenized in 4 volumes (vol/wt) of 0.05 M sodium phosphate (pH 7.0) and filtered through Miracloth, as in the above-mentioned milder virus purification steps  mRNA (P. janthinellum β-tubulin mRNA (benA) were employed as internal controls for RT-PCR, in which primer sets EF-1 and EF-2  mRNA  and P. jand EF-2  and Bt2aand EF-2  were useLC519840 to LC519850.The complete nucleotide sequence of the HadV1 genome was deposited in the EMBL/GenBank/DDBJ database under accession numbers


The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of this variability is the accumulation of mutations in viral genes and reassortment enabled by its segmented genome. The latter process can induce major changes and the production of new strains with pandemic potential. However, not all genetic combinations are tolerated and lead to the assembly of complete infectious virions. Reports have shown that viral RNA segments co-segregate in particular circumstances. This tendency is a consequence of the complex and selective genome packaging process, which takes place in the final stages of the viral replication cycle. It has been shown that genome packaging is governed by RNA–RNA interactions. Intersegment contacts create a network, characterized by the presence of common and strain-specific interaction sites. Recent studies have revealed certain RNA regions, and conserved secondary structure motifs within them, which may play functional roles in virion assembly. Growing knowledge on RNA structure and interactions facilitates our understanding of the appearance of new genome variants, and may allow for the prediction of potential reassortment outcomes and the emergence of new strains in the future. Orthomyxoviridae. Its viral genome is organized in eight, negative-sense RNA segments [Influenza A virus (IAV) is responsible for common respiratory infection in human, spreading as seasonal epidemics and sporadic pandemics. IAV belongs to the family segments . The virsegments ,3.The vRNA segments in the virion are organized in ribonucleoprotein complexes (vRNPs) ,5. Each The correlation between the structure and function of RNA has been the subject of intensive research for years . NumerouResults of mapping experiments performed in cellulo, in vivo, and ex virio—conditions in which interactions with proteins and other factors occur—are often difficult to interpret. The availability of nucleotides may be affected by the existence of additional interactions, rather than their involvement in the formation of secondary structure motifs. Although RNA structure mapping has been conducted in vivo in several studies, their conclusions were modest and careful, focusing on thermodynamically stable, small local motifs. This is reasonable when interpretation is hard, due to the many factors that could influence chemical mapping results. These factors include RNA–RNA interactions, such as with viral RNA and with proteins . The other difficulty are the limitations of RNA folding programs, which expand with the length of the studied RNA. Incorporation of experimental data can greatly facilitate RNA structure prediction; however, not all of the RNA folding rules are known and, even though many are known, some of the defined rules are chosen by different prediction programs, in order not to be incorporated in parallel during calculation, due to the complexity of such folding. At present, researchers may routinely choose several individual options: prediction without pseudoknot, pseudoknot prediction separately, choosing pairing distance , or no pairing distance. These options generate different secondary structures, which all need to be considered and coupled with other experiments.Recently, a work has been published on the vRNA structure in purified A/WSN/1933 (H1N1) (WSN) virions (in virio) and in vitro transcribed RNA segments or deproteinized virus particles (ex virio), as analyzed by selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) coupled with next-generation sequencing (NGS) . A numbeIt has been found that NP binds to vRNA in a non-uniform manner ,45. ThisAnother study defined the binding profile of NP for each IAV vRNA segment in the infected cell culture supernatant using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) coupled with NGS . The NP–Recent reports have also shown that vRNPs may present heterogeneous conformation and dynamic structure . In cryoOnly a complete virion, containing exactly one copy of each RNA segment, is able to infect host cells. Selective packaging of segments is a topic which has been thoroughly investigated by researchers. Electron microscopy images have provided information on the overall genome architecture, where the seven vRNPs are arranged around a central one, regardless of the tested strain ,48,49,50The mutagenesis of regions or residues selected as candidates for functional packaging signals and successive investigations into their effects on viral production provide a valuable source of information. The role of packaging signals within coding regions has been investigated through the introduction of synonymous mutations, which altered genome assembly ,43. The Research on packaging signals has also been performed using viruses carrying premature termination codons in HA or M2 (matrix protein 2) and containing random sequences (10–12 nucleotides) in the 3′ bundling signals of these segments . These eSimilar conclusions concerning packaging sequences have been drawn in experiments conducted on the A/SC35M H7N7 (SC35M) influenza strain . To compMutational studies of vRNA5 terminal packaging regions defined as low-NP binding in PAR-CLIP analysis have been carried out, in order to prove the RNA structure-function correlation . These rRecently, RNA–RNA interactions have been investigated in virio in WSN using the sequencing of psoralen-crosslinked, ligated, and selected hybrids (SPLASH) method and validated by qPCR . A complMultiple intersegment interactions of WSN concentrated around hotspots have also been detected in virio in a study using dual crosslinking, immunoprecipitation, and proximity ligation (2CIMPL) . About 1Recently, a comprehensive study of RNA-RNA interactions using high-throughput sequencing techniques has been reported . CrossliThese recent findings were also compliant with previous reports concerning human A/Moscow/10/99 (H3N2) (Moscow), avian A/Finch/England/2051/91 (H5N2) (England), and WSN strains ,49,56. TThe segmented genome of the influenza virus is a source of genetic diversity, obtained through a process of antigenic shift. This phenomenon is responsible for significant antigenic changes. The reassortment of segments between animal and human strains may lead to the production of strains with pandemic potential. Certain exchange and composition of segments enables transmission from animals to humans, sustained person-to-person transmission, and other features carried by new strains . Co-circFrom the calculated 256 possible genetic sets which may theoretically appear during segment exchange between two strains, only a small number have been identified in natural or laboratory conditions ,62. SomeA valuable source of data comes from vaccine production procedures, where a so-called seed strain is generated through the reassortment of a seasonal influenza virus with an egg-adapted strain . The maiReassortation has also been studied through the use of reverse genetics. Segment exchange between the aforementioned seasonal Udorn and the high-growth PR8 was investigated in a competitive plasmid transfection models ,67. The Site–directed mutagenesis was applied to exchange PB1 gene fragments between strains and reveal which part of the segment is important for the incorporation into the progeny virion. The effect was studied through competitive plasmid transfection . These eResearch carried out on reassortant viruses has indicated that the network of RNA interactions is inherited from parental strains. One of the reports focused on reassortant virus strain obtained under laboratory conditions, which acquired segments PB1 and NA from Udorn and the other vRNAs from PR8 strain (the co-segregation of vRNAs have been reported previously and described above) . The strAn experiment has also been conducted concerning the reverse-engineering of viruses, in which plasmids containing vRNA segment sequences from PR8 were transfected together with plasmids containing vRNA segment sequences of other seasonal H3N2 strains . The co-The experimental data was also consistent with the observation that avian segment PB1 and segment NA from N2 subtype tend to co-segregate in natural environments. Such an event led to the production of the Asian influenza pandemic strain in 1957 and the Hong Kong pandemic in 1968. In the first case, the (at that time) seasonal strain H1N1 acquired avian H2N2 segments PB1, HA, and NA. In the second event, the seasonal H2N2 virus reassorted with avian-origin segments PB1 and HA, through the interaction of segment NA from seasonal subtype N2 with avian segment PB1. Thorough studies on this co-segregation tendency have emphasized the influence of RNA sequence and structural changes on the fate and evolution of the virus. Further analyses of such correlations may lead to better prediction of strain reassortment, improved vaccine design, and allow for assessment of the epidemiological risks related to the production of new viral strains.Another example of gene selection bias is segment HA of avian, swine or equine origin which, according to thorough analyses, is always incorporated into a human virus, together with other non-human segments . The setAnother group of interesting processes is related to the so-called incomplete viral genomes (IVGs) present within the infected cell ,70,71. RReassortation events and the expansion of new viral strains among poultry has elevated the risk of avian-human IAV gene exchange . A recenNP is a viral protein which, apart from its structural role, fulfills important functions in the replication cycle, from transcription and replication, through nuclear import and export of vRNP complexes, to genome packaging . The proThe published data provide a consistent picture of the influenza RNA structure and interactions that influence genome assembly and progeny virion production . A numbe


The SLC22 family of OATs, OCTs, and OCTNs is emerging as a central hub of endogenous physiology. Despite often being referred to as “drug” transporters, they facilitate the movement of metabolites and key signaling molecules. An in-depth reanalysis supports a reassignment of these proteins into eight functional subgroups, with four new subgroups arising from the previously defined OAT subclade: OATS1 , OATS2 (SLC22A7), OATS3 , and OATS4 . We propose merging the OCTN  and OCT-related (SLC22A15 and SLC22A16) subclades into the OCTN/OCTN-related subgroup. Using data from GWAS, in vivo models, and in vitro assays, we developed an SLC22 transporter-metabolite network and similar subgroup networks, which suggest how multiple SLC22 transporters with mono-, oligo-, and multi-specific substrate specificity interact to regulate metabolites. Subgroup associations include: OATS1 with signaling molecules, uremic toxins, and odorants, OATS2 with cyclic nucleotides, OATS3 with uric acid, OATS4 with conjugated sex hormones, particularly etiocholanolone glucuronide, OCT with neurotransmitters, and OCTN/OCTN-related with ergothioneine and carnitine derivatives. Our data suggest that the SLC22 family can work among itself, as well as with other ADME genes, to optimize levels of numerous metabolites and signaling molecules, involved in organ crosstalk and inter-organismal communication, as proposed by the remote sensing and signaling theory. The SLC (solute carrier) gene family includes 65 families with over 400 transporter genes. In humans, 52 of these families are expressed, encompassing more than 395 genes and it has been estimated that 2000 (10% of the genome) human genes are transporter-related . SeTo visualize these transporter-metabolite interactions, which were acquired from a combination of GWAS, in vivo, and in vitro studies, we created networks using Cytoscape . These nWhile there is no single metabolite that is associated with all SLC22 transporters, some are linked to multiple family members, and thus may be a hallmark of the subgroup or family as a whole. These metabolites are prostaglandin E2, prostaglandin F2, estrone sulfate, uric acid, carnitine, and creatinine, which are each linked to at least five different SLC22 members, respectively . This reSeveral metabolites have been identified as substrates of SLC22A6 (OAT1) and SLC22A8 (OAT3). While many are unique, there is notable overlap. Both OAT1 and OAT3 interact with uremic toxins  and gut microbiome derived products (indolelactate and 4-hydroxyphenylacetate), as well as many of the more general SLC22 metabolites, like prostaglandin E2, prostaglandin F2, uric acid, and creatinine ,28,29,30SLC22A7 (OAT2) is the only member of the OATS2 subgroup and is associated with prototypical SLC22 substrates, such as prostaglandins, carnitine, creatinine, and uric acid ,35,36,37In humans, SLC22A11 (OAT4) and SLC22A12 (URAT1) share only two substrates, uric acid and succinate  39,40].,40.39,40p-value of 4.12 × 10−27 or lower for all members  has been well characterized as a transporter of both urate and nicotinate, but SLC22A14 has no available transport data . However+-antiporter family  and BOCT2 (SLC22A23), it was noted that they both show high expression levels in the brain, as well as a nonconserved amino terminus that may negate prototypical SLC22 function . SLC22A1r family  10]. Fu. Fu+-antThe OCT subclade of SLC22A1 (OCT1), SLC22A2 (OCT2), and SLC22A3 (OCT3) has ample data to support its formation and has been widely accepted and utilized as the prototypical subgroup of organic cation transporters. All three members of this subgroup transport monoamine neurotransmitters, carnitine derivatives, creatinine and the characteristic OCT substrates, MPP+, and TEA  34,37,6,62,63,64The OCTN/OCTN-Related subgroup is a combination of two previously established subclades, OCTN and OCTN-related . PreviouOur new subgroupings are primarily based on the endogenous function of the transporters, but they are also supported by additional analyses. These analyses are necessary, as structural and evolutionary similarities can predict functional traits that have yet to be discovered. Though the previously established phylogenetic subclades remain sound, our re-analysis includes new and updated amino acid sequences that support the proposed subgroups with more confidence, especially when investigating similarities within functional regions . MSA proFull length sequence analysis via Clustal-Omega, MAFFT, and ICM-Pro v3.8-7 supported the division of SLC22 into eight subgroups . While tThe branching pattern of OATS3 member Oat-pg (Slc22a22) differs between tree variations. These analyses consistently indicate a similar relationship between Oat-pg and OATS3, as well as OATS4. However, in an analysis of the SLC22 ECDs, it is most closely associated with OATS3 over any other subgroup. This, in conjunction with shared substrate specificity with both SLC22A12 and SLC22A11, and not OATS4 members, supports its membership within the OATS3 subgroup ,39,40,46In full-length sequence alignments, the grouping of SLC22A4, SLC22A5, and Slc22a21 is consistently conserved, while the topology of both SLC22A15 and SLC22A16 is irregular. Despite this, analysis of the large ECD shows similarity between all OCTN/OCTN-related members other than SLC22A15. Previous analyses have noted the large difference between the ECD of SLC22A15 and all other SLC22 members, which is supported by our analysis in Genomic clustering within the SLC22 family has been previously described . SpecifiMotif analyses revealed subgroup specific motifs within functionally important regions, such as the large ICD, large ECD, and the region spanning TMD9 and TMD10, for all novel OAT subgroups . HoweverOATS2 (SLC22A7) is a Systemically-Expressed Transporter of Organic Anions and Cyclic Nucleotides”. This pattern is also seen in OATS1, which consists of multi- and oligo-specific transporters OAT1, OAT3, and OAT6. In addition to having few subgroup-specific motifs, the multi/oligo-specific nature of this subgroup is reflected by the shared evolutionary conservation of the large extracellular domain with other OAT subclade members  in mouse, rat, cow, and human. This analysis also showed a 37% sequence identity (pP = 5.5) shared between the ECD of hSLC22A31 and human heterochromatinization factor BAHD1. These and other interesting sequence similarities to proteins, including those involved in signaling, are noted in In the years following the establishment of the previous SLC22 subclades, there has been a notable increase in functional data, particularly with respect to endogenous substrates, concerning these transporters and their substrates . With thAlthough the functional data were inherently biased due to the high level of interest in some SLC22 members, particularly the “drug” transporters OAT1, OAT3, OCT1, and OCT2, for the majority of the transporters, there are enough data to create functional subgroups that play distinct and overlapping roles in metabolism . GenomicProtein families are established based on shared ancestry and structural similarity, which is commonly considered grounds for shared functionality. This is exemplified amongst SLC22 members with the generally shared structural characteristics of 12 TMDs, a large extracellular loop between TMD1 and TMD2, and a smaller intracellular loop between TMD6 and TMD7. Despite these shared features, we show here that there are many functional differences between these transporters. Although our analyses mostly align with previous evolutionary studies when considering ancestry, here, we show that phylogenetic grouping is not always reflective of a similar structure and function. For example, although the previously established OCTN subclade of SLC22A4, SLC22A5, and Slc22a21 does not share common ancestry with Slc22a16, the newly proposed group shares functional similarity and ECD homology. Thus, by expanding our investigation beyond phylogenetic relationships, we can now more appropriately group proteins from the same family and better understand their roles in endogenous physiology.An important concept in the remote sensing and signaling network is that of multi-specific, oligo-specific, and relatively mono-specific transporters working in a coordinated function . Multi-sOne of the best examples of multi-specific transporters working in concert with oligo, and mono-specific transporters is the regulation of uric acid ,18. HandWhen considering such a large number of proteins, the function on both local and systemic levels of metabolites is likely to be impacted. The SLC22 family is a central hub of coexpression for ADME -related genes in non-drug treated conditions, which underscores their importance in regulating endogenous metabolism through the transport of small molecules . In the The RSST proposes that a network of ADME genes  regulates the levels of hundreds if not thousands of small organic molecules with “high informational content” including key metabolites and signaling molecules involved in intra-organ, inter-organ, and inter-organismal remote communication. The RSST would seem to imply that organisms are constantly solving a multi-objective optimization problem, where balancing each particular compound’s serum concentration represents a single objective. Each compound present in the blood has a range of healthy concentrations, and when the concentration is outside of that range, the body must address it, in part through the regulation of transporters and enzymes. Due to their wide range of tissue expression and diverse functional roles at body fluid interfaces, the particular combination of transporters and enzymes are critical variables necessary for solving this multi-objective optimization problem. Transporters regulate the entry and exit of substrates to and from cells, but enzymes are responsible for the altering of these compounds. To use a simple hypothetical example, if a metabolite’s serum concentration is too high, a transporter with high affinity for that metabolite can move it into the cell, where an enzyme with high affinity for the substrate can change it so that it may re-enter the circulation or be more readily cleared from the body. The existence of abundant multi-specific and oligo-specific transporters and enzymes, in addition to relatively mono-specific ones, expressed differentially in tissues and at body fluid interfaces, allows for a highly flexible and responsive complex adaptive system that not only maintains homeostasis in blood, tissue, and body fluid compartments , but also helps restore it after acute or chronic perturbations.Thus, together, transporters and enzymes have tremendous potential to manage levels of metabolites, signaling molecules, and antioxidants in the circulation and in specific tissues. By developing functional groupings for the SLC22 family, we could better understand the metabolic networks in which they function and how their expression is utilized to regulate concentrations of metabolites, signaling molecules , antioxidants , and other molecules affecting diverse aspects of homeostasis . Although this analysis focuses on the SLC22 family, a similar approach can be applied to develop a deeper understanding of other families of transporters and enzymes.In the past, the majority of functional data have come from transport assays using cells overexpressing a specific SLC22 transporter and a single metabolite of interest. These assays lack uniformity and, as the OAT knockouts have shown, are not necessarily reflective of endogenous physiology ,28,30. Rhttps://genome.ucsc.edu/cgi-bin/hgGateway) [https://www.ebi.ac.uk/gxa/home) [SLC22 human and mouse sequences were collected from the National Center for Biotechnology Information (NCBI) protein database. Sequences were confirmed and genomic loci were recorded using the University of California Santa Cruz (UCSC) genome browser by searching within each available species on the online platform (Gateway) . The NCBGateway) . Tissue xa/home) . Tissue https://www.ebi.ac.uk/Tools/msa/clustalo/), as well as MAFFT  and ICM-Pro v3.8-7c [http://itol.embl.de/) [http://www.tcdb.org/) with the Cyanate Porter (CP) Family (2.A.1.17) as an outgroup to further investigate the belongingness of SLC22A18 in either the SLC22 or DHA1 family [Sequences for SLC22 were aligned using Clustal-Omega with default parameters via the online platform provided by the European Bioinformatics Institute (EMBL-EBI)  . Topologmbl.de/) . SSearch1 family ,85. SSea1 family .ICM-Pro v3.8-7c was used to align sequences in FASTA format as well as perform homology searches of all human and mouse SLC22 sequences against ICM-Pro’s curated database of all known proteins . Threshohttp://meme-suite.org/tools/meme) suite [n = 57) as well as a set of evolutionarily conserved motifs for each of the four proposed OAT subgroups. These motifs were then mapped onto 2D topologies of one member from each of the newly proposed OAT subgroups . A separate motif analysis was also performed for the rodent expansion consisting of Slc22a19, and Slc22a26-30 and was mapped onto mouse Slc22a27. Transmembrane domains (TMDs) of these transporters were predicted by the constrained consensus topology prediction server  [http://www.sacs.ucsf.edu/TOPO2/) to visualize the 2D representation of the transporters with the OAT subclade’s evolutionarily conserved motifs shown in blue and each subgroup’s evolutionarily conserved motifs shown in red [Motif comparisons were performed on the subgroups of the OAT subclade using the multiple expectation-maximum for motif elicitation  suite . A thresmta.hu/) . TMD locn in red .http://www.metabolomix.com/list-of-all-published-gwas-with-metabolomics/) were also utilized in searching for SNPs and their effect on metabolite transport by SLC22 members [https://www.ncbi.nlm.nih.gov/gene/about-generif) was used to search for non-synonymous mutations that did not affect protein expression yet affected transport of metabolites and/or drugs. These methods were accompanied by an extensive literature search for in-vitro transport and knockout data. Most in-vitro data come from tissue culture assays from a variety of cell lines while most in-vivo data comes from genetic or chemical knockout mice. Metabolite data were abstracted from the aforementioned databases and confirmed via literature review. The import from table feature on Cytoscape 3.7.2 was used to generate functional networks for the entire SLC22 family and the subgroups [To determine the diversity of substrate specificity, the number of drugs that list SLC22 members as a target on DrugBank were recorded ,90. The  members . Currentubgroups . A sprin


Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3′ protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.Type III CRISPR–Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in  The ribN domain . BindingN domain ,41,42. CN domain ,44 or asN domain ,46, haveN domain .Anti-virus or anti-plasmid immunity afforded by Type III crRNPs, is transcription-dependent as these systems specifically recognize an RNA target having a crRNA interaction region (i.e. the RNA protospacer) ,14,19,472 > p) in certain organisms  in controlling Type III activities. When PFS sequences are complementary to the 5′ crRNA tag (as would be the case if the cell produced antisense crRNAs), the DNase and cOA production activities fail to become activated ,14,53,54rganisms ,46. Howerganisms ,44. AddiPyrococcus furiosus (Pfu) is a hyperthermophilic archaeon that contains three distinct functional CRISPR–Cas effector crRNPs: two DNA-targeting systems (Types I-A (Csa) and I-B (formerly known as I-G) (Cst)   system of Pyrococcus furiosus (Pfu) and further define how the Csx1 RNase and Cmr2 DNase activities are regulated by target RNA elements and second messenger signaling events.G) (Cst) ) and a TG) (Cst)  shown toG) (Cst) ,11,17,27by Cmr4)  and non-by Cmr4) . The tartruction ,11. In ctruction ), was crcleavage . Whetherimmunity . These fP. furiosus strains utilized in this study are listed in pyrF marker gene.All P. furiosus strains were produced using homologous recombination of transformed SOE-PCR (splicing by overlap extension-polymerase chain reaction) constructs as previously reported  restriction site was introduced into cmr2 and a NruI-HF (5′-TCGGGA-3′ → 5′-TCGCGA-3′) restriction site was introduced into csx1. Primers used to make the complementation strains can be found in Pfu gene of interest and DNA sequencing (Eurofins Genomics).reported . ComplemP. furiosus Cmr1–6 and Csx1 proteins were amplified via PCR and cloned into modified versions of pET24-D , pET101-D (Cmr1–1) and pET200-D  as previously detailed . Expression cultures for wildtype and mutant proteins were grown in 1 l , 2 l  or 4 l (Cmr6) cultures at 37°C. Luria broth ) or Terrific broth  medium was used for cultures and supplemented with either 50 μg/ml kanamycin sulfate  or 100 μg/ml ampicillin (Cmr1–1) for plasmid selection. Cultures were grown to an OD600 of 0.7 at 37°C then induced with 0.5 mM iso-propyl-d-thiogalactopyranoside (IPTG) at 24°C overnight. Cells were pelleted then resuspended in lysis buffer (40 mM Tris–HCl (pH 7.5), 500 mM NaCl, 10 mM Imidazole) containing one protease inhibitor tablet (Roche) and lysed via sonication. Cell lysates underwent a thermal precipitation by incubating in a 75°C bead bath for 20 minutes. Insoluble material was removed by centrifugation at 14,000 rpm for 20 minutes at 4°C and filtered through a 0.8 μM syringe filter (Corning Incorporated). Proteins were purified using gravity affinity chromatography and either Ni-NTA resin  or Talon Cobalt resin . Cell lysates were rotated with pre-rinsed and equilibrated resin for 1 h at 4°C. The proteins were then washed with the lysis buffer and wash buffer (40 mM Tris–HCl (pH 7.5), 500 mM NaCl, 20 mM Imidazole). The proteins were then eluted using four different elution buffers containing increasing amounts of imidazole . Wildtype and HEPN mutant Csx1 proteins were dialyzed and underwent a second round of gravity affinity chromatography. Buffer exchange was performed using Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific) in elution buffer lacking Imidazole. Protein concentrations were assessed using Qubit protein concentration assays (Invitrogen) and purity was assessed via SDS-PAGE and Coomassie blue staining analysis.The genes encoding detailed ,37. All m) contains a H436A mutation and was created as previously described (m). Quikchange mutagenesis was used to create site-specific mutations within the predicted cOA binding pocket of the CARF domain of Pfu Csx1. Two cOA binding mutants were created: Csx1-INAA  and Csx1-INQQ . Successful mutagenesis for all mutant plasmids was confirmed via DNA sequencing (Eurofins Genomics).The wildtype gene encoding Csx1 was subcloned into a modified pET24D vector. The Csx1 HEPN mutant  were purchased from Integrated DNA Technologies and DNAs from Eurofins Genomics. The RNA and DNA sequences can be found in escribed . 7.01 crescribed  and gel in vitro transcription using T7 RNA polymerase and the MEGAshortscript T7 kit (Invitrogen) as described  or No Target (pJE47) control plasmid. The transformations were incubated for 15–45 min at room temperature prior to plating. Each transformation mixture was split between two plates and spread onto solid defined media lacking uracil. The plates were incubated at 90°C in an anaerobic chamber. Plates were observed for colony growth and counted after 64 h of incubation. Results shown represent two replicates.Plasmid transformation interference assays were performed as previously described . Liquid 2), and 12.5 nM of 7.01 crRNA for 25 min at 70°C. After the preincubation, 0.5–1.5 nM of radiolabeled synthetic 7.01 target RNA was added to the reaction and incubated for one hour at 70°C. One unit of Proteinase K (NEB) was then added to each reaction and incubated for 30 min at 37°C prior to gel electrophoresis. For DNase assays, the Cmr crRNP was assembled in DNA assay buffer (20 mM Tris–HCl (pH 7.5), 250 mM NaCl, 1.5 mM MgCl2, 200 μM NiCl2) and 50 nM of 7.01 crRNA for 25 min at 70°C. After the preincubation, 100 nM of 7.01 target RNA and 1 nM of radiolabeled dsDNA was added to the reaction and incubated for one hour at 70°C. Unless otherwise indicated, DNase assays were completed with target RNA containing a 5′-GGG-3′ PFS sequence. One unit of Proteinase K was then added to each reaction and incubated for 30 min at 37°C prior to gel electrophoresis. Cyclic oligoadenylate production assays were performed by assembling the Cmr crRNPs as described above using RNA assay buffer and 50 nM of 7.01 crRNA. After assembly, 0.5 mM of ATP (NEB), 5 nM of α-32P-ATP , and 100 nM of target RNA was added to the reaction and incubated for 1 h at 70°C. Unless otherwise indicated, cOA production assays were completed with target RNA containing a 5′-GGG-3′ PFS sequence. All Reactions were stopped by adding Gel Loading Buffer II (Life Technologies) and visualized by using 7M urea denaturing 15% polyacrylamide gels followed by autoradiography. Cyclic oligoadenylate reactions were also ran on 8M urea denaturing 20% polyacrylamide sequencing gels. Decade Markers (Life Technologies) and partial alkaline hydrolysis ladders (Ambion) of poly A19 RNA were generated as previously described  and incubated with 20 nM of Csx1, 5′-end labeled target RNA, and assay buffer for 1 h at 70°C.Unlabeled cOA production assays were performed as described above except the reactions were incubated for 2 h. Liquid chromatography high resolution mass spectrometry (LC-HRMS) analysis was performed on a Thermo Scientific Velos Pro instrument equipped with HESI source and Dionex UltiMate 3000 chromatography system as previously described .in vivo work with the Pfu Cmr (III-B) system, we found that individual mutations of either the HD (H13A/D14A) or Palm domain GGDD (D673A/D674A) motifs of Cmr2  or a single deletion of the csx1 gene (Csx1Δ), did not interfere with anti-plasmid immunity but double mutations in the Cmr2 HD and GGDD Palm motifs  prevented immunity  DNase and/or Csm6/Csx1 RNase activities for anti-plasmid and anti-viral immunity ,38,47,62immunity   or empty plasmid control  or Palm GGDD motif , or if the csx1 gene was deleted from the genome (Csx1Δ)  in conjunction with a second mutation within the DNase catalytic site of Cmr2 (Cmr2-HD mutation) . Cmr-mediated immunity was rescued by restoring either wildtype cmr2 or csx1 genes in the Cmr2-HDm/Csx1Δ double mutant strains  Figure ). As exp) Figure ). These Pfu Cmr system functioned through generating cOA signaling molecules as has been observed for other bacterial and archaeal Type III systems , were assembled in vitro . ,17. Pfu o Figure  and testo Figure . Moreoved Figure . To testn Figure . These fPfu Cmr crRNPs, are capable of stimulating the ribonuclease activity of Pfu Csx1 in vitro  of the Csx1 enzyme is used  and cyclic-tetraadenylate (cA4) species  of the generated cOA leads to the activation of Csx1 ribonuclease activity, varying concentrations of commercially available synthetic cA4 and cA6 compounds were incubated with Pfu Csx1 in the presence of 5′-end labeled substrate RNA . cA4 but not cA6 stimulated the ribonuclease activity of Csx1  disrupted the ribonuclease activity of Pfu Csx1  or high (500 nM) concentrations and with or without cOA addition   products  that could lead to host cell toxicity or death during the immune response. Recently, a new class of CARF-domain containing proteins called ring nucleases, were discovered in activity . S. solfolecules . In conti (A2>p) . Previouse motif . That knA Figure . Indeed,s Figure ,45). Then Figure . Moreoven Figure . The restruction , also utPfu Cmr complexes revealed the important role of three nucleotides within the PFS immediately adjacent to the crRNA/target RNA protospacer interaction  in vitro required a PFS with a NGN, NNG, or NAA sequence (Pfu crRNPs (via Cmr4 backbone subunit) occurs independent of the PFS  effector crRNP was the first example of a CRISPR system that identifies and pairs with RNA transcripts rather than DNA strands of invaders  and DNase (Cmr2 HD domain) activities, respectively  activator to switch off the signaling pathway and to limit the activity of Csx1 through autoregulation  or RNase activity  leads to robust anti-plasmid immunity. Our finding that the DNase and cOA generation capacities are activated by distinct 3′ protospacer flanking sequences (PFS) of the target RNA highlight that the two target-RNA stimulated enzymatic processes are differentially regulated by distinct allosteric control mechanisms. Taken together with results obtained from other investigated bacterial or archaeal Type III-B (Cmr) and III-A (Csm) systems, our results contribute to making a compelling case for Type III systems as the most complex and highly regulated CRISPR systems discovered thus far.The invaders . A decadr crRNPs ,30,64,65r crRNPs . Previouectively ,11,17,27 Figures , also dePfu Csx1 in anti-plasmid immunity through single csx1 gene mutational analysis, failed to uncover its role (Pfu crRNP (via mutation of the HD motif of Cmr2), the RNase activity of Csx1 (via mutation of the HEPN motif or csx1 gene deletion), or the ability to generate cOA second messengers  needed to activate Csx1 RNase activity  and/or by destroying host RNA transcripts (leading to cellular dormancy or death)  while sparing cellular RNAs (The exact mechanism of action of ) Figure . We are residues ) and tharesidues ,21–23,34lar RNAs , or to dlar RNAs . It is clar RNAs .Pfu Cmr complex generates primarily cA4 signaling molecules  cleaves and inactivates cOA signaling molecules . Previos Figure ,44,46,51Pfu Csx1, can cleave and inactivate cOA signaling molecules expands the number of mechanisms capable of down-regulating Type III-induced cOA signaling pathways. For example, Sulfolobus islandicus Csx1 appears to lack the ability to itself degrade cOA4 molecules and instead utilizes extrinsic CARF domain-containing factors  to switch off Csx1 RNase activation , was found to be capable of autoregulating RNase activity through binding and cleaving cA4  depended upon the CARF rather than HEPN domain despite evidence from high resolution structural studies showing that cOA4 binds to both CARF and HEPN domains of Ton Csm6  thenPfu ,54,67,68Pfu ,11   and Csm4/Cmr3 (5′ tag interacting) subunits ,48,49. WPfu PFS elements that triggered activity, shared a purine-rich character within position +2 and +3 of the target RNA PFS appears to responsible for differentially activating the DNase or cOA synthesis. These results have provided insight that the two important activities for immunity are differentially controlled by distinct molecular mechanisms. Additional evidence supporting the notion that distinct conformational changes triggered by PFS/protein interactions separately influence either DNase or cOA production were observed with S. thermophilus Type III-A system where Csm1 mutations at residues thought to transmit allosteric effects of the PFS were found to impair DNase activity only, cOA synthesis only, or both activities   ,71. In ts Figure .in vivo role of the RNase activity of Cmr4 on anti-plasmid immunity. Cmr4 is the backbone subunit of the III-B crRNP . Given B crRNPs ,56. ConsB crRNPs ,10,13. F3 mutant . Taken tP. furiosus III-B system is robust when only employing DNase activity (mediated by the HD domain of Cmr2) or RNAse activity   in stimulating the DNase and RNase activities of the crRNP (Figure S. epidermidis III-A system (In addition to these functional differences, the regulatory mechanism that govern spatiotemporal control of the activities of type III systems have also shown to be quite different. For example, we show here clear evidence that the P Figure . In contA system . CollectPfu Type III-B crRNPs in the presence and absence of substrate target RNAs and DNAs and with and without Csx1, would provide important insight into specific molecular mechanisms that control the timing and specificity of Type III crRNP-mediated immunity.In summary, our findings significantly contribute to our understanding of the multiple mechanisms that ensure that Type III CRISPR systems become appropriately activated and then rapidly deactivated to achieve maximum benefit to host cells experiencing infection from invading mobile genetic elements. In future studies, a detailed biochemical and structural analyses of gkaa176_Supplemental_FileClick here for additional data file.


RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to bleached fluorophore replacement. This property makes the arrays readily compatible with structured illumination super-resolution microscopy. Fluorogenic RNA aptamers have been used for RNA imaging, but folding and fluorescence stability often limited their use in high resolution applications. Here the authors present an array of stably folding Mango II aptamers for imaging of coding and non-coding RNAs at single-molecule resolution, in both live and fixed cells. For two decades, genetically tagging RNA molecules with MS2 stem loops (MS2-SL) able to bind fluorescent protein (FP)–MS2 coat protein (MCP) fusions have helped visualise individual RNA molecules in live cells during transcription, translation and transport in cell lines, yeast and even mouse models10. However, the recent observation that MS2 arrays block 5′- and 3′-exonucleases12 and the requirement for nuclear localisation of the FP–MCP protein fusions for enhanced imaging contrast in the cytoplasm places restrictions on the utilisation of this technology. Despite new generations of MS2-SLs (MS2v5 and MS2v6) that reduce effects on RNA processing14, there remains an advantage to developing high contrast imaging strategies that do not rely on fluorescent FP–MCP proteins and that improve imaging contrast within the cell.In the cell, RNA molecules act as the messenger for genetic information, regulate key RNA processing events and are catalysts in translation and splicing. Imaging the dynamics of single RNA molecules in live cells has been a long standing challenge that if solved holds the promise for routine study of cellular RNAs24. Their fluorogenicity enables the induction of fluorescence upon RNA expression and can facilitate high-contrast imaging and offer the potential to use shorter RNA tags. These RNA aptamers after being transcribed, induce fluorescence by binding a fluorogenic dye making it thousands of times brighter upon binding25. A wide range of fluorogenic RNA aptamers have been selected and shown to induce aptamer specific fluorescence in live cells. Two of the most promising families of small monomeric aptamers are Spinach and Mango19. It has long been thought that aptamer arrays, similar to the MS2 cassettes, could enable single-molecule imaging with improved sensitivity. However, both the folding and fluorescence stability of the Spinach aptamer have hindered high-resolution imaging of such arrays20. More recently, arrays of the Pepper aptamer have been used to image RNAs with improved resolution26, but the use of fluorescently labelled proteins is still required for single-molecule detection27. The brightness, thermodynamic stability and high-affinity of Mango aptamers bodes well to the accurate detection of RNAs abundance in a cellular environment. The recent isolation of brighter and more photostable Mango aptamers, as well as the detection of as low as five tagged RNAs within an individual foci, suggest the potential to image single molecules of RNA using Mango arrays28. Here we show the development of stably folding Mango II repeats and their ability to directly image both coding and non-coding RNAs at single-molecule resolution without affecting their known localisation patterns. By making dual Mango and MS2 arrays of exactly the same size we directly compare the imaging capabilities of the MS2 and Mango systems in a head to head fashion. Furthermore, exchange of the Mango fluorogenic dye in fixed samples extends imaging times, which also benefits super-resolution techniques such as structured illumination microscopy (SIM).Since their development, fluorogenic RNA aptamers have been poised as a potential complementary alternative to the MS2 system28 to develop an RNA imaging array. We chose the Mango II aptamer  as a reporter for mRNA transcription (Mango fluorescence) and translation (mCherry fluorescence). An M2x24 array was inserted downstream of the mCherry CDS/stop codon and the construct was placed under the expression of a doxycycline (dox) inducible promoter Fig. . Cos-7 c28  have a slow diffusive behaviour with a mean MSD = 0.062 ± 0.019 µm2/s and a mean intensity ~6-fold greater than that expected for a single mRNA molecule suggesting that they correspond to transcription sites , show a broader distribution of MSD sharing similarities of both nuclear and cytosolic distributions described previously with a mean MSD = 0.122 ± 0.077 µm2/s. Further confirmation of slow diffusing molecules was observed with data acquired at a 3.6 s time frame rate (black distribution) which have a mean MSD = 0.089 ± 0.010 µm2/s. As expected, the difference in MSD between M2/MS2-SLx24 and M2x24 arrays imaged in the presence of TO1-B was negligible . Both MS2v5-EGFP and M2x24 labelled β-actin mRNAs show a significantly higher PI than β-actin mRNAs lacking the 3’UTR and the mCherry-M2x24 construct negative control  and Quazar® 670 (Q670) channels for probes against the M2 array and mCherry CDS respectively, in cells expressing mCherry-M2x24 (ncell = 27). The data shows a positive correlation close to a slope of 1 (0.88) and an r2 = 0.7, indicating that the majority of foci correspond to full-length transcripts  to acquire Mango tagged RNA images at super-resolution (~100 nm)41. To first test the recovery of Mango array fluorescence, the mCherry-M2x24 construct was imaged with constant wave illumination for 5 s followed by a 5 s pause to allow for fluorophore recovery. Images were acquired in either PKM buffer , Vectashield liquid mounting media or ProLong Diamond hard setting mounting media  showed a diffuse colocalization of both signals  was chosen and labelled with the M2x24 array. NEAT-1 is an interesting candidate due to its well-known nuclear coordination of paraspeckles, which has been well characterised using SIMals Fig. . Upon SIals Fig. . Given t10, single-molecule imaging of mRNAs has provided an important window into the dynamic life of RNA molecules during transcription, splicing, post-transcriptional modification, translation and degradation49. However, the stability of the capsid bound RNA tag within rapidly degraded mRNAs in yeast has highlighted the need to develop alternative labelling strategies to improve both imaging contrast and reduce aberrant degradation products. In addition to the recently modified MS2-SL sequences13, fluorogenic RNA aptamers represent an interesting and complementary alternative approach. Fluorogenic RNA aptamers give the added benefit of producing a specific fluorescent signal only in the presence of tagged RNA. To date, fluorogenic RNA aptamers have been used to image short non-coding RNAs with attempts at improving the resolution of imaging with tandem aptamer arrays20. However, the fluorescent stability of these arrays both in vitro and in bacterial cells has hindered the observation of single molecules. Recently, two new aptamer-based RNA imaging technologies have emerged both naming their aptamers Pepper. The first using RNA-stabilised fluorescent proteins able to image single mRNAs in live cells27. The second, is a fluorogenic RNA aptamer that has been shown to enable RNA imaging in cells, but not at the single molecule level26. RNA Mango is likely compatible with these technologies while enabling direct RNA imaging at single molecule resolution without the need for fluorescent proteins.Since the development of fluorescently tagged bacteriophage coat protein to image RNA molecules in cells20 . Dual labelling individual transcripts with both Mango and MS2 arrays demonstrates clear improvements in signal-to-noise, detection, and tracking for Mango imaging over MS2 imaging, particularly in the nucleus where nuclear localised FP–MCP dominates the signal observed  and non-coding RNAs (NEAT-1 v1) could be resolved and demonstrated RNA specific localisation patterns previously observed28 we have routinely been able to detect low Mango copy number in cells suggesting that array size minimisation is likely. As widefield microscopy uses relatively intense laser light to image live cells, the photobleaching conditions were quite harsh. Further improvements in live-cell imaging methodology are therefore easily imagined as a consequence. Specifically, further optimisation of fluorophore concentration and Mango array size could allow continuous RNA imaging via fluorophore exchange within living cells. This combined with the use of increasingly standard microscope imaging methodologies such as light sheet52, orbital tracking53, laser pulse sequence, or laser scanning methodologies54 appear highly likely to further improve this already compelling approach to the routine imaging of single RNAs within live cells via the use of RNA Mango.While the long Mango arrays used in this study allowed us to directly compare with existing MS2 arrays in live cells, there appears to be no fundamental reason to use such long repeat lengths with the Mango system. In this and previous studiesoC containing 40 mM Tris-HCl, 30 mM MgCl2, 2 mM spermidine, 1 mM dithiothreitol, 5 mM NTPs, 1 U/μl E. coli inorganic pyrophosphatase (NEB), 4 U/μl T7 RNA polymerase (NEB - pH 7.9) in a total volume of 150 µl. Varied lengths of the transcription products were purified from an 8 M Urea, 18% 29:1 Polyacrylamide:Bis-acrylamide gel run at 20 W for ~4 h in 1X TBE buffer. RNA was extracted from the gel by a crush and soak method using an RNA elution buffer  followed by ethanol precipitation and re-suspension in nuclease free water.The conserved stem of the Mango II aptamers was mutated to enable efficient folding of adjacent aptamers. Mango aptamers were mutated by transposing or flipping 6 of the 8 bp in the stem. The first and last G-C pair of the stem were maintained so not to affect the stacking of the G-quadruplex. To create a Mango II x3 construct (M2 x3) 5 nt polyT spacers were used to separate adjacent aptamers and were synthesised as ssDNA by Integrated DNA Technologies (IDT). Similarly, M2 x3 constructs were multimerized 8 times to obtain a total of 24 aptamers with each triple again being separated by a 5 nt polyT sequence and synthesised by Genewiz Ltd. RNA encoding the tandem Mango arrays was made using the T7 promoter site within the pBSII-SK(-) plasmid to initiate run-off transcription of the MunI linearised plasmid. In vitro transcription used 4 µg of DNA incubated for 4 h at 372, pH 7.2). The modified Thiazole Orange dye (TO1-3PEG-Biotin) was synthesised by Applied Biological Materials Inc. Fluorescence measurements were taken using a Varian Cary Eclipse fluorometer (Agilent) at 22 °C. Mango excitation and emission wavelengths were 505 nm and 535 nm, respectively. TO1-B titrations were conducted by acquiring the peak fluorescence from emission scans at each concentration. Fluorescence measurements were then normalised to the point of saturation, plotted on a log scale (X-axis [dye]) and were fit with a Hill equation  was denatured and refolded in 10 mM Sodium Phosphate and 140 mM KCl (pH 7.2) with a temperature ramp of 1 °C/min from 15–95 °C and returning to 15 °C. The differential of the raw data was calculated and was smoothed using a binomial calculation in the Igor Pro 7 software using 9 consecutive data points.The mammalian expression vectors for the mCherry-M2x24 construct were made by direct digestion of the pUC-57 plasmid synthesised by Genewiz Ltd which, contained an mCherry coding sequence followed by a stop codon and the M2x24 array in the 3’UTR followed by the SV40 poly-adenylation sequence. The doxycycline inducible pLenti-mCherry-M2x24 vector was made by performing QuikChange mutagenesis (Agilent) of the BamHI site in-between the mCherry coding region and Mango array into a PvuI site following the manufacturer’s instructions. KOD Hot Start polymerase (Merck Millipore) was used amplify the mCherry-M2x24 sequence to incorporate a 5′’ AgeI site and a 3’ BamHI site. This product was gel-purified, digested and ligated into a linearised and phosphatase treated pLenti-puro vector . The plasmid is a modified version of the plasmid pCW-Cas9 Addgene plasmid 50661. A pLenti-mCherry-MS2v5x24 plasmid was made similarly by amplification and QuickChange mutagenesis of an mCherry-MS2v5x24 sequence and its insertion into the pLenti backbone using the 5’ AgeI and 3’ BamHI sites. To image β-actin mRNA the coding region was obtained from a plasmid containing the β-actin and an N-terminal fusion with the BFP derivative mAzurite. The plasmid mAzurite-Actin-7 (a gift from Michael Davidson Addgene plasmid 55227) was modified by incorporating the M2x24 array with 5’ XbaI and 3’ BclI sites. The minimal β-actin 3’UTR sequence (~500 bp) which encodes the Zipcode sequence was amplified from the plasmid pUBC-HaloTag-bActinCDS-bActinUTR-MS2V5  and cloned into the mAzurite-β-actin-M2x24 plasmid with 5’ BamHI and 3’ XbaI sites. Similarly, the M2x24 array was cloned into the pUBC-HaloTag-bActinCDS-bActinUTR-MS2V5 using 20 bp of 5’ and 3’ homology to replace the MS2 cassette using the Hifi builder enzyme mix (NEB). This was later followed by further Gibson assembly of the Halo-bActinCDS-bActinUTR-M2x24 insert into the pLenti-puro backbone using NEB Hifi builder. The short isoform of human NEAT-1 lncRNA (hNEAT-1 v1) was amplified from the plasmid pCRII_TOPO_hNEAT1  and incorporated into an AgeI/BamHI digested pmax-ponA backbone vector, pmax-ponA 12xTRICK - 24xMS2SL was a gift from Jeffrey Chao (Addgene plasmid 64542) which also contained the M2x24 array between the BamHI and MunI sites in replacement of the 24xMS2-SL array. To label the MS2 arrays used in this study, either tdMCP-EGFP or mCherry was expressed from the phage-ubc-nls-ha-tdMCP-gfp plasmid (a gift from Robert Singer - Addgene plasmid 40649) with mCherry incorporated digestion and ligation into XbaI and ClaI sites. Phage-cmv-cfp-24xms2 (a gift from Robert Singer—Addgene plasmid 40651) was also used in intial experiments for imaging MS2-EGFP foci in live cells (Supplementary Movie 2 in a humidified incubator. Cells used for imaging were cultured in Ibidi glass bottomed 8-well chamber slides (Ibidi GmbH). Approximately 15–20,000 cells were seeded on to poly-d-Lysine (Sigma) coated dishes and were left to adhere overnight prior to imaging experiments.HEK293T (293T-ATCC® CRL-3216™) and Cos-7 cells (ATCC® CRL-1651™) were grown in Dulbecco Modified Eagle’s Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 2 mM D-Glucose, 2 mM L-Glutamine, 1 mM Sodium Pyruvate and 100 U/ml Penicillin/Streptomycin (Thermo Fisher) and maintained at 37 °C with 5% CO2, pH 7.2) containing 4% paraformaldehyde (Thermo Fisher) for 10 min at room temperature followed by permeabilization in 0.2% Triton X-100 for 5–10 min. Cells not requiring immunostaining were washed three times for 5 min each with PKM buffer followed by a 10 min incubation in 200 nM TO1-B diluted in PKM buffer before replacing with imaging media . For immunostaining, cells were first blocked (2% BSA in PKM) for 30 min followed by primary antibody (1:500 dilutions) incubation for 120 min in blocking solution. Primary antibody Anti-p54-nrb (NONO) (ab50952—5 µg/ml). Secondary antibodies used were Donkey Anti-Rabbit Alexa Fluor 647 (Molecular Probes). Primary antibodies were washed three times for 20 min each in blocking solution followed by incubation with secondary antibody at 1:500 dilution for 60 min, which was subsequently washed as above. After immunostaining, the cells were washed and stained in TO1-B and Hoechst 33258 as described previously. HaloTag expressing cells were labelled with HaloTag-TMR ligand (Promega-G8251) following the manufacturers instructions for live cell labelling prior to fixation and permeabilization.Cells were fixed in PKM buffer , 488 nm (Green), 561 nm (Red) and 642 nm (Far-Red). Emission was detected with a 63× oil immersion objective with a high numerical aperture of 1.4 and band-pass filters at 420–480 nm, 495–550 nm, 570–640 nm and a long-pass filter > 650 nm, respectively. Image acquisition for the fixed samples used 5 mW of power and 200 ms exposure time for each channel, except in photobleaching-assisted microscopy experiments and fast acquisition live-cell experiments .Plasmids were transfected directly into 8-well chamber slides using Fugene HD following manufacturer’s instructions in the culture medium for 16–24 h prior to induction. pLenti-tet-ON plasmids were induced with 1 µg/ml doxycycline for ~2 - 4 h and imaged whereas, pmax-ponA plasmids were induced for ~2–4 h with 1 µM ponasterone A. To facilitate expression of RNA from the pmax-ponA plasmids each construct was cotransfected with the equal amounts of the plasmid pERV-3 which encodes the inducible RXR-VgEcR-VP16 transcription coactivator modules that initiates tight expression of the responsive element encoded in the plasmid upon addition of ponA. Following induction of plasmid expression, the cells were washed once with PKM and either replaced with live-cell imaging media  for longer imaging sessions, or in 20 mM HEPES buffer containing 140 mM KCl and 1 mM MgClTM labelled DNA oligos, synthesised by IDT (Supplementary Table 2 pH 7.2) containing 4% paraformaldehyde (Thermo Fisher) for 10 min at room temperature followed by permeabilization in 0.2% Triton X-100 for 5–10 min. FISH probes were hybridised for 16 h at 37˚C directly in ibidi 8-well chamber slides using Stellaris® hybridisation buffer supplemented with 10% deionized formamide. Cells were washed twice at 37 ˚C for 30 min with Stellaris® wash buffer A supplemented with 10% formamide, followed by a 5 min wash with Stellaris® wash buffer B at room temperature. Cells were then imaged in Vectasheild® antifade mounting medium with DAPI (H-1200).Single molecule RNA FISH samples were prepared using Stellaris® Design Ready probes Human ACTB with Quasar® 670 Dye (VSMF-2003-5), mCherry with Quasar® 670 Dye VSMF-1031-5) and custom sets of MS2v5 and M2 probes. MS2v5 probes were synthesised by Biosearch™ technologies using their custom probe designer and labelled with Fluorescein dyes (FAM). M2 probes were designed and ordered as HPLC purified AlexaFluor 488031-5 andTM bead sample was used to create a bead map to which the chromatic aberrations were corrected following manufacturer instructions. The SIMcheck ImageJ plugin was used to determine the quality of the reconstructed images42, chromatic aberration/drift and most notably the rates of bleaching throughout the acquisition.Structural illumination images were acquired using a Zeiss Elyra microscope. Blue (405 nm) Green (488 nm) and Far Red (642 nm) channels were acquired by sequentially alternating the excitation whilst retaining the same setting with respect to phase, rotation and multi-cube filterset. Images were acquired with 5 phases, 3 rotations and a grating of 32 µm. Images were processed using the automatic reconstruction settings within the Zen software. A TetraSpek57. Due to the fluorescent plateau caused by TO1-B exchange at later time points in the bleaching curves, the step sizes from the initial 100 frames of each bleaching curve were used. The bleaching steps from 70 single-molecule trajectories were subsequently binned and the histogram was fit with a Gaussian function.In order to obtain the appropriate signal-to-noise ratio and time resolution for the analysis of single-step photobleaching, foci were imaged in fixed cells at 20 fps, and 10 mW of widefield laser illumination at 488 nm for ~300 frames. Maximum likelihood estimation was used to determine each of the photobleaching steps within a trace as previously described30. Foci detection in fixed samples was limited to foci at least 1.5-fold brighter than background signal and ≤500 nm in diameter. FISH-quant was used to acquire foci intensity values for both positive and negative control constructs. To obtain the distribution of intensity in the MS2x24 control in the presence of TO1-B, the intensity threshold for foci detection was lowered to detect the overall background fluorescence of the cell. The TrackMate ImageJ plugin31 was used for single particle tracking in live cells. Similar to FISH-quant analysis in fixed samples, a LoG filter was used to enhance the ability to detect single particles in live cells. Foci ~350 nm in diameter with a signal-to-noise ratio ≥ 1 were detected. Frame rates were taken directly from the metadata as compiled in the Zeiss software and ranged from 226 ms–3.6 s. To alleviate some of the non-specific particle tracking caused by excessive nuclear signal from the NLS-tdMCP-mCherry protein, the cytosolic mCherry signal was filtered to leave the lower population of signal based on total foci intensity. To obtain the population of bright nuclear foci in the mCherry channel, an opposing filtering method was used based on total foci intensity and foci signal-to-noise ratio to obtain the high intensity foci. Due to the absence of intense nuclear signal using M2x24 + TO1-B, this data was not filtered based on total foci intensity and both nuclear and cytosolic foci detected and tracked. Following filtering the observed foci tracks were built given a maximum of 2 µm for linking frame to frame displacement and gap-closing. Similarly, a maximum frame gap of 2 was used to recover the partial loss of significant trajectories. Trajectories were finally subjected to a final step of thresholding based on the mean track quality being >30 and each trace having ≥5 localisations. The.csv data output from TrackMate was used to calculate the maximum foci intensity, signal-to-noise ratio and mean square displacement as computed using Matlab.Fixed cell images acquired as z-stacks were processed using a 3D Laplacian of Gaussian (LoG) filter within the FISH-quant Matlab software37 In this publication, they used Eq. The polarisation index (PI) was calculated with a similar method as in described in Park et al.xcell, ycell,zcell) was computed from the xy coordinates from a given binary mask of the cell and the mid-point of the z-axis range (zcell). The xyz coordinates of each RNA localisation  was obtained using the ImageJ plugin FociPicker3D using a threshold above the observed cellular background. The PI for a given localisation was then normalised for cell size by dividing by the radius of gyration of the cell (Rgcell) which is the root-mean-square distance of all the pixels in the mask with respect to the centroid. The average values of PI and the deviation as standard error in the mean were used to represent global changes in RNA localisation.Modifications were made to this process using a custom MATLAB image processing pipeline, in which individual polarisation indices were computed for each observed RNA localisation and its relative distance from the centroid of the cell. The centroid of the cell , images were first processed using the FISH-quant LoG filter followed by background subtraction. The overlapping fraction was then calculated using the ImageJ plugin, Just Another Colocalisation Plugin (JACoP)Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Reporting Summary


Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts.In eco-epidemiological studies, Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve.Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for −3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of  Leishmania, which are transmitted during the blood-feeding of female phlebotomine sand flies. The infection can be manifested in three major clinical forms, cutaneous (CL), mucocutaneous, and visceral leishmaniasis . Ea. EaL. aemexicana  corroboria assay .Fig. 5AnL. infantum) mouse spleen and liver samples, detecting down to 10−3 parasite equivalents per mg tissue [Although only a few positive hyrax tissue samples were tested, the JW kDNA qPCR could identify most of the true-positive samples under the used sample storage conditions. A sample was considered positive if identified by two different assays. The SL RNA assay identified one false-negative sample and showed generally higher Cq values compared to the JW kDNA PCR than for sand fly screening, which is probably due to the fact that the samples had been stored in ethanol for two years before DNA/RNA extraction was performed. Most likely, proper RNA storage conditions and/or immediate RNA isolation would result in a substantially improved performance of the SL RNA qPCR on tissue samples , 34. Favg tissue .versus DNA in positive samples can be easily assessed by comparison with a no-reverse transcription control.Considering the large sample size that needs to be screened in search for positive field specimens, a low-cost, efficient nucleic acid extraction method is preferred . We founL. major promastigotes. The determined theoretical LoD of 10−3 parasite equivalents per reaction of our assay is similar to findings of Bezerra-Vasconcelos et al. [−3 parasites per reaction with a kDNA qPCR assay on L. infantum-spiked L. longipalpis sand flies. This substantiates that the sensitivity of qPCR assays targeting SL RNA and kDNA are comparable, which corroborates the comparative assessment performed in the present study. Moreover, congruence of the assays appears very good, indicating that both can achieve reliable quantification. Based on the standard curve, it can be concluded that SL RNA qPCR can quantify down to 10 parasites per sand fly with high PCR efficiency, which is sufficient for determination of biologically relevant parasite loads.Determination of the parasite load in sand flies can be highly informative, especially for studies that investigate, e.g. the vectorial capacity. Previously, the LoD of the SL RNA qPCR on cultured promastigotes has been established at 0.0002 parasite equivalents . We asses et al. , who couLeishmania parasites in field-collected and laboratory-infected sand flies, even in combination with a crude, low-cost extraction method. The SL RNA qPCR assay is inexpensive, sensitive and pan-Leishmania specific, which can be a major advantage for eco-epidemiological studies including identification of vectors and reservoirs.Overall, to the best of our knowledge, this study shows for the first time that the SL RNA target can be used for detection and quantification of Additional file 1: Table S1. qPCR results of sand fly and hyrax samples from the laboratory and field in four different assays. For each assay the mean quantification cycle threshold value (± standard deviation) are presented.Additional file 2: Data S1. Dataset assay comparison on laboratory-infected (Leishmania major) Lutzomyia longipalpis sand flies.Additional file 3: Data S2. Dataset assay comparison on field-collected (Ethiopia) Phlebotomus pedifer sand flies (some L. aethiopica-infected).Additional file 4: Data S3. Dataset assay comparison on field (Ethiopia) collected Heterohyrax brucei tissue samples (some L. aethiopica-infected).Additional file 5: Data S4. Data of extraction method and copy number comparison.Additional file 6: Data S5. Data of SL RNA qPCR with and without the use of reverse transcriptase.Additional file 7: Data S6. Data of standard curve of the SL RNA qPCR based on sand flies spiked with promastigotes.


RNA viruses are the fastest evolving known biological entities. Consequently, the sequence similarity between homologous viral proteins disappears quickly, limiting the usability of traditional sequence-based phylogenetic methods in the reconstruction of relationships and evolutionary history among RNA viruses. Protein structures, however, typically evolve more slowly than sequences, and structural similarity can still be evident, when no sequence similarity can be detected. Here, we used an automated structural comparison method, homologous structure finder, for comprehensive comparisons of viral RNA-dependent RNA polymerases (RdRps). We identified a common structural core of 231 residues for all the structurally characterized viral RdRps, covering segmented and non-segmented negative-sense, positive-sense, and double-stranded RNA viruses infecting both prokaryotic and eukaryotic hosts. The grouping and branching of the viral RdRps in the structure-based phylogenetic tree follow their functional differentiation. The RdRps using protein primer, RNA primer, or self-priming mechanisms have evolved independently of each other, and the RdRps cluster into two large branches based on the used transcription mechanism. The structure-based distance tree presented here follows the recently established RdRp-based RNA virus classification at genus, subfamily, family, order, class and subphylum ranks. However, the topology of our phylogenetic tree suggests an alternative phylum level organization. RNA polymerases are key enzymes in RNA virus replication and transcription. All currently known viral RNA-dependent RNA polymerases (RdRps) share a right-hand-shaped appearance with palm, fingers and thumb subdomains ,2,3,4, sRiboviria , phage phi6 (dsRNA virus) and vesicular stomatitis Indian virus [(+)RNA virus] RdRp structures in None of the amino acid residues assigned to be equivalent by HSF were chemically identical. However, the conserved catalytic aspartates RNA viruses included in our dataset. Cluster II contains all the RdRps of (+)RNA viruses and the remaining dsRNA virus RdRps RNA viruses, descend from a common ancestor.In order to obtain further insights into the structural relationships among viral RdRps, the identified common structural cores were compared between different polymerase structures using HSF  and a stIn our structure-based distance tree, the viral RdRps form two major branches RNA virus RdRps appear to be a monophyletic group (In the case of dsRNA viruses (including picobirnaviruses and partitiviruses), the polymerase is an integral part of the viral capsid and it needs to interact correctly with the capsid protein during the virion assembly and the subsequent intra-capsid genome replication and transcription processes ,51,52. TA simplified jackknifing test was carried out to evaluate the robustness of the structure-based distance tree. The overall topology of the tree  was mainPicobirnaviridae, Coronaviridae, and Permutotetraviridae. Consequently, none of the individual RdRp brings substantial additional structural diversity to the dataset, implying that the structural diversity in the core structure is relatively constant across the studied viral RdRps. Notably, the overall structural architecture of the palm subdomain appears to be unaffected by the different connectivity in the birnavirus and permutotetravirus RdRps [The jackknifing test also provides information on the stability of the core size as the equivalent residues, i.e., the common structural core is calculated separately for each of the partial dataset as part of the test ,21; see  was obseus RdRps ,43,53, aBirnaviruses and permutotetraviruses represent dsRNA and (+)RNA viruses, respectively. Yet, their RdRps are structurally similar and contain a unique sequence permutation, not seen in any other known structurally characterized viral RdRps, suggesting shared origin ,43. The laevo arrangement [laevo shell which has a similar role during virus entry, we favor the evolutionary pathway from a cysto/reovirus ancestor to birnavirus. Interestingly, the birnavirus and permutotetravirus capsid proteins also display substantial structural similarity, despite their different icosahedral arrangements in virions  [Birnaviruses are unique among dsRNA viruses as they lack the characteristic 120-subunit capsid organization of other dsRNA viruses. Instead, birnaviruses have a 780-subunit capsid organized into T = 13 icosahedral lattice as trimers of the major capsid protein . Interesangement ,55,56. Tangement . The birangement . Based oangement . Considectively) . This liAuthors of previous studies have suggested alternative scenarios for the development of birnavirus and permutatetravirus RdRps, favoring the development of birnavirus RdRp from an ancestral permutotetravirus RdRp ,58. TakiBirnaviridae and Permutotetraviridae of the kingdom Orthornavirae  and theaviridae ,70, beloPolyploviricotina and Haploviricotina, respectively, in our data set; Reoviridae family. The observation is also in line with the earlier sequence-based phylogenetic analysis [The RdRps of (−)RNA viruses with segmented and non-segmented genomes  and segmented (−)RNA viruses (subphylum Polyploviricotina) may thus represent an intermediate structure between these two groups  of Branch II , these De Farias et al.  proposedOverall, our structure-based phylogenetic tree reflects key functional properties imposing structural constraints. Interestingly, these differences can be detected by comparison of the conserved core structure which does not contain features that contribute to these different functions . Such observation suggests that a protein structure evolves little by little over generations similarly to amino acid or nucleotide sequences. The results obtained here also correlate well with earlier sequence-based analyses on viral RdRps. This indicates that the evolutionary structural signal in the three-dimensional structure of the proteins is sufficiently robust to capture differences, even between relatively similar protein structures , although structure-based comparison methods are generally expected to be more powerful in the identification of longer distance relationships, e.g., between protein families or superfamilies , than se