A list of these genes can be found at the HUGO Gene Nomenclature Committee (HGNC) website (see www.genenames.org/genefamilies/SLC). This special issue features mini-reviews for each of these SLC families written by the experts in each field. The existing online resource for solute carriers, the Bioparadigms SLC Tables (www.bioparadigms.org), has been updated and significantly extended with additional information and cross-links to other relevant databases, and the nomenclature used in this database has been validated and approved by the HGNC. In addition, the Bioparadigms SLC Tables functionality has been improved to allow easier access by the scientific community. This introduction includes: an overview of all known SLC and “non-SLC” transporter genes; a list of transporters of water soluble vitamins; a summary of recent progress in the structure determination of transporters (including GLUT1/SLC2A1); roles of transporters in human diseases and roles in drug approval and pharmaceutical perspectives.The field of transport biology has steadily grown over the past decade and is now recognized as playing an important role in manifestation and treatment of disease. The SLC ( Transporters have been called the gatekeepers of small molecules and can be divided based on passive or active mechanisms of function . Passive+, K+, H+, Ca2+ and Cu2+ out of cells or into organelles  transporter family and ion pumps (ATPases). Mammalian ABC transporters  bind and hydrolyze ATP for the transport of a variety of substances such as ions, carbohydrates, lipids, xenobiotics and drugs out of cells or into cellular organelles . Ion pumganelles . These pSimilar to transporters, channels allow movement of solutes down their electrochemical gradients . TranspoIn the past, the molecular identification of transporters has lagged compared to other protein classes because of their membrane localization, hydrophobic nature, and relatively low abundance, although physiological data for a variety of transporters have been reported extensively. It has now been over two decades since the expression cloning approach for transporters was developed , when th1.1The SLC gene nomenclature system was originally established in the 1990s by Matthias Hediger in collaboration with Phyllis McAlpine, the first chair of the HGNC, and has since been maintained and extended in collaboration with Elspeth Bruford who now leads the HGNC. In 2004, a special mini-review series was commissioned to provide an overview of the different types of mammalian transport systems belonging to the SLC series . Since t1.2SLC3A1). Transporters are assigned to a specific family if the encoded protein has at least 20% amino acid sequence identity to other members of that family. A few exceptions to the classifications above exist within the SLC series, including the SLC51 family which consists of two members, SLC51A and SLC51B, that are not related by sequence similarity but instead encode the two subunits  of the organic solute transporter. Another naming deviation is the SLC21 organic anion transporter family which now uses the root symbol SLCO, an update made to accommodate a unique species-independent classification system that is necessary because this family has been the subject of rapid evolution (Slc7a12 and multiple members of the SLC22 family) or even in insects (slc18a4 has been identified in Drosophila and honey bee). While orthologs of human genes commonly use the same designation as the human gene, though the case of the symbol may vary , we anticipate that as more animal genomes are sequenced and annotated, the SLC series will expand appropriately to encompass novel genes that are not orthologs of human solute carriers.The list of currently approved SLC human gene families is shown in volution . Indeed,1.3http://www.bioparadigms.org/slc/intro.htm) and additional SLC transporters are still being identified and their physiological roles elucidated. Besides transporter genes belonging to the SLC series, genes related to membrane transport include those encoding ion channels & ionotropic receptors, ABC transporters and ATP-driven transporters  of all human genes are transporter-related, consistent with the biological significance of transporters and their roles in cell homeostasis. The SLC families represent a considerable portion of these genes: almost 400 different human genes fall into the solute carrier classification  gene bsu1+ has been associated with pyridoxine (vitamin B6) uptake in yeast cells and the encoded protein  works as a proton cotransporter, its human homologue remains to be elucidated , representing less than 1% of all available 3D-resolved structures. The under-representation comes from the hydrophobic segments of integral membrane proteins that are embedded in phospholipid bilayers, making membrane proteins difficult to express and crystallize. Due to this issue, the structure determination of membrane proteins has long been considered to be a very difficult or even impossible task. However, recently developed new strategies and techniques have helped to improve the situation. Indeed, over the past decade, a large and increasing number of high-resolution structures have been solved, including numerous homologues of SLC family members   from its bacterial homologue (XylE) described below. From a pharmaceutical point of view, knowledge of the 3D-structures of human membrane proteins provides the starting point for structure-based drug design. This computational approach defines the topographies of complementary surfaces to which ligands bind. The obtained information can be used to assist development of novel compounds with improved potency and selectivity. This is nicely illustrated by the recent structure of a bacterial SLC1 homologue bound by the inhibitor DL-TBOA (SLC1 mini-review ). FinallEscherichia coli homologue (XylE) of human GLUT1-4 (SLC2A1-4) that shares about 30% sequence identity and 50% similarity was obtain in complex with d-xylose (PBD ID: 4GBY), d-glucose (PDB ID: 4GBZ) and 6-bromo-6-dexoxy-d-glucose (PDB ID: 4GCO) by X-ray crystallography methods (d-xylose symporter belonging to the major facilitator superfamily (MFS). It is composed of 12 transmembrane segments (TMs) separated into two distinct protomers (N- and C-domain) that are connected by an intracellular domain comprising four helices , Q288/Q289/N294 (TM7), W392 (TM10) and Q415 (TM11) (d-glucose and its derivative 6-bromo-6-dexoxy-d-glucose (6-BrGlc) were obtained at resolutions of 2.9 and 2.6 Å, respectively. However, d-glucose was not transported and was shown to inhibit d-xylose uptake. Interestingly, d-glucose bound around the same position as d-xylose. All amino acids comprising the d-xylose binding site are conserved with the exception of N325 (TM8) and involved new residues such as I171/Q175 (TM5) and F383/G388 (TM10).Each SLC mini-review includes a section describing structural aspects of its family members. A very recent publication on counterpart structures of the SLC2 family could not be added in this SLC mini-review series and therefore should be mentioned here. The high resolution structures of an  methods . XylE is helices A. TM7 and-glucose binding site in the human homologues.The structure of XylE permitted the modelling of a predictive structure of human GLUT1 (SLC2A1) which differs drastically from previous models. Sequence alignment analysis shows that all amino acids are conserved in human GLUT1 except Q175 which is replaced by I168. The corresponding amino acids are listed in 4www.bioparadigms.org). What follows is a summary of recent examples of pathologies associated with genetic defects of SLC genes:Given the increased understanding of the roles of transporters in normal physiology and disease, a focus of many of the mini-reviews in this series is to elaborate the latest information in this area. In addition to the following mini-reviews, an up to date summary of the knowledge can also be found by visiting the Bioparadigms website (4.1SLC1A2 (GLT1) is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) as well as Alzheimer disease (AD). Large-scale genetic analyses for disease-associated genes have indicated a link between the SLC1A2 (GLT1) gene and autism. The SLC1 family member SLC1A3 (GLAST) has been linked to the pathogenesis of schizophrenia. Under pathological conditions such as ischemia after a stroke, the neuronal glutamate transporter SLC1A1 (EAAC1) is likely to run in reverse. Therefore, blockage of “reversal glutamate transport” by EAAC1 under ischemic conditions, using a glutamate transporter subtype-specific inhibitor may lead to a possible therapeutic strategy to prevent excitotoxicity under ischemic conditions.As stated in detail in the corresponding mini-review by Kanai et al. , glutama4.2SLC2A9 are linked to these pathologies.Mueckler and Thorens explain in their SLC2 mini-review  that alt4.3In their mini-review on SLC6 transporters, which transport substrates such as serotonin, dopamine, norepinephrine, GABA, taurine, creatine, as well as amino acids, 4.4+-coupled di- and tri-carboxylate/sulfate transporters by Bergeron et al. (SLC13A2 (NaDC1), including renal handling of citrate and nephrolithiasis. In addition SLC13A3 (NaDC3) has been suggested to participate in the pathogenesis of the two inborn metabolic diseases glutaric aciduria type 1 (GA1) and Canavan disease (CD).As summarized in the SLC13 family mini-review about Nan et al. , major . Abnormal zinc metabolism has also been shown to be associated with the risk of diabetes, breast cancer and prostate cancer   has been5Due to the known roles of many SLC family members in drug disposition, transporters are now being evaluated as a routine part of the drug development process and are receiving attention by regulatory authorities around the world. The recognition of the roles of transporters in both drug distribution and drug-drug interactions has become a topic around which the US-FDA launched an expert panel review leading to a white-paper from which recommendations and guidelines have now been issued by both the US-FDA and the European Medicines Agency (EMA). In this series, Maeda and Sugiyama  review t6In addition to the roles of transporters in the diseases noted above, several transporters are also of great importance from a pharmaceutical perspective. For example, transporters can serve as drug targets or as a mechanism to facilitate drug delivery to cells and tissues. Recently exploited drug transporter targets include neurotransmitter transporters (SLC6 family), intestinal bile acid transporters (SLC10 family) and cation-Cl cotransporters (SLC12 family). In a very recent announcement, dapaglifloxin (Forxiga), the first of a class of inhibitors of the SGLT2 (SLC5A2) sugar transporters, has been approved in Europe for treatment of Type 2 diabetes. The intestinal oligopeptide transporter PepT1 (SLC15A1) or transporters at the blood–brain barrier (various SLC families) are proving to be important drug delivery systems. Due to the competitive nature of many of these drug discovery programs, it is difficult to fully assess the current activity of pharmaceutical research centered around transporters. However the examples cited above are likely only the “tip of the iceberg” of opportunity.7It is now clear that our understanding of the roles of transporters in disease, drug disposition, and toxicology is evolving quickly, driven by technical advances in assay development, protein structure determination, and evaluation of the roles physiology in both health and disease. Although we now understand many of the gene families, the interplay between the substrates and ligands with which they interact will remain a challenge to understand more fully. Each of the mini-reviews in this series provides, based on the information known today, a prospective look at the direction of research and opportunities. It is clear that the upcoming years will see us rounding out our understanding of all the transporter genes, and their functions and roles in physiology will elucidate exciting and possibly unexpected opportunities for the understanding and treatment of human diseases. As protein structures become ever more available, improved strategies emerge using this information, along with improvements in rational drug discovery approaches, to facilitate development of both new drugs as well as research tools to more deeply understand the biological roles transporters play. The future challenge for the scientific community will be the biochemical, biophysical, physiological and pharmacological assessment of all these novel gene products in a manner that can be used to improve our understanding of transporter biology, with a focus on human physiology, pathophysiology and drug discovery.


Steroid receptors are a subfamily of nuclear receptors found throughout all metazoans. They are highly important in the regulation of development, inflammation, and reproduction and their misregulation has been implicated in hormone insensitivity syndromes and cancer. Steroid binding to SRs drives a conformational change in the ligand binding domain that promotes nuclear localization and subsequent interaction with coregulator proteins to affect gene regulation. SRs are important pharmaceutical targets, yet most SR-targeting drugs have off-target pharmacology leading to unwanted side effects. A better understanding of the structural mechanisms dictating ligand specificity and the evolution of the forces that created the SR-hormone pairs will enable the design of better pharmaceutical ligands. In order to investigate this relationship, we attempted to crystallize the ancestral 3-ketosteroid receptor (ancSR2) with mifepristone, a SR antagonist. Here, we present the x-ray crystal structure of the ancestral 3-keto steroid receptor (ancSR2)-progesterone complex at a resolution of 2.05 Å. This improves upon our previously reported structure of the ancSR2-progesterone complex, permitting unambiguous assignment of the ligand conformation within the binding pocket. Surprisingly, we find mifepristone, fortuitously docked at the protein surface, poised to interfere with coregulator binding. Recent attention has been given to generating pharmaceuticals that block the coregulator binding site in order to obstruct coregulator binding and achieve tissue-specific SR regulation independent of hormone binding. Mifepristone’s interaction with the coactivator cleft of this SR suggests that it may be a useful molecular scaffold for further coactivator binding inhibitor development.  Steroid hormones play a crucial role in all but the most basic metazoans, orchestrating the cell-cell communication required to coordinate development, growth, metabolism, immunity and more . These hSRs have a modular domain architecture consisting of a highly variable N-terminal domain, a DNA-binding domain (DBD), a short hinge region, and a ligand binding domain (LBD) . ApoSRs Recent efforts to achieve tissue-specific SR-mediated regulation has been focused on developing compounds to block the SR-coactivator interface to modulate certain SR-mediated gene activity. These small molecules, dubbed Coactivator Binding Inhibitors (CBIs), are effective at competing for coactivator binding space and altering downstream transcription . These c In the absence of HSPs, SRs are inherently unstable, complicating efforts to identify the mechanisms driving ligand specificity and our ability to build robust structure-function relationships. Recent studies have utilized ancestral steroid receptors (ancSRs) to identify the molecular mechanisms that dictated the evolution of ligands specificity among SRs -13. AncS Here we report the structure of the ancestral 3-ketosteroid receptor, ancestral steroid receptor 2 (ancSR2), the ancestor of the AR, PR, MR, and GR. This structure shows the ancSR2—progesterone complex with the SR antagonist mifepristone bound at two surface sites . This stChemicals were purchased from Sigma  or Fisher . Mifepristone was purchased from Tocris Biosciences . Progesterone was purchased from MP Biomedicals . The vector for His tagged TEV was a gift from David Waugh . The pLIC_MBP vector was a gift from John Sondek . The ancSR2 LBD was resurrected using well-established protocols and was kindly provided by Dr. Joseph Thornton  . E. coli. Cultures (1.0 L in TB) were grown to an OD600 of 0.8 and induced with a final concentration of 400 μM IPTG and 50 μM progesterone at 30 °C for 4 hours. Ancestral SRs, like the extant receptors are inherently unstable in the absence of ligand and adding ligand at induction is required to for soluble overexpression of the recombinant protein. Cell mass was collected by centrifugation at 4 krpm for 20 minutes, resuspended in 150 mM NaCl, 20 mM Tris HCl (pH 7.4), 5 % glycerol, 25 mM imidazole, 0.1 % PMSF and lysed using sonication on ice. The 6xHis-MBP-ancSR2-MBP was initially purified using Ni2+ affinity chromatography . Fractions containing ancSR2-MBP were identified by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), pooled and dialyzed against 150 mM NaCl, 20 mM Tris HCl (pH 7.4), 5 % glycerol and 1 mg TEV. Following TEV cleavage, the 6xHis-tagged MBP was removed by an additional Ni2+ affinity column. The flow though containing untagged ancSR2 LBD was concentrated using an Amicon Ultra 10K centrifugal filter device (Millipore), concentrated to 3-5 mg ml-1, and dialyzed against 150 mM sodium chloride, 20 mM Tris HCl (pH 7.4), and 5 % glycerol. The final purity of the ancSR2 LBD was assessed using SDS-PAGE. In an attempt to exchange progesterone for mifepristone in the ligand binding pocket (LBP), mifepristone  was added to the ancSR2-progesterone complex for 30 minutes at 4 °C and then centrifuged at 14 K rpm for 1 minute to clarify the solution prior to crystallization trails.AncSR2 LBD was expressed as a 6xHis-MBP fusion protein in BL21(DE3) -1 protein and 1.0 μL of the following crystallant 0.8 M MgSO4, 10 % glycerol, 0.1 M MES (pH 6.0)  22-BM at the Advanced Photon Source (APS) at Argonne National Laboratory in Chicago, IL using a wavelength of 0.97 Å. Data were processed and scaled with HKL2000  . Crystal HKL2000  18]. In. In-1 pr-12 to 10-6 M for 24 hours, then assayed using Dual-Glo luciferase (Promega). Firefly luciferase activity was normalized by Renilla luciferase activity. Luminescence was read using a Synergy 4 microplate reader (BioTek). Dose-response relationships were estimated using nonlinear regression in Prism4 software ; fold increase in activation was calculated relative to vehicle-only (DMSO) control.AncSR2 LBD, was cloned into the Gal4-DBD-pSG5 vector; 31 amino acids of the glucocorticoid receptor hinge containing the nuclear localization signal-1 were inserted between the DBD and the LBD to ensure nuclear localization and conformational independence of the two domains . The hor50 of 56 nM  shows that the receptor maintains the canonical steroid receptor fold consisting of a three-layered alpha-helical bundle with four beta strands . Our prece group . Despiteo-Fc omit electron density shows clear evidence for the presence of progesterone within the LBP –deoxycorticosterone–small heterodimer partner  NRBox1 peptide complex (PDB accession code 2Q3Y), the most closely-related SR, reveals that mifepristone occupies the same position as a coregulator peptide  13]. Th. Th13]. 2 . Twelve  Unlike site-one mifepristone, site-two mifepristone does not make any crystal contacts, suggesting its binding may be biologically significant. However, there is a very low predicted free energy of binding  between the ligand and receptor, indicating low affinity binding . FurtherThe structure overlays very closely with the structures of both the ancSR2–progesterone complex (PDB accession code: 4FN9) and the PR–progesterone complex (PDB accession code: 1A28); the root mean squared deviation values for all atoms between these structures are 0.3 Å and 0.6 Å, respectively . ProgestThis structure is higher resolution than the previously published 2.75 Å ancSR2–progesterone structure . Proper in vitro. Despite ancSR2’s ability to respond to a wide array of ligands in mammalian cells [in vitro were not successful, even upon the addition of recombinant HSP90 (data not shown). It is possible that the full HSP90-HSP70-p23-FKBP52-p60 complex is required for ligand binding or exchange in vitro [in vitro appears unique to ancSR2 since other ancestral SRs, including ancCR, ancGR1, and ancGR2, as well as the modern SRs are ligand exchangeable [off preventing in vitro ligand exchange. It is well known that SRs display a narrow thermal window of activity and that their active versus inactive states are dictated by subtle thermodynamic changes. This is a consequence of both natural selection and neutral drift permitting the fine balance needed to allow the relatively small energetics of hormone binding to drive allosteric changes within the protein to propagate a signal. While too little stability prevents protein folding, too much stability may drive constitutive activation or prevent a dynamic response to ligand. It is possible that the ancSR2 is overstabilized (i.e. samples the active conformation too frequently when complexed to a ligand). This would explain its activation in the presence of mifepristone and its inability to exchange ligand in vitro. In line with these observations, ancSR2 is also unable to bind to coregulator peptides in vitro. It is well known that there is allosteric communication between the ligand binding sites and coregulator biding site [Attempts to determine the crystal structure of the ancSR2–mifepristone complex via ligand exchanged resulted instead in a crystal structure of ancSR2–progesterone with mifepristone bound at two surface sites. While the inability of mifepristone to exchange for progesterone was unexpected, it prompted us to examine the ability of ancSR2 to bind ligands and coregulators an cells ,17, attein vitro . This inangeable . AncSR2 ing site . Despiteing site . Thus, aing site . in vitro, it was surprising to see a weak secondary mifepristone interaction site at the AF-H surface. It is unclear whether this site is physiologically relevant in vivo however, the binding of mifepristone to the coactivator cleft in the crystal structure suggests there may be potential to use mifepristone as a scaffold for designing coactivator binding inhibitors. These novel pharmaceuticals would be instrumental in the treatment of a range of diseases. Currently, there is a struggle to design effective peptidomimetic CBIs due to their inability to permeate the cell membranes [Given the inability of ancSR2 to bind coactivator or ligand embranes . MifepriThis structure is only the second to show a small molecule bound to the coactivator cleft of a steroid receptor. The first showed the anti-cancer drug 4-hydroxytamoxifen (HT) bound at the coactivator cleft of estrogen receptor beta (ERβ) (PDB accession code 2FSZ) . OverlayOur present structure suggests that this may extend to small molecules targeting the coactivator cleft binders. The AF-H surface presented in ancSR2 may represent a less dynamic, low energy target for such molecules. Future work should be devoted to investigating whether mifepristone shows potential for acting as a scaffold for further CBI development. Adaptations of previously approved drugs may be a successful avenue for development of pharmaceuticals that are otherwise difficult to craft.Given their increased stability, ancestral steroid receptors are ideal tools for the extensive mutagenesis required to build robust structure-function relationships for both endogenous and synthetic ligands ,16. This


Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5′ cap structure and 3′ poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5′-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5′-end of a reporter mRNA having a short 5′ untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free,  Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is the causative agent of SARS. The nsp1 protein of SCoV blocks host protein synthesis, including type I interferon, a general inhibitor of virus replication, in infected cells. This finding suggests that SCoV nsp1 protein plays a key role in the severe symptoms that accompany SARS infection. Nsp1 binds to the 40S ribosome subunit, which is an essential component for protein synthesis, and inactivates the translation activity of the ribosome. Furthermore, nsp1 binding to the 40S ribosome induces the modification of host mRNAs, leading to the accelerated decay of these RNAs in SCoV-infected cells. We found that the nature of nsp1-induced RNA modification was RNA cleavage and that nsp1 did not recognize specific nucleotides in host mRNAs to induce this cleavage. Interestingly, nsp1 did not induce RNA cleavage in SCoV mRNAs. These data indicate that nsp1 induces RNA cleavage of host mRNAs to suppress the expression of host genes, including those having antiviral functions; yet viral mRNAs are spared from such cleavage events, which, most likely, facilitate efficient SCoV protein synthesis and virus replication in infected cells. Severe acute respiratory syndrome (SARS) coronavirus (SCoV) is the causative agent of SARS, which was first recognized in southern China in 2002 and spread to different areas of the world in a 2002-2003 epidemic Coronaviridae, is an enveloped RNA virus carrying a long (∼30 kb), single-stranded, positive-sense genomic RNA. The 5′-proximal ∼22 kb-long gene 1 region of SCoV genomic RNA has two partially overlapping open reading frames (ORFs) 1a and 1b  of nsp1 on the integrity of SCoV mRNAs primarily by using cell-free SCoV nsp1 induces endonucleolytic RNA cleavage near the 3′-region of the EMCV IRES of dicistronic RNA transcripts, Ren-EMCV-FF, in which expression of the upstream rluc ORF is mediated by cap-dependent translation and the translation of downstream firefly luciferase (fluc) ORF is driven by the EMCV IRES in both RRL E. coli. The GST tag was subsequently eliminated To determine the molecular basis for the nsp1-induced, template-dependent endonucleolytic RNA cleavage, we tested whether nsp1 induced RNA cleavage in the IRES region of a series of dicistronic RNA transcripts, each containing an IRES derived from different picornaviruses, including TMEV (Ren-TMEV-FF), poliovirus (Ren-PV-FF), CVB (Ren-CVB-FF), and HRV 2 (Ren-HRV2-FF) or a flavivirus, classical swine fever virus (CSFV) (Ren-CSFV-FF); the latter IRES has an HCV-like IRES structure. In all transcripts, expression of the upstream rluc ORF was mediated by cap-dependent translation and the translation of downstream fluc ORF driven by the IRES. The Ren-TMEV-FF or Ren-CSFV-FF transcripts were incubated in RRL with a recombinant nsp1 protein, which was initially expressed as glutathione S-transferase (GST)-nsp1 fusion protein in in vitro and in vivo.We previously reported that co-transfection of a plasmid encoding nsp1 and one that encoded dicistronic RNA transcripts carrying the EMCV IRES resulted in RNA cleavage of the expressed dicistronic RNA transcripts, demonstrating that expressed nsp1 exerts RNA cleavage in cultured cells We next determined the nsp1-induced RNA cleavage sites in Ren-EMCV-FF and Ren-PV-FF RNA. We took advantage of the fact that the RNA structure as well as the structural and functional relationships of the EMCV IRES and the poliovirus IRES are well characterized Northern blot analysis indicated that the nsp1 induced the RNA cleavage roughly 100-200 nt upstream of the initiation codon of the downstream fluc ORF of Ren-PV-FF ; hence, in vitro-synthesized capped and polyadenylated mRNAs encoding the rluc gene which carried the 5′ UTR of human β-actin mRNA or that of rabbit β-globin mRNA to characterize nsp1-mediated RNA modification of capped, monocistronic mRNAs. Incubation of ALA mRNA (containing the β-actin 5′ UTR) or GLA mRNA (containing the rabbit β-globin 5′ UTR) with nsp1, but not GST or nsp1-mt, in RRL resulted in the efficient suppression of rluc protein expression  in SCoV mRNAs that protects the viral mRNAs from the nsp1-induced endonucleolytic RNA cleavage, we prepared five different SCoV mRNA 9-like RNAs . m9LN3 Ranalysis . Similaranalysis . We alsoanalysis . As expeanalysis . These d7GpppAUAU- - -”, while SCoV mRNA 9 mt 1 and SCoV mRNA 9 mt 2 start with “m7GpppGAUAU- - -” and “m7GpppAGAU- - -”, sequences, respectively . A computer-assisted structural analysis showed that the 5′ UTR of SCoV mRNA 9 and the two mutants had the same RNA secondary structure  containing RNAs were extracted from SCoV-infected or mock-infected cells and subjected to The present study was aimed at identifying the nature of the nsp1-induced host mRNA modification, exploring the basis of the RNA modification site selection, and further understanding template-dependent, nsp1-induced RNA modification by examining several different RNA templates, including SCoV mRNAs and their derivatives.Nsp1 induced an endonucleolytic RNA cleavage within the IRES region of different dicistronic RNAs containing a picornavirus type I IRES or type II IRES We identified the nsp1-induced RNA cleavage sites of several mRNAs. For Ren-EMCV-FF and Ren-PV-FF, RNA cleavage mainly occurred near the 3′-end of the IRES elements, where the 40S ribosome is recruited  and 3. M32P cap-labeled ALA mRNA that had been incubated with nsp1 showed a discrete ∼29 nt 32P cap-labeled RNA fragment .Vero E6 cells were infected with the Urbani strain SCoV or SCoV-mt Replacing the rluc gene of pRL-SV40 (Promega) with the PCR product containing a T7 promoter sequence, 5′ UTR of human β-actin mRNA, rluc gene and a 50-nt-long poly(A) tail resulted in pALA-SV40 encoding ALA mRNA. A similar method was used to generate pGLA-SV40 encoding GLA mRNA, except that the 5′ UTR of human β-actin mRNA in pALA-SV40 was replaced by the 5′ UTR of rabbit β-globin mRNA. By using pGLA-SV40 as a template, we employed a QuikChange site-directed Mutagenesis Kit (Stratagene) to generate plasmids encoding GLA mRNA mutants. A reporter plasmid, pRL-HL, carrying in the following order: a CMV promoter, T7 promoter, rluc ORF, HCV IRES and fluc ORF, was described previously in vitro by using the mMESSAGE mMACHINE T7 kit or T7 Ultra kit (Applied Biosystems) according to the manufacturer′s protocol. Synthesis of SCoV mRNA 9 was performed as previously described -CGGAGTAATACGACTCACTATTATATTAGGTTTTTACCTACCC-OH, and SARS 3′ UTR primer, 5′-TTTTTTTTTTTTTTTTTTTTGTCATTCTCCTAAGAAG-OH; to ensure that the 5′-proximal sequence of the in vitro synthesized RNA was identical to that of authentic SCoV mRNA 9, the T7 class II Φ2.5 promoter (underlined sequence) was used to initiate transcription by ATP, instead of GTP TTAATACGACTCACTATAG AACAAATTAAAATGTCTGATA ATGG-OH (T7 promoter sequence underlined); 5′-AATTAATACGACTCACTATAG ATATTAGGTTTTTACCTACC-OH (T7 promoter sequence underlined); and 5′-CGGGATCCGAG TAATACGACTCACTATT AGATTAGGTTTTTACCTACCC-OH (the T7 class II Φ2.5 promoter sequence underlined). The amplicon, purified on an agarose gel, served as a template for the synthesis of uncapped SCoV mRNA 9 using the MEGAscript T7 kit (Applied Biosystems). The in vitro synthesized SCoV mRNA 9 was purified with the RNeasy mini kit (Qiagen), capped with vaccinia virus capping enzyme using the ScriptCap m7G Capping System (Epicentre Biotechnology) and further purified with the RNeasy mini kit (Qiagen). Similar procedures were used for the generation of m9LN and m9LrLuc3 RNAs, except that the PCR products, as shown in Transcription of dicistronic RNA transcripts was described previously In vitro translation was performed using the Retic Lysate IVT kit (Applied Biosystems). RRL was initially incubated with 1 µg GST protein, nsp1 protein or nsp1-mt protein purified from E. coli at 4oC for 10 min 32P labeled primers , primer extension was performed by using the Primer Extension System (Promega) according to the manufacturer's protocol. The RNAs, primer sequence, and primer-binding site and conditions are as follows: GLA and ALA mRNAs, 5′-TTTTTCTGAATCATAATAATTAA-3′, ∼100 nt downstream of rluc translation initiation codon, incubation at 42°C for 1 h and subsequent incubation at room temperature for 10 min; Ren-EMCV-FF, 5′-AGCAATTGTTCCAGGAACCAGGG, ∼100 nt downstream of fluc translation initiation codon, incubation at 55°C for 1 h and subsequent incubation at room temperature for 10 min; Ren-PV-FF, 5′-GGGCCTTTCTTTATGTTTTTGGCG, ∼6 nt downstream of AUG translation initiation codon of fluc gene, incubation at 55°C for 1 h and subsequent incubation at room temperature for 10 min; SCoV mRNA 9, m9LN3, and m9LN, 5′-GGGTCCACCAAATGTAATGC-3′, ∼44 nt downstream of N gene translation initiation codon, incubation at 50°C for 1 h and subsequent incubation at room temperature for 10 min; SCoV mRNA 3, 5′-TGTAGCATGAACAGTACTTGC-3′, ∼75 nt downstream of 3a gene translation initiation codon, incubation at 50°C for 1 h and subsequent incubation at room temperature for 10 min; m9LrLuc3, 5′- TTTTTCTGAATCATAATAATTAA-3′, ∼100 nt downstream of rluc translation initiation codon, incubation at 42°C for 1 h and subsequent incubation at room temperature for 10 min; and m9N3, 5′-GTCCTCCATTCTGGTTATTGTC-3′, ∼76 nt downstream of N gene translation initiation codon, incubation at 50°C for 1 h and subsequent incubation at room temperature for 10 min; m9Lactin3 and actin, 5′- AGCGCGGCGATATCATCATC-3′, ∼1 nt downstream of the AUG translation initiation codon of β-actin gene, incubation at 55°C for 1 h and subsequent incubation at room temperature for 10 min. AMV reverse transcriptase was used for primer extension. After ethanol precipitation, the primer extension products were resolved in 8% polyacrylamide/7M Urea DNA sequencing gels.After incubation of RNA samples with the 5′-end in vitro transcription kit (Applied Biosystems). Cap labeling was performed by incubation of 30 µg of uncapped RNA with vaccinia virus capping enzyme (ScriptCap m7G capping system) in the presence of α-32P GTP  at 37°C for 1 h. Cap-radiolabeled ALA mRNA was purified with the RNeasy mini kit and 0.75 µg RNA  was used for in vitro translation in RRL. Then the sample was incubated with proteinase K, and RNA was extracted by phenol/chloroform.Uncapped and polyadenylated ALA mRNA was prepared by using the MEGAscript 32P GTP . After ethanol precipitation, the cap-labeled marker RNA was dissolved in a solution containing 8 µl H2O, 1 µl 10X kinase buffer  and 1 µl 10X cleavage reagent . The full-length RNA marker was cleaved into an RNA ladder by incubation at room temperature for 5 min. The produced RNA ladder contained a set of cap-labeled RNA molecular weight markers of 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 and 10 nt in length. An equal volume of 2x proteinase K digestion buffer was added to the prepared Decade Marker RNA ladder to adjust the salt concentration to a level similar to that of RNA samples extracted from RRL. The RNA samples were analyzed on 10% polyacrylamide/7M Urea DNA sequencing gels.The RNA size marker (ranging from 10 nt to 150 nt) was prepared with the Decade Marker system (Applied Biosystems) according to the manufacturer's instruction with modifications. Full-length Decade Marker RNA (0.5 µg) of 150 nt in length was cap labeled with vaccinia virus capping enzyme in the presence of α-Figure S1Susceptibilities of Ren-CVB-FF and Ren-HRV2-FF to nsp1-mediated RNA cleavage. (A) Schematic diagram of the structures of full- length dicistronic RNA transcripts (Full length), RNA 1 containing the 5′ rluc gene and intercistronic IRES sequence and RNA 2 containing only the 5′ rluc gene. (B and C) Ren-CVB-FF (B) and Ren-HRV2-FF (C) were independently incubated with GST, nsp1, or nsp1-mt or without any protein (mock) in RRL+HeLa at 30°C for 10 min. RNA samples were extracted and analyzed by Northern blotting using an 5′ rluc probe (left panel) and 3′ fluc probe (right panel). Marker represents a mixture of in vitro-transcribed full length RNA transcripts, RNA 1 and RNA 2.(TIF)Click here for additional data file.Figure S2Susceptibilities of discistonic RNA transcripts carrying different IRESes to nsp1-mediated RNA cleavage in cells. Plasmids pCAGGS-CAT encoding the CAT gene, pCAGGS-nsp1 encoding nsp1 and pCAGGS-nsp1 mt encoding nsp1-mt were co-transfected with each of the CMV promoter-driven dicistronic reporter plasmids encoding the 5′ rluc gene, IRES and 3′ fluc gene in HEK293 cells. The IRESes included in the RNA expression plasmid were: TMEV IRES (A), poliovirus IRES (B), HCV IRES (C) or CSFV IRES (D). After 24 hr, total RNA was extracted from the cells by using Trizol reagent. Samples were treated with DNase I and RNAs were purified with RNeasy (Qiagen). The RNAs were subjected to Northern blot analysis using an rluc probe.(TIF)Click here for additional data file.Figure S3Susceptibility of actin mRNA to nsp1-induced RNA modification.In vitro-synthesized actin mRNA was incubated with GST, nsp1 or nsp1-mt in RRL. RNAs were extracted and subjected to primer extension analysis by using the same primer described in (TIF)Click here for additional data file.Figure S4Effect of nsp1 on translation of SCoV mRNA in RRL and SCoV-like mRNA encoding a reporter protein in HeLa S10 extract. (A) Intracellular polyA+ RNAs obtained from SCoV-infected Vero E6 cells (SCoV-infected) were subjected to an in vitro translation reaction in the presence of 35S-methionine along with GST, nsp1 or nsp1-mt in RRL (left panel). As a control, intracellular poly A+ RNA from mock-infected Vero E6 cells was subjected to in vitro translation in the presence of GST (Mock). A similar in vitro translation assay was performed using in vitro-transcribed capped GLA mRNA (right panel). The radiolabeled proteins were analyzed by SDS-PAGE and autoradiography. SCoV major structural protein (N protein) and rluc protein are indicated by arrows. (B) In vitro synthesized m9Lrluc3, a SCoV mRNA 9-like reporter mRNA carrying an rluc gene in the place of N gene ORF (in vitro translation assay in HeLa S10 extract in the presence of GST (GST) or nsp1 proteins (nsp1). After 1.5 h incubation, the rluc activity was measured and represented as the average of three independent experiments (+/- SD).gene ORF , was sub(TIF)Click here for additional data file.


We assessed how the photosynthesis and respiration of different reef habitats is controlled by light and hydrodynamics. Numerous fluxes (over a 0.25 h period) were as high as 4500 mmol O2 m−2 d−1, which can only be explained by efficient light utilization by the phototrophic community and the complex canopy structure of the reef, having a many-fold larger surface area than its horizontal projection. Over diel cycles, the reef crest was net autotrophic, whereas on the reef slope oxygen production and respiration were balanced. The autotrophic nature of the shallow reef crests implies that the export of organics is an important source of primary production for the larger area. Net oxygen production on the reef crest was proportional to the light intensity, up to 1750 µmol photons m−2 s−1 and decreased thereafter as respiration was stimulated by high current velocities coincident with peak light levels. Nighttime respiration rates were also stimulated by the current velocity, through enhanced ventilation of the porous framework of the reef. Respiration rates were the highest directly after sunset, and then decreased during the night suggesting that highly labile photosynthates produced during the day fueled early-night respiration. The reef framework was also important to the acquisition of nutrients as the ambient nitrogen stock in the water had sufficient capacity to support these high production rates across the entire reef width. These direct measurements of complex reefs systems yielded high metabolic rates and dynamics that can only be determined through in situ, high temporal resolution measurements.Quantitative studies of coral reefs are challenged by the three-dimensional hard structure of reefs and the high spatial variability and temporal dynamics of their metabolism. We used the non-invasive eddy correlation technique to examine respiration and photosynthesis rates, through O Close to a billion people are to some extent dependent on reefs, and 275 million people live less than 30 km from the coast whose ‘The total surface area of reefs can be 15 times the planar reef area 2 efflux from photosynthesizers and in increasing respiration due to the flushing of permeable sediments Reef metabolic rates are highly affected by the variable hydrodynamics and the numerous micro-habitats created by the reef's 3D structure. This makes it very difficult to extrapolate observations from a small area or a single species to a complete reef system. The 3D structure also creates micro-habitats that receive different amounts of light throughout the day 2 across the water-air interface which is difficult to define accurately −2 y−1Many of the in situ analyses of coral reefs have been done on shallow, low energy reefs with a unidirectional current, as most of the evaluations were done by flow respirometry techniques that require these conditions 2 production and consumption for the entire reef environment representing corals, algae, and other benthic organisms. The EC technique has proven very useful in a variety of environments including: muddy deep-sea sediments In this study we used the eddy correlation (EC) technique 2 exchange in different reef habitats, including highly productive reef crests and less productive reef slopes and (2) to examine how production and respiration are affected by light, hydrodynamics, and community structure. To answer these questions we applied EC to the high surface area, rough hydrodynamic and heterogeneous reef environments. We finally discuss the suitability of EC for coral reef studies and show that examining O2 exchange rates at a high temporal resolutions on an ecosystem-scale is important to revealing reef metabolism dynamics.The goals of this research were (1) to quantify O2 quadrats by SCUBA diving at 0, 2, 5, 10, and 15 m, with the coverage of benthic communities determined by Coral Point Count with Excel extensions software The research was done in the Florida Keys National Marine Sanctuary 7 km offshore within Grecian Rocks Sanctuary Protection Area  at the southern tip of Florida, USA. Research was conducted in June through August of 2009 and 2010. Two reef crest (RC) sites were located on the 2.0 m average depth reef crest and two reef slope (RS) sites were located 125 m away off the reef crest at an average depth of 4.5 m . The ben2 concentration with a high-resolution custom-made picoamp amplifier . Fast responding (<0.3 sec) Clark-type O2 microsensors 3 measuring volume 2 concentration were measured at 64 Hz over 0.25 h long measuring periods. Each deployment was >24 h to capture diel fluctuations. All instrumentation was self-contained and attached to a triangular frame that was designed to minimize hydrodynamic interferences. At all sites the instrument was located in a small sand patch with the measuring volume of the instrument located about 0.8 m above the bottom on the RC and 0.6 m above the bottom on the RS. A number of supporting instruments were also deployed to examine the drivers and controls of the 0.25 h averaged O2 fluxes. From the ADV measurements the current direction, mean velocities, and significant wave height were determined. Current direction was determined from the velocity field and a known instrument heading. Significant wave height was determined from the pressure data using QuickWave software . Temperature was also recorded by the ADV. O2 optodes  were positioned at the same height as the measuring volume and were used as a validation and calibration of the Clark-type microsensors. Photosynthetically active radiation (PAR) was evaluated at the same height as the instrument measuring volume using HOBO pendant light intensity loggers  or LI-COR Spherical Quantum PAR Sensors  that were all site-specifically inter-calibrated as described by Long et al. (2012) The EC instrument consists of an Acoustic Doppler Velocimeter , which measures the three dimensional velocity field and records the O2 concentration as:The time-averaged flux across the reef-water interface was derived from high-resolution measurements of the vertical velocity and the O2 concentration, respectively 2 data were carefully examined for sensor malfunctions due to particles or organisms interfering with the sensors which were evident by signal jumps, anomalous noise, variation from the stable optode, and loss of sensor response. Daily averaged rates of gross primary production (GPP), respiration (R), and net ecosystem metabolism (NEM) were calculated from 24 h long data sets where NEM = GPP+R. The daily rates were weighted by the hours of light and dark and assumed that the respiration rates were the same during the day and night where the bars symbolize the averaging over time, and The 0.25 h fluxes were examined for corrleations with measured environmental parameters using linear regression analysis. The daytime fluxes were also correlated to light using standard P-I curves which account for respiration p = 0.01 level were used to determine if differences existed between the different data treatments. Rotation of the velocity field, where the X velocity is aligned with the mean current direction, was not done as Reimers et al. (2012) Subsets of EC data were also examined for variances due to the averaging method used to determine the means in the Reynolds Decomposition including: mean removal, linear detrending, and filtering The transect analysis revealed that the reef crest (RC) sites were dominated by gorgonians and soft corals (51%), algae (24%), and sand, pavement, and rubble (20%) where the percentages are the percent cover of the reef surface. The reef slope (RS) sites were dominated by sand, pavement, and rubble (47%), algae (41%), and gorgonians and soft corals (9%). Hard coral coverage averaged 3% for the RC sites and 1% for the RS sites. The RC had a well-developed hard reef structure that was ∼0.5 m high over the underlying permeable sand, while the RS had scattered coral heads and rubble with permeable sand separating them .2 m−2 d−1 over a 0.25 h interval. When all the fluxes from multiple 24 h periods were binned and plotted versus light (2 m−2 d−1 (determined from the maximum measured PAR), a respiration rate of −590 mmol O2 m−2 d−1, and a light compensation point of 300 µmol photons m−2 s−1. However, on the RC sites we observed a decrease in the flux with the highest irradiances  or the RS .An example of a 24 h EC data set for the RC is presented in us light  a hyperbadiances  that weradiances . On the fternoon , when thfternoon . When di2 = 0.285, p = 0.275) or the RS  when binned and correlated in a similar way. The R rates decreased gradually during the night on both sites (2 concentration (2 concentrations decreased by only 5–15%. The flow velocities and the wave heights did not change significantly across the night (data not shown). Thus, neither the small decrease in the O2 concentration, nor hydrodynamic effects were the cause for the decrease in respiratory activity.Nighttime respiration (R) rates were similarly increased by the velocity on both the RC and RS sites . Signifith sites . This dentration . HoweverOver a diel cycle, the RS had a net balance between respiration (R) and gross primary production (GPP), and a net ecosystem metabolism (NEM) of approximately 0, while the RC had a net positive NEM . All dai2 microsensors reduced the response times of electrodes after 48 h.A total of 23 RC and 8 RS deployments were conducted, resulting in 7 complete RC 24 h data sets and 5 complete RS 24 h datasets. Due to the rough hydrodynamics and faunal activity at the RC sites microsensor breakage was common. Interference and breakage from large debris such as seagrass blades occurred periodically at both sites and microorganism biofouling on the glass OThe footprint of the EC technique changed with the current direction and therefore the flux was examined as a function of direction on the RC . Using tVariations in the EC data due to how the mean (used in the Reynolds Decomposition) was determined 2 fluxes were up to 4500 mmol m−2 d−1  2 m−2 d−1 and a light saturation constant of 240 µmol m−2 d−1. However, when surface area is considered the light is spread out over a much larger area on a complex reef structure (per m−2 of planar reef area), and much higher rates of production are possible compared to that of a flat surface. For example, based on this first-order calculation, a reef with a surface area of 7 times that of the m−2 reef planar area may produce an average flux of 4550 mmol O2 m−2 d−1 at the peak light levels of 2750 µmol photons m−2 s−1 measured in this study , with the daytime fluxes tightly coupled to the irradiance (2 m−2 d−1 (2 m−2 d−12 m−2 d−12 m−2 d−12 m−2 d−12 for comparison purposes). These studies were conducted using flow respirometry techniques which, like the EC technique, integrate over a large reef area and incorporate the complex surface area of coral reefs but do not have the spatial or temporal resolution of EC. The high-resolution and high-magnitude fluxes reported here can only be resolved by using the EC technique.The shallow, well developed RC produced large fluxes during high irradiances  and a conservative C:N ratio of 10∶1 for the reef benthos dominated by soft corals and macroalgae −2 d−1. Using our average measured velocity of 0.04 m s−1, a 2 m deep and fully mixed water column, and a total N concentration measured over a nearby reef at the same time of year of 7 µmol L−1−1 m−1. While the inorganic N fraction represents only 2% of the total available N −2 basis. This is equivalent to the removal of 36% of the total N across the entire reef width (∼175 m). Further, the high rates of plankton depletions reported for reefs of up to 90% A substantial nutrient supply is required to support these large production values in the oligotrophic subtropical waters. A GPP rate of 1000 mmol C m−2 s−1, The relationship between the irradiance and the daytime fluxes produced ecosystem-wide P-I curves with the RS displaying an expected production maxima at high irradiance levels . However2 from organisms 2 production during periods of high flow in the afternoon. Further, the fact that no increase in O2 production was found with increasing flow velocities suggests that the flow stimulation of R masked any concurrent increase in O2 production due to flow that may have occured. Compared to studies conducted on the surfaces of individual species, it is likely that the stimulation of R by flow seen here was much greater than the concurrent effects of flow stimulating photosynthesis when these processes were examined in situ on an ecosystem-scale.The reduced canopy structure on the RS may allow for photoinhibition which has been shown to reduce afternoon production rates as the photoreceptive capacities of phototrophs are reduced with increased light While there were not significant differences between the morning and afternoon fluxes, a hysteresis was seen in the fluxes with morning fluxes increasing linearly with irradiance . With thRespiration is stimulated by flow due to the flushing of permeable sediments 2 and photosynthates as they are consumed across the night or due to diel changes in the activity of reef heterotrophs 2 concentrations on the RC and RS decreased by only 5–15% across the night while the R rate decrease by 60%. This decrease in R across the night is similar to that found by Falter et al. (2011) Respiration rates also decreased across the night at both sites  which ca2 fluxes represent the most accurate in situ analysis that can currently be conducted on reef ecosystems.The higher rates of R immediately after dark , 7 also The RS had reduced GPP and R rates compared to those of the RC  due to t2 flux rates by EC are reported per m2 of planar surface, but incorporate the total 3D surface and structure of complex environments such as coral reefs. Through these measurements, high-temporal resolution O2 fluxes can be correlated to changes in environmental conditions while incorporating all of the organisms within and on the reef structure.This research shows the EC technique as an excellent approach for the analysis of reef systems, as it can be deployed at any depth, is totally self-contained, and can be deployed over any bottom structure. The EC technique measures over a large reef area, or footprint, 2 flux rates for a complete reef system. For these reasons, these first eddy correlation data from reefs likely represents the most accurate representation of the O2 exchange rates of complete reef ecosystems.Sampling at this community level is also important due to the difficulty of scaling up measurements done on individuals to the community level, as they do not account for varying community compositions, self-shading, densities of individuals, 3D structures, or are done on only part of an individual 2 flux with changes in current direction   indicate2 measurements were also examined and did not produce any significant differences in the flux (If the velocity sensor is tilted relative to mean flow field of velocity, or if waves are present, a wave or tilt bias may occur the flux . This inThese results highlight the amount and quality of data that can be measured with EC while showing that it can be applied in complex and challenging environments. These data revealed high production rates on autotrophic reef crests due to high SA that created numerous microenvironments relative to light and flow. This high primary production and 3D structure of reefs also supports a large range and quantity of organisms, enabling reefs to be hotspots of biodiversity and a source of primary production to surrounding environments. These ecosystem-scale measurements also show how high production rates are possible, even in nutrient-replete environments, when the reef canopy and framework are considered. Furthermore, when working in complex environments, ecosystem-scale measures that include all variability in the system are needed to determine the net effects on production, respiration and ultimately carbon cycling.


PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations:  However, the majority of somatic alterations in cancer genomes are a consequence of genomci instability and appear to be passenger or bystander mutations that are unlikely to be involved in oncogenesis Recently, the first cancer genomes to be completely sequenced have revealed an unanticiapted breadth and complexity of somatic alterations Sequencing cancer genomes is a formidable task that requires expensive technologies and computational support to assemble large portions of the genome. Because of the intense interest in identifying key somatic alterations, investigation has focused on techniques for screening or analyzing regions of interest. Most studies have concentrated on coding regions and adjacent intronic or putative regulatory regions HRM specific PCR products are generated to interrogate conformational differences, also known as dissociation curve analysis, using conventional real-time PCR platforms. It is utilized in combination with a double stranded DNA binding dye in order to characterize primer-related non-specific amplification (or primer dimer) for detection of a specific target. Single-base changes in PCR products are detected by altered HRM properties monitored through the release of fluorescent double strand DNA binding dye The aim of this study was to assess the sensitivity and specificity of an inexpensive HRM analysis platform for mutation scanning of single-base variation in a range of tumor samples: frozen pediatric small rounded blue-cell tumors and paraffin-embedded tumors from breast, endometrium and ovarian cancers. Bi-directional sequence analysis was performed to determine the accuracy of this DNA HRM technology.The Institutional Review Board for the Polish Breast, Ovarian, and Endometrial Cancer Study were approved by the National Cancer Institute (NCI), at Bethesda, MD, the M. Sklodowska Institute of Oncology and Cancer Center in Warsaw, and the Institute of Occupational Medicine (IOM) in Lodz, both in Poland http://chtn.nci.nih.gov/), were anonymized, and our protocol was reviewed by the Office of Human Subjects Research at National Institutes of Health, Bethesda, MD, and deemed exempt.All frozen samples from pediatric small rounded blue-cell tumors and obtained from Cooperative Human Tissue Network (http://chtn.nci.nih.gov/). Neuroblastoma cell lines and their culture conditions are described elsewhere Snap frozen tumor samples were obtained from Cooperative Human Tissue Network , the M. Sklodowska Institute of Oncology and Cancer Center in Warsaw, and the Institute of Occupational Medicine (IOM) in Lodz Single 0.6 mm tissue cores targeted to areas with tumor that had been identified and marked by a pathologist (MES) were obtained from each tumor block for DNA extraction, using a tissue microarray coring needle for each sample. Microdissection was performed for a small proportion of the samples, making it difficult to accurately assess the percentage of tumor material. Nucleic acid extraction was performed with the Agencourt® FormaPure™ kit  according to the manufacturer's instructions. To avoid interferences with the PCR we removed RNA from purified total nucleic acid during the extraction method. After extraction and purification, DNA concentration and purity were quantified using NanoDrop . Total genomic DNA extracted with this method yielded an averaged of 2.07 µg (range 0.03 to 7.69 µg). The purity of DNA for each extraction method was assessed by measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm (A260) and 280 nm (A280).PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3) in published reports, with a particular emphasis on breast, ovarian and endometrium cancers The set of exons selected for this mutation scanning analysis were drawn from cancer genes frequently mutated  design of PCR primers to keep GC content under or as close as 60% as possible, product size around 200 bp and avoid known variants within the primer region; 2) selection of optimal annealing temperatures with gradient PCR; 3) and design of PCR experiments in a consistent manner: same assay, with same sample batch and same machine run.PIK3CA, ERBB2, KRAS at 66°C; TP53, EGFR, BRAF at 68°C; GATA3, FGFR3 at 70°C). After PCR, the samples were heated to 93°C for 30 seconds and then cooled to 25°C before HRM.PCR-based analyses for the different genes were performed in 96-well format with 10 µL volumes and included 10 ng of genomic DNA for frozen samples, and 1 µL of solution containing genomic DNA for paraffin-embedded tissue samples, with mean concentration of 25.8 ng/µL (SD = 21.7), and ranging from 2 to over 55 ng/µL . Master Mix that included all deoxynucleoside triphosphates, Taq polymerase, and the LCGreen® PLUS  was used for the pre-HRM PCR. PCR was performed using a MJ Research PTC 225 Thermal Cycler  with an initial denaturation at 95°C for 2 minutes, followed by 45 cycles of 2 step temperature cycling of 95°C for 30 seconds, and 66 to 70°C for 30 seconds  or minimal variations (n = 99) in the normalized HRM curve, and also in 20% randomly chosen negative samples (n = 45). A second round of HRM was performed to assess the reproducibility of the method, using known negative controls and positive controls. Frank variations were defined as those HRM curves interpreted by the software to be suspicious of harboring a nucleotide change or a mutation/variant, and were represented in red in the graphics . Depending on the assay combination on the plate, HRM range was set to accommodate each assay individual profile with at least 4°C prior to the first melt transition on the plate, with a slope of 0.3°C/s, and at least 3 degrees after the last fragment has completely melted.Since HRM was performed in this study as the screening technology, the curves were analyzed using custom LightScanner™ software . Normalization and background subtraction were first performed by fitting an exponential to the background surrounding the HRM transitions of interest. The normalized HRM curves were temperature-overlaid, to eliminate slight temperature errors between wells or runs. Difference plots of these normalized and temperature-overlaid curves were obtained by taking the fluorescence difference of each curve from the average wild-type curve at all temperature points TGTAAAACGACGGCCAGT) or M13 reverse (CAGGAAACAGCTATGACC). PCR conditions for sequencing analysis were performed in 96-well format with 10 µL volumes and included 1 µL of amplified DNA from the pre-HRM PCR reaction. Genomic DNA was used only when the sequence reaction failed with amplified DNA. PCR products were sequenced using a modified ABI Prism® BigDye Terminator protocol . Unincorporated dyes terminators and salts were removed utilizing a Sephadex G-50  spin columns in a MultiScreen®-HV 96-well filter plate . The reactions were run on an ABI 3730XL . Sequence traces were analyzed and compared using two software packages  and reviewed by two independent reviewers TP53 and GATA3), PCR conditions as well as their primers were modified to improve sequencing. This included generating primers that extended across more of the genomic regions or slide 20–30 bp up or down stream. But we noted that the new, specific assays failed to optimize while testing different regions would alter the purpose of the study. In summary, sequencing error rate was 2.5% for frozen specimens and 20.0% for paraffin-embedded.Bi-directional sequence analysis was performed with primers that were designed by extending each oligonucleotide used in the pre-HRM PCR with a universal sequencing primer: M13 forward (www.ncbi.nlm.nih.gov/projects/SNP/), using Genewindow (genewindow.nci.nih.gov) Nucleotide changes detected by sequencing were classified as novel alterations or known SNPs (or Single Nucleotide Polymorphisms) if found in dbSNP, Build 130, from the NCBI  and a successful pre-HRM PCR assay, measured either by the presence of a single amplicon in agarose gel or the presence of a DNA product at the HRM analysis, was performed by logistic regression analysis. Agreement between 2 variables (or reliability) was determined by a Kappa test. Kappa values less than 0.40 indicate low association, between 0.40 and 0.75 indicate medium association, and values greater than 0.75 indicate high association between two measures. Screening capabilities of HRM and the consistency of the analysis were measure using classical metrics, such as sensitivity, specificity, false negative and positive rates, considering sequencing analysis as the standard measurement for both frozen and paraffin-embedded extracted samples.www.r-project.org/).Statistical analyses were conducted using Microsoft® Excel  and R statistical package . We observed 59 HRM positive samples, 2510 HRM negative, and only 23 of these measurements were not evaluable (less than 1%). For the repeat HRM experiments, 47 were positive, 156 negative, and only 1 out of 204 was not evaluable. In total, 2,772 out of 2,796 , or 99.1%, experiments were evaluable for screening of mutations/variants by HRM analysis.ERBB2 exon 25, the distal region of TP53 exon 5, GATA3 exon 5, and the proximal region of GATA3 exon 6. For the remainder tested exons, between 1 to 9 putative nucleotide substitutions were detected; notably exon 13 of FGFR3 had the highest number, 30. The results of mutation screening by HRM technology in both experiments, initial and repeat, as well as the validation with sequencing for frozen samples are displayed in In the initial round of analysis, 4 assays had no mutation detected: TP53), by AA.HRM experiments were repeated on all the samples with evidence of a putative mutation on the HRM curve, and also in a subset of negative samples. The agreement between the initial and repeat screen of HRM experiments was 91%, with a kappa test value of 0.77, or high concordance between both. In general, HRM curves presented similar shapes in both independent analyses . The majFGFR3, rs7688609  for all breast cancer samples, a mean of 1.82 (SD = 0.12) for endometrial cancer samples, and a mean of 2.0 (SD = 0.27) for ovarian cancer samples. There was a direct association between the concentration of DNA (in ng/µL) extracted from the paraffin-embedded samples, the DNA amount used for the pre-HRM PCR, and the presence of a single band in the agarose gel (p<0.001). Also there was an association between extracted DNA concentration and the presence of an adequate HRM curve for analysis (p<0.001). HRM was successful 96% of the time when the quantity of paraffin-embedded extracted DNA used for this technique was more than 30 ng in comparison with 92% when the quantity was ≤30 ng .Overall, 93%  of the measurements of paraffin-embedded samples by HRM analysis were evaluable. This technique was more successful when frozen DNA specimens were analyzed than when DNA extracted from paraffin-embedded samples was used, 99.1% versus 93.3% (p<0.001).TP53  and GATA3 , affecting the comparison between sequence analysis and HRM curves. Sequencing of these amplicons failed in 341 out of 397 reactions, which accounted for 86% of all failed sequencing. When these failed assays were excluded from the comparison between HRM curves and sequencing, sensitivity increased to 92%, with an accuracy of 80%. Paraffin-embedded sequencing details are described in The results of the mutation screening by HRM technology and its validation with bi-directional sequence analysis for the paraffin-embedded samples are displayed in HRM analysis using the LightScanner™ represents a moderate-throughput screening test for mutations among candidate genomic regions. The comparison with bi-directional sequencing analysis provides strong evidence for its accuracy despite the low prevalence mutation/variant rate, particularly since selected exons harbored no mutations. Our results are consistent with earlier reports KRAS and EGFRTP53 assays Sensitivity and specificity of HRM analysis was higher in frozen samples compared to paraffin-embedded samples, with an observed sensitivity of 97% for DNA extracted from frozen samples whereas it is 88% for DNA extracted from paraffin-embedded tissue. Lower performance of some assays when comparing DNA from paraffin-embedded specimens versus frozen samples has also been described in previous studies for In order to eliminate the subtle differences in the reagent components between the final elution buffers from multiple extraction platforms and to minimize variability within samples our approach was to perform the DNA extraction using a common extraction platform, conditions and protocol. With optimized sample handling and standardized DNA extraction, it is possible to screen paraffin-embedded samples with higher sensitivity DNA extracted from paraffin-embedded tissues was also more difficult to sequence than DNA from frozen tissue Based on these considerations, our recommendations to maintain and, perhaps, enhance the screening capabilities of HRM analysis for paraffin-embedded extracted samples with a LightScanner™ would include the following:Inclusion of ≥30 ng total genomic DNA may increase HRM analysis success rate up to 96%.Pre-HRM PCR optimization should include careful primer selection to reduce GC content, adequate amplicon size, and optimal annealing temperature.Amplicons that failed sequencing over 50% of the times also performed poorly during HRM analysis. So it may be worthwhile to test the selected amplicons by sequencing a few samples at the beginning of the experiment.The false positive rate of HRM analysis in paraffin embedded samples was approximately 20%, which implies that additional sequencing is needed to improve accuracy in the subset of samples with a putative mutation In conclusion, HRM analysis with the LightScanner™ is a promising screening tool for mutation/variant in somatic DNA extracted from either frozen or paraffin-embedded samples, although overall accuracy is better in frozen specimens, probably related to DNA quality. This method is able to detect mutations as well as known SNPs, even in genomic regions with a low mutation prevalence rate in the range of 5% or perhaps lower.Figure S1BRAF exon 15 from genomic DNA extracted from frozen samples. Red arrow: HRM curves with a plot interpreted by the software to be suspicious of harboring a nucleotide change or a mutation/variant. HRM was repeated for all these samples, and all of them were sequenced. Green arrows: HRM curves with minimal variations with respect to the averaged wild-type curve. All these samples also were sequenced and HRM was repeated. Black arrow: All normalized HRM curves considered to have a wild-type sequence. 20% of these samples were randomly chosen to be repeated and sequenced as negative controls.Representation of HRM curve of (2.49 MB TIF)Click here for additional data file.Figure S2KRAS. Each square represents a well: brown squares are negative controls; grey squares represent samples with no mutation/variant; red squares represent possible mutation/variant; and green are unknown for mutation/variant. B. Normalized HRM curve from the same samples in the exon 2 of KRAS initial analysis. C. Normalized HRM curve of the repeated KRAS analysis.Example of HRM output from genomic DNA from tumor frozen samples set. A. Output of one of the 3 plates used for the initial analysis of exon 2 of (0.68 MB TIF)Click here for additional data file.Figure S3EGFR) performed in paraffin-embedded samples from breast cancer: 1. Normalized HRM curves of the assay; 2. Segment of sample assembled trace after sequencing, with the presence of a variant, where AA has replaced both alleles GG. B. Endometrial paraffin-embedded samples for KRAS. 1. Normalized HRM curves of the assay with elevated number of positives samples observed in the HRM curves from paraffin specimens compare to frozen samples; 2. Genotype GG has been substituted by GA.Samples of mutation screening with HRM technology and its validation with sequencing from paraffin-embedded samples. A. One of the assays ((9.83 MB TIF)Click here for additional data file.Table S1Mutation details from the sequencing analysis of paraffin-embedded samples.(0.09 MB DOC)Click here for additional data file.Table S2New nucleotides changes found on the study samples submitted to dbSNP (build 131).(0.09 MB DOC)Click here for additional data file.


Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3′-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3′ initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family  T = 3 icosahedral capsid, and belongs to genus Alphanodavirus of the family Nodaviridae. FHV was originally isolated from grass grubs in the former Flock House agricultural station in Bulls, Rangitikei District, New Zealand. Like other nodaviruses, FHV contains a bipartite genome of (+)-RNAs, RNA1 (3.1 kb) and RNA2 (1.4 kb), both of which are capped but not polyadenylated Drosophila melanogaster, the model insect, and can also abundantly replicate in mammalian Nodaviridae, and an attractive experimental model for the studies of various aspects of RNA virology, including viral RNA replication, assembly and structure, evolution, and the RNAi-based antiviral immunity Flock House virus (FHV) is a nonenveloped positive-strand (+) RNA viruses with a (+)-RNA viruses encode their own RdRPs, usually in concert with other viral and host factors, to catalyze RNA-templated viral RNA synthesis de novo) initiation. The primer-dependent initiation of RNA synthesis requires the involvement of either an oligonucleotide or a protein primer de novo initiation does not require primer, but uses the starting nucleotide to provide the 3′ hydroxyl group (3′-OH) for adding the next nucleotide. In the case of de novo initiation, RNA synthesis can be initiated at the terminal or an internal site of the template RNA For viral RNA synthesis, a proper initiation is required for the integrity of viral RNA genomes and the efficiency of RNA synthesis Nodaviridae originally isolated from cabbage fly (Pieris rapae) by our group de novo mechanism. Moreover, we found that WhNV protein A possesses a terminal nucleotidyl transferase (TNTase) activity, which can function to restore one or two 3′-proximal nucleotides of template RNA as a potential mechanism for rescuing 3′-terminal nucleotide loss For this issue, the previous studies of Wuhan nodavirus (WhNV), another member of GAATTCA TGACTCTAAAAGTTATTCTTGGAGC) and Pro A-Rev (GTCGACTCACTTC CGGTTGTTGGAAGGCTGTGC). The EcoR I and Sal I nuclease sites were introduced during PCR amplification. The polymerase active site mutagenesis GDD to GAA, containing the double substitution of both Asp692 and Asp693 to alanine, was also carried out by PCR-mediated mutagenesis TTTAATCGGACC GAAGTGCGGTGCCGCCGGTCTTTCCCGGGC and TTTGAATGATAGCCCGG GAAAGACCGGCGGCACCGCACTTCGGTC). After digested with EcoR I and Sal I , the PCR amplified fragments were cloned into the EcoR I -Sal I site of the pMAL-c2X vector . Transformants were analyzed by restriction enzyme mapping and confirmed by DNA sequencing . All the primers used were purchased from Invitrogen .To construct the bacterial expression vector for MBP-Pro A or its derivative, the FHV protein A coding sequence was PCR amplified from the cDNA of the FHV RNA1 genome plasmids FHV1 The recombinant protein MBP-Pro A, its mutant and MBP alone were expressed and purified as previously described in vitro from PCR products using T7 RNA polymerase  for 4 h. DNA templates were removed by RQ1 RNase-free DNase I (Promega) at 37°C for 1 h. After that, TRIzol reagent (Invitrogen) was used to extract the RNAs according to the manufacturer’s protocol. The blocking of RNA template was conducted as previously described The DNA fragment of the 3′-end of the (+)RNA1, (–)RNA1 was amplified from the cDNA of the FHV RNA1 genome plasmids FHV1. Following the manufacturer’s instructions , each pair of the primers was designed to include one primer, which contains a portion of 5′-end of expected RNA product plus the T7 promoter region (TAATACGACTCACTATAGg), and the other primer whose sequence is reverse-complementary to the 3′-end of the expected RNA product. RNA was transcribed To get rid of T7 RNA polymerase and NTP, all the RNA templates were electrophoresed in a 6% polyacrylamide–7 M urea gel and further purified by Poly-Gel RNA Extraction Kit  according to the manufacturer’s instruction. Purified RNAs were quantified with Bio-Rad Quantity One using known amounts of RNA as controls after Northern blots.2, 100 mM NaCl, 4 mM DTT, 250 µM DIG RNA Labelling Mix , 40 units of RNasin (Promega), 0.3 µg template RNA (∼100 nM) and 500 nM purified MBP-Pro A or its mutant. Reactions were incubated at 30°C for 90 min. The reaction mixture was supplemented added with isopyknic RNA loading mix  and then incubated at 65°C for 5 min. The 10×loading buffer contains 1% SDS, 50% glycerol and 0.05% bromophenol blue. Then, the products were analyzed by electrophoresis on 1.8% agarose-formaldehydegel denaturingn gels and transferred to N+ nylon membranes (Millipore) and fixed. The transferred nylon membrane was incubated with anti-DIG-AP antibody (Roche), and then incubated with CDP-Star™ (Roche), a kind of chemi-luminescent alkaline phosphatase substrates, at 37°C for 10–15 minutes. The signal is then visualized by radiography on an X-ray film. The RNAs were quantified with Bio-Rad Quantity One software.Standard RdRP reaction mixtures  contained 25 mM HEPES (pH 8.0), 1 mM MnClGliotoxin were purchased from EMD Chemicals , and phosphonoacetic acid (PAA) were purchased from Sigma . Gliotoxin or PAA was supplemented into the standard RdRP reaction mixtures. To explore the effects of primers in the RdRP reaction, the specific RNA primer (GUUUUCGAAACAAAU ) were synthesized by Takara Bio.in vitro transcription according to our standard procedures All reactions were stopped by adding the RNA loading mix, and analyzed by Northern blots as previously described GGAGAAACCC AGCGTGGTGGCATAC) and (−)3′RNA1-Rev (ACTCGGTTCCCTCCACTCTGA GGA), respectively associated with the synthesized plus- and minus-strand RNAs, were used for RT-catalyzed cDNA synthesis, followed by PCR amplification of cDNAs. PCR was performed with (+)3′RNA1-For/(+)3′RNA1-Rev (ACCTCTGCCCTTTCGGGCTAGAACGGGT) or (−)3′RNA1-For (GTTTTCGAA ACAAATAAAACAG)/(−)3′RNA1-Rev primers for amplification of cDNAs. The RT reactions in the absence of any primer were set as controls. Results were detected using UV transillumination after ethidium bromide staining. The cDNAs were then cloned into pMD-18T vector (Takara Bio), followed by DNA sequencing (BioDev-Tech. Co).Purified RNAs extracted from RdRP reaction mixtures was subjected to RT with M-MLV (Promega) according to the manufacturer’s protocol. The strand-specific oligodeoxynucleotide primers (+)3′RNA1-For , which would normally disrupt the RdRP activity GAA were efficiently expressed at expected molecular weights (∼155 kDa).To study the activity of FHV protein A, we first expressed wild-type and replication-defective mutant forms of soluble recombinant protein A as MBP-fusion proteins in in vitro, we used the 3′ end 191 nucleotides of (+)-RNA1 and 201 nucleotides (−)-RNA1, indicating as (+)-RNA11–191 and (−)-RNA11–201, as the templates for RNA synthesis , while the negative controls, MBP alone and MBP-Pro AGAA, could not initiate RNA synthesis . Similar results were obtained when the (−)-RNA11–201 template was used for (+)-RNA synthesis  in the standard RdRP reaction, and the RNA products were then detected via Northern blots using DIG-labeled probes specific for FHV (+)-RNA1 or (−)-RNA1 . As shown in Taken together, our results show that FHV protein A possesses the RdRP activity to initiate the RNA synthesis from RNA1-specific templates in the absence of an exogenous primer.de novo initiation is the mechanism by which FHV protein A initiates RNA synthesis. To this end, (−)-RNA11–201 with its 3′-end being blocked (oxidized) was used as the template, and the RNA primer specific to 3′-end of (−)RNA1 was added to the RdRP reaction at different concentrations (50 nM-1.5 µM) to determine if the RNA primer could promote (+)-RNA synthesis. As shown in After determining that FHV protein A possesses the RdRP activity in the absence of an exogenous primer, we sought to examine if de novo mechanism.In the absence of an exogenous primer, some RNA template can use its 3′-terminal sequence to fold back and form a hairpin structure. The folded back sequence can serve as an endogenous primer to initiate RNA synthesis, and this mechanism is called “copy-back” initiation, which is also primer dependent de novo mechanism, we determined the optimal conditions for the de novo RNA synthesis using (−)-RNA11–201 as the template. Our results showed that the RdRP activity of FHV protein A exhibited the highest activity at ∼30°C  and/or other NTPs. As shown in TNTase activity has been found in several viral RNA polymerases to add nucleotides to the 3′ end of RNAs 1–201 or (+)-RNA11–191 as the substrate/template, the GDD-to-GAA mutation (MBP-Pro AGAA) resulted in the complete loss of both TNTase -RNA11–191, in the presence or absence of 3′-OH oxidation. As shown in The TNTase-catalyzed addition of nucleotide to the 3′-end of RNA requires the presence of 3′-OHs in RNA substrates. To determine if this also applies to the TNTase activity of FHV protein A, we examined the TNTase reaction using the RNA substrates, (−)-RNA11–201 and (+)-RNA11–191 on RNA synthesis -RNA synthesis. The RNA synthesis initiation could tolerate the loss of two nucleotides at the 3′ end -RNA11–201 on the RdRP-catalyzed (+)-RNA synthesis. As shown in Next, we evaluated the effects of 3′ nucleotide loss of (−)-RNA15′-GAAAAC-3′, 5′-GUUUAC-3′, and 5′-GGUAAC-3′, respectively. Previous study has already reported that the U at the position 2 of FHV RNA2 is essential for its replication 1–201 template with the A at the position 3 being mutated to G, C, or U, which was indicated as (−)-RNA11–201A3G, (−)-RNA11–201A3C, or (−)-RNA11–201A3U, respectively. Our results showed that these mutations at the (−)-RNA11–201 template only resulted in minor reduction of the RNA synthesis -RNA13–201 template resulted in minor reduction of the RNA synthesis -stranded RNA1, RNA2, and sgRNA3 are ynthesis , lane 6.Since the RdRP and TNTase activities of a given viral RNA polymerase share the same catalytic motif (GDD), it was very hard to dissect the two activities and study the role of one activity in the absence of the other. To solve this issue, we have designed a set of experiments in our previous study 1–201 on the TNTase-mediated nucleotide addition. When one, two or three nucleotides were lost at the 3′ end of the (−)-stranded substrate, the nucleotide addition could be detected using a standard TNTase assay -RNA14–201 and (−)-RNA13–201A3U, were efficiently rescued  mechanism. Moreover, we found that FHV protein A possesses the TNTase activity, which was able to restore the nucleotide loss at the 3′-end initiation site of RNA template to rescue RNA synthesis initiation in vitro.FHV is a well recognized model for studying RNA replication of (+)-RNA viruses. However, although many progresses have been achieved by studying FHV RNA replication, the mechanism employed by FHV replicase, protein A, to initiate RNA synthesis had not been determined before this study. Here, we uncovered that FHV protein A can initiate RNA synthesis via a de novo initiation mechanism is primer-independent, and RdRPs do not require primer, but normally synthesize dinucleotides as the starting nucleotides on the 3′-end of RNA templates for adding next nucleotides de novo mechanism.An accurate initiation is required for the proper replication of viral RNA, and the mechanisms of RNA synthesis initiation by virus-encoded RdRPs are principally classified as two major types based on their dependence on the involvement of primers. The de novode novo and polyadenylation-independent characteristic of nodaviral or noroviral RdRP de novo initiation may be a general mechanism in the family Nodaviridae.Our previous study of WhNV protein A has determined that this nodaviral replicase protein also initiates RNA synthesis de novo initiation of RNA synthesis in vitro study found that deleting 13 or more nucleotides only resulted in a partial loss of RNA synthesis. This discrepancy is probably due to the different experimental conditions, as the former study used an FHV RNA1 replicon in baby hamster kidney 21 (BHK21) cells, while our in vitro experiment was conducted using purified recombinant FHV protein A and RNA1 template in the absence of other viral and cellular factors. Considering the importance of cellular factors such as intracellular membranes and host proteins on viral RNA replication The 3′-ends of RNA templates are of great importance for in vitro. Similarly with the RdRP activity, the 3′-end of RNA is also critical for the TNTase activity, as FHV protein A required the 3′-terminal 4 nucleotides for its TNTase activity (The 3′-ends of viral RNAs may be subjected to degradation by cellular exonucleases activity , which ide novo, and also possesses TNTase activity. Although the exact role of TNTase is hard to be determined in cells due to technical limitations, we found that FHV protein A’s TNTase activity can repair the 3′ initiation site and rescue synthesis initiation at least in vitro. The current study, together with our previous study of WhNV protein A, suggests that the de novo initiation mechanism of RNA synthesis as well as the TNTase activity may generally apply to the family Nodaviridae, and represents an important progress in understanding the RNA replication of FHV, the well recognized model virus.In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that FHV protein A initiates RNA synthesis