Trypsin-like Serine Protease Research Articles

Purification and Identification of the Nematicidal Activity of S1 Family Trypsin-Like Serine Protease (PRA1) from Trichoderma longibrachiatum T6 Through Prokaryotic Expression and Biological Function Assays

Background/Objectives: Heterodera avenae is a highly significant plant-parasitic nematode, causing severe economic losses to global crop production each year. Trichoderma species have been found to parasitize nematodes and control them by producing enzymes that degrade eggshells. The T. longibrachiatum T6 (T6) strain has been demonstrated the parasitic and lethal effects on H. avenae cysts and eggs, associated with the increased serine protease activity and trypsin-like serine protease gene (PRA1) expression. Methods: Our present study aimed to purify the recombinant PRA1 protease through a prokaryotic expression system and identify its nematicidal activity. Results: The recombinant PRA1 protease was identified as S1 family trypsin-like serine protease, with a molecular weight of 43.16 kDa. The purified soluble protease exhibited the optimal activity at 35 °C and pH 8.0, and also demonstrating higher hydrolytic ability toward casein and skimmed milk. Meanwhile, the Ca2+ and Mg2+ enhanced its activity, while the inhibitor PMSF significantly reduced it. The contents of H. avenae eggs leaked out after treatment with the recombinant PRA1 protease, with egg hatching inhibition and relative hatching inhibition rates at 70.60% and 66.58%, respectively. In contrast, there was no sign of content dissolution, and embryos developed normally in the control group. Conclusions: Our present study revealed that the PRA1 protease of T6 strain has a lethal effect on H. avenae eggs, which providing a theoretical basis for developing biocontrol agents to control nematodes.

Structural Catalytic Core in Subtilisin-like Proteins and Its Comparison to Trypsin-like Serine Proteases and Alpha/Beta-Hydrolases

Subtilisin-like proteins are serine proteases that use two types of catalytic triads: Ser-His-Asp and Ser-Glu-Asp. Here, we investigate the two known families of subtilisin-like proteins, the subtilases (Ser-His-Asp triad) and the serine-carboxyl proteinases (Ser-Glu-Asp triad), and describe the local structural arrangements (cores) that govern the catalytic residues in these proteins. We show the separation of the cores into conserved structural zones, which can be repeatedly found in different structures, and compare the structural cores in subtilisin-like proteins with those in trypsin-like serine proteases and alpha/beta-hydrolases.

In vitro, in silico and crystallographic-based identification of serine protease inhibitors

Serine proteases are involved in various ailments, including pancreatitis, and colon cancer. Based on substrate recognition serine proteases are classified into different groups. Trypsin and trypsin-like serine proteases are among most studied group of serine proteases. Trypsin is among the chief hydrolysing enzyme involved in the pathogenesis of pancreatitis. Its inhibition can help to manage the disease. Herein, we investigated the trypsin inhibitory effect of some arginine-based small molecules, through in vitro, in silico, and crystallographic methods. Compounds 1-3 were evaluated against bovine pancreatic trypsin (BPT). Compound 1 was found to be active against trypsin with IC50 value of 247.98 ± 2.44 μM. Molecular docking studies were used to investigate the binding energy and binding conformation of inhibitor. All three compounds were subjected to crystallisation with trypsin. Compounds 1-2 were successfully crystallised with BPT The crystal structures of trypsin in complexed with compounds 1, and 2 were determined at 2.30 and 2.50 Å resolution, respectively. Both molecules showed their binding affinity with the active site residues of trypsin. This study will provide insight into the binding mechanism of E-64 and arginine and might be useful in designing effective inhibitors of serine proteases.

Macrocyclic Inhibitors Targeting the Prime Site of the Fibrinolytic Serine Protease Plasmin.

Two series of macrocyclic inhibitors addressing the S1 pocket and the prime site of the fibrinolytic serine protease plasmin have been developed. In the first series, a P1 tranexamoyl residue was coupled to 4-aminophenylalanine in P1' position, which provided moderately potent inhibitors with inhibition constants around 1 μM. In the second series, a substituted biphenylalanine was incorporated as P1' residue leading to approximately 1000-fold stronger plasmin inhibitors, the best compounds possess subnanomolar inhibition constants. The most effective compounds already exhibit a certain selectivity as plasmin inhibitors compared to other trypsin-like serine proteases such as trypsin, plasma kallikrein, thrombin, activated protein Ca, as well as factors XIa and Xa. For inhibitor 28 of the second series, the co-crystal structure in complex with a Ser195Ala microplasmin mutant revealed that the P2' residue adopts multiple conformations. Most polar contacts to plasmin and surrounding water molecules are mediated through the P1 tranexamoyl residue, whereas the bound conformation of the macrocycle is mainly stabilized by two intramolecular hydrogen bonds.

Granzyme K provides protection for Nile tilapia (Oreochromis niloticus) from acute bacterial infection.

Granzyme K (GzmK) is a trypsin-like serine protease that functions as a pro-apoptotic protease, has non-cytotoxic functions, and is involved in various physiopathological processes. However, the impact of GzmK on the immunological response of Nile tilapia against bacterial challenge is still poorly explored. In this study, we identified the GzmK gene from Nile tilapia (On-GzmK) and investigated its immunologic roles through in vivo experiments. On-GzmK has an open reading frame of 786 bp and encodes 255 amino acids. On-GzmK has over 60 % genetic similarity with other fish and over 30 % similarity with mammals. Its transcript levels were highest in the liver and vastly increased after being challenged with Streptococcus agalactiae and Aeromonas hydrophila. In vivo experiments implied that On-GzmK might promote inflammatory responses and regulate apoptosis and pyroptosis by participating in the activation of multiple signaling pathways, such as JAK-STAT, MAPK, and NF-κB. The present observations offer additional theoretical support for investigating the processes by which GzmK shields fish against bacterial infections.

Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca2+-Mediated Activation of Esterase Activity.

The presence of Ca2+ ions is known to facilitate the activity of trypsin-like serine proteases via structural stabilization against thermal denaturation and autolysis. Herein, we report a new and hidden regulatory role of Ca2+ in the catalytic pathways of trypsin and α-chymotrypsin under physiological conditions. We discovered that macromolecular crowding promotes spontaneous homotypic condensation of trypsin via liquid-liquid phase separation to yield membraneless condensates over a broad range of concentrations, pH, and temperature, which are stabilized by multivalent hydrophobic interactions. Interestingly, we found that Ca2+ binding in the calcium binding loop reversibly regulates the condensation of trypsin and α-chymotrypsin. Spontaneous condensation effectively prevents autolysis of trypsin and preserves its native-like esterase activity for a prolonged period of time. It has also been found that phase-separated trypsin responds to Ca2+-dependent activation of its esterase activity even after 14 days of storage while free trypsin failed to do so. The present study highlights an important physiological aspect by which cells can spatiotemporally regulate the biocatalytic efficacy of trypsin-like serine proteases via Ca2+-signaling.

A sperm-activating trypsin-like protease from the male reproductive tract of Spodoptera litura: Proteomic identification, sequence characterization, gene expression profile, RNAi and the effects of ionizing radiation

Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate) sperm. Formed in the testis, both kinds of sperm are released into the male reproductive tract in an immature form and are stored in the duplex region of the tract. Neither type of sperm is motile at this stage. When stored apyrene sperm from the duplex are treated in vitro with an extract of the prostatic region of the male tract, or with mammalian trypsin, they become motile; activation is greater and achieved more rapidly with increasing concentration of extract or enzyme. The activating effect of prostatic extract is blocked by soybean trypsin inhibitor (SBTI), also in a dose-dependent way. These results suggest that the normal sperm-activating process is due to an endogenous trypsin-like protease produced in the prostatic region.Proteomic analysis of S. litura prostatic extracts revealed a Trypsin-Like Serine Protease, TLSP, molecular weight 27 kDa, whose 199-residue amino acid sequence is identical to that of a predicted protein from the S. litura genome and is highly similar to predicted proteins encoded by genes in the genomes of several other noctuid moth species. Surprisingly, TLSP is only distantly related to Serine Protease 2 (initiatorin) of the silkmoth, Bombyx mori, the only identified lepidopteran protein so far shown to activate sperm. TLSP has features typical of secreted proteins, probably being synthesized as an inactive precursor zymogen, which is later activated by proteolytic cleavage.cDNA was synthesized from total RNA extracted from the prostatic region and was used to examine TLSP expression using qPCR. tlsp mRNA was expressed in both the prostatic region and the accessory glands of the male tract. Injection of TLSP-specific dsRNA into adult males caused a significant reduction after 24 h in tlsp mRNA levels in both locations. The number of eggs laid by females mated to adult males that were given TLSP dsRNA in 10 % honey solution, and the fertility (% hatched) of the eggs were reduced. Injecting pupae with TLSP dsRNA caused the later activation of apyrene sperm motility by adult male prostatic extracts to be significantly reduced compared to controls.Exposure of S. litura pupae to ionizing radiation significantly reduced expression of tlsp mRNA in the prostatic part and accessory gland of irradiated males in both the irradiated generation and also in their (unirradiated) F1 progeny. The implications of these findings for the use of the inherited sterility technique for the control of S. litura and other pest Lepidoptera are discussed.

Airborne indoor allergen serine proteases and their contribution to sensitisation and activation of innate immunity in allergic airway disease.

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.

Fragment-Based Design, Synthesis, and Characterization of Aminoisoindole-Derived Furin Inhibitors.

A 1H-isoindol-3-amine was identified as suitable P1 group for the proprotein convertase furin using a crystallographic screening with a set of 20 fragments known to occupy the S1 pocket of trypsin-like serine proteases. Its binding mode is very similar to that observed for the P1 group of benzamidine-derived peptidic furin inhibitors suggesting an aminomethyl substitution of this fragment to obtain a couplable P1 residue for the synthesis of substrate-analogue furin inhibitors. The obtained inhibitors possess a slightly improved picomolar inhibitory potency compared to their benzamidine-derived analogues. The crystal structures of two inhibitors in complex with furin revealed that the new P1 group is perfectly suited for incorporation in peptidic furin inhibitors. Selected inhibitors were tested for antiviral activity against respiratory syncytial virus (RSV) and a furin-dependent influenza A virus (SC35M/H7N7) in A549 human lung cells and demonstrated an efficient inhibition of virus activation and replication at low micromolar or even submicromolar concentrations. First results suggest that the Mas-related G-protein coupled receptor GPCR-X2 could be a potential off-target for certain benzamidine-derived furin inhibitors.

YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

Prostate cancer is a leading cause of cancer-related mortality in males. Dysregulation of RNA Adenine N-6 methylation (m6A) contributes to cancer malignancy. m6A on mRNA may affect mRNA splicing, turnover, transportation and translation. m6A exerts these effects, at least partly, through dedicated m6A reader proteins, including YTHDF2. YTHDF2 is necessary for development while its dysregulation is seen in various cancers including prostate cancer. However, the mechanism underlying the dysregulation and function of YTHDF2 in cancer remains elusive. Here, we find that the deubiquitinase OTUB1 increases YTHDF2 protein stability by inhibiting its ubiquitination. With in vivo and in vitro ubiquitination assays, OTUB1 is shown to block ubiquitin transfer to YTHDF2 independent of its deubiquitinase activity. Furthermore, analysis of functional transcriptomic data and m6A-sequencing data identifies PRSS8 as a potential tumor suppressor gene. OTUB1 and YTHDF2 decrease mRNA and protein levels of PRSS8, which is a trypsin-like serine protease. Mechanistically, YTHDF2 binds PRSS8 mRNA and promotes its degradation in an m6A-dependent manner. Further functional study on cellular and mouse models reveals PRSS8 is a critical downstream effector of the OTUB1-YTHDF2 axis in prostate cancer. We find in prostate cancer cells, PRSS8 decreases nuclear β-catenin level through E-cadherin, which is independent of its protease activity. Collectively, our study uncovers a key regulator of YTHDF2 protein stability and establishes a functional OTUB1-YTHDF2-PRSS8 axis in prostate cancer.

House dust mite allergens induce Ca2+ signalling and alarmin responses in asthma airway epithelial cells

Type 2 inflammation in asthma develops with exposure to stimuli to include inhaled allergens from house dust mites (HDM). Features include mucus hypersecretion and the formation of pro-secretory ion transport characterised by elevated basal Cl− current. Studies using human sinonasal epithelial cells treated with HDM extract report a higher protease activated receptor-2 (PAR-2) agonist-induced calcium mobilisation that may be related to airway sensitisation by allergen-associated proteases. Herein, this study aimed to investigate the effect of HDM on Ca2+ signalling and inflammatory responses in asthmatic airway epithelial cells. Primary bronchial epithelial cells (hPBECs) from asthma donors cultured at air-liquid interface were used to assess electrophysiological, Ca2+ signalling and inflammatory responses. Differences were observed regarding Ca2+ signalling in response to PAR-2 agonist 2-Furoyl-LIGRLO-amide (2-FLI), and equivalent short-circuit current (Ieq) in response to trypsin and 2-FLI, in ALI-asthma and healthy hPBECs. HDM treatment led to increased levels of intracellular cations (Ca2+, Na+) and significantly reduced the 2-FLI-induced change of Ieq in asthma cells. Apical HDM-induced Ca2+ mobilisation was found to mainly involve the activation of PAR-2 and PAR-4-associated store-operated Ca2+ influx and TRPV1. In contrast, PAR-2, PAR-4 antagonists and TRPV1 antagonist only showed slight impact on basolateral HDM-induced Ca2+ mobilisation. HDM trypsin-like serine proteases were the main components leading to non-amiloride sensitive Ieq and also increased interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) from asthma hPBECs. These studies add further insight into the complex mechanisms associated with HDM-induced alterations in cell signalling and their relevance to pathological changes within asthma.

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

Serine protease mediates Ovomermis sinensis-inhibited host immune responses by inducing apoptosis: implications for successful parasitism and host mortality.

Mermithid nematodes are entomopathogens that parasitize and kill insect hosts and are used for biological control. It is widely believed that mermithid nematodes kill their host upon nematode emergence, unlike other parasites that depend on virulence factors. In this study, we disproved this theory by demonstrating that the mermithid nematode Ovomermis sinensis mediates host mortality by serine protease-induced apoptosis. Successful parasitism of O. sinensis increased with the infection rate, and the inhibition of host immunity by O. sinensis increased with the parasitic load. A serine protease was identified from the host hemolymph. This protease belongs to the trypsin-like serine protease family, which is an apoptosis-inducing serine protease. Specifically, Os-sp was highly expressed only during the parasitic stage and could be induced by host hemocytes and the fat body. Importantly, host immune effectors (melanization, phenoloxidase activity, and encapsulation) were suppressed by the recombinant protein rOs-sp that induced apoptosis of hemocytes and fat body in a dose-dependent manner, which contributes to host death. Serine protease mediates O. sinensis-inhibited host immune responses by inducing apoptosis that is lethal to the insect host. Our findings have broader implications for understanding the mechanism of successful parasitism and killing of host by nematodes. © 2023 Society of Chemical Industry.

Cloning, expression, and characterization of a surfactant-stable alkaline serine protease (KNBSSP1) from Bacillus safensis PRN1 with remarkable applications in laundry and leather industries

Proteases represent an important category of proteolytic enzymes with vast biotechnological applications in various industries like detergent, leather, agriculture, poultry, pharmaceutical, food, etc. The present study proposes an eco-friendly enzyme-mediated approach as opposed to conventional chemical approaches in detergent and laundry industries that are hazardous to human health and the environment. Accordingly, the knbsSP1gene, which encodes the trypsin-like serine protease KNBSSP1, from Bacillus safensis PRN1 was isolated, cloned into an expression vector (PET-28a), and expressed in the Escherichia coli cells BL21 (DE3). A single Ni-NTA affinity chromatography step was utilized to purify the recombinant protease (rKNBSSP1). The molecular weight of the rKNBSSP1 was approximately ∼33 kDa determined in Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The rKNBSSP1 showed optimal activity at 60 °C and pH 8. It retained above 70% of its enzyme activity at pH 10 and 70 °C. The protease was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate suggesting its inclusion in the serine family of protease. The rKNBSSP1 demonstrated outstanding compatibility and stability with metal ions, commercial detergents, and surfactants, exhibiting 97.8 ± 2.5% stability with Surf Excel, a commercial laundry detergent, and 96.9 ± 2.0% stability with SDS. Interestingly, the protease demonstrated blood-stained removal and goat skin dehairing (8 h) properties. All these remarkable characteristics make this protease a potential eco-friendly candidate in the detergent formulation and leather processing industries.

Composition characterization of various viperidae snake venoms using MS-based proteomics N-glycoproteomics and N-glycomics

Viperidae snake species is widely abundant and responsible for most envenomation cases in Turkey. The structural and compositional profiles of snake venom have been investigated to study the venom component variation across different species and to profile the venom biological activity variation against prey. In this context, we used proteomics, glycoproteomics and glycomics strategies to characterize the protein, glycoproteins and glycan structural and compositional profiles of various snake venoms in the Viperidae family. Moreover, we compared these profiles using the downstream bioinformatics and machine learning classification modules. The overall mass spectrometry profiles identified 144 different proteins, 36 glycoproteins and 78 distinct N-glycan structures varying in composition across the five venoms. A high amount of the characterized proteins belongs to the glycosylated protein family Trypsin-like serine protease (Tryp_SPc), Disintegrin (DISIN), and ADAM Cysteine-Rich (ACR). Most identified N-glycans have a complex chain carrying galactosylated N-glycans abundantly. The glycan composition data obtained from glycoproteomics aligns consistently with the findings from glycomics. The clustering and principal component analyses (PCA) illustrated the composition-based similarities and differences between each snake venom species' proteome, glycoproteome and glycan profiles. Specifically, the N-glycan profiles of M. xanthina (Mx) and V. a. ammodytes (Vaa) venoms were identical and difficult to differentiate; in contrast, their proteome profiles were distinct. Interestingly, the variety of the proteins across the species highlighted the impact of glycosylation on the diversity of the glycosylated protein families. This proposed high throughput approach provides accurate and comprehensive profiles of the composition and function of various Viperidae snake venoms.

Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

Prostasin and matriptase are extracellular membrane serine proteases with opposing effects in solid epithelial tumors. Matriptase is an oncoprotein that promotes tumor initiation and progression, and prostasin is a tumor suppressor that reduces tumor invasion and metastasis. Previous studies have shown that a subgroup of Burkitt lymphoma have high levels of ectopic matriptase expression but no prostasin. Reducing the matriptase level via small interfering RNAs in B lymphoma cells impeded tumor xenograft growth in mice. Here, we report a novel approach to matriptase regulation in B cancer cells by prostasin via exosomes to initiate a prostasin-matriptase protease activation cascade. The activation and shedding of matriptase were monitored by measuring its quantity and trypsin-like serine protease activity in conditioned media. Sustained activation of the protease cascade in the cells was achieved by the stable expression of prostasin. The B cancer cells with prostasin expression presented phenotypes consistent with its tumor suppressor role, such as reduced growth and increased apoptosis. Prostasin exosomes could be developed as an agent to initiate the prostasin-matriptase cascade for treating B lymphoma with further studies in animal models.

Coumarins as factor XIIa inhibitors: Potency and selectivity improvements using a fragment-based strategy

Previously, we described weak coumarin inhibitors of factor XIIa, a promising target for artificial surface-induced thrombosis and various inflammatory diseases. In this work, we used fragment-based drug discovery approach to improve our coumarin series. First, we screened about 200 fragments for the S1 pocket. The S1 pocket of trypsin-like serine proteases, such as factor XIIa, is highly conserved and is known to drive a major part of the association energy. From the screening, we selected fragments displaying a micromolar activity and studied their selectivity on other serine proteases. Then, these fragments were merged to our coumarin templates, leading to the generation of nanomolar inhibitors. The mechanism of inhibition was further studied by mass spectrometry demonstrating the covalent binding through the formation of an acyl enzyme complex. The most potent compound was tested in plasma to evaluate its stability and efficacy on coagulation assays. It exhibited a plasmatic half-life of 1.9 h and a good selectivity for the intrinsic coagulation pathway over the extrinsic one.

Comparative kinetic characterization of the activity of glycosylated and non-glycosylated trypsin-like serine protease isolated from adults of Rhyzopertha dominica (Coleoptera: Bostrichidae) reared on the grain of three different cultivars of wheat

Comparative kinetic characterization of the activity of glycosylated and non-glycosylated trypsin-like serine protease isolated from adults of Rhyzopertha dominica (Coleoptera: Bostrichidae) reared on the grain of three different cultivars of wheat

Mouse skin peptidomic analysis of the hemorrhage induced by a snake venom metalloprotease.

Hemorrhage induced by snake venom metalloproteases (SVMPs) results from proteolysis, capillary disruption, and blood extravasation. HF3, a potent SVMP of Bothrops jararaca, induces hemorrhage at pmol doses in the mouse skin. To gain insight into the hemorrhagic process, the main goal of this study was to analyze changes in the skin peptidome generated by injection of HF3, using approaches of mass spectrometry-based untargeted peptidomics. The results revealed that the sets of peptides found in the control and HF3-treated skin samples were distinct and derived from the cleavage of different proteins. Peptide bond cleavage site identification in the HF3-treated skin showed compatibility with trypsin-like serine proteases and cathepsins, suggesting the activation of host proteinases. Acetylated peptides, which originated from the cleavage at positions in the N-terminal region of proteins in both samples, were identified for the first time in the mouse skin peptidome. The number of peptides acetylated at the residue after the first Met residue, mostly Ser and Ala, was higher than that of peptides acetylated at the initial Met. Proteins cleaved in the hemorrhagic skin participate in cholesterol metabolism, PPAR signaling, and in the complement and coagulation cascades, indicating the impairment of these biological processes. The peptidomic analysis also indicated the emergence of peptides with potential biological activities, including pheromone, cell penetrating, quorum sensing, defense, and cell-cell communication in the mouse skin. Interestingly, peptides generated in the hemorrhagic skin promoted the inhibition of collagen-induced platelet aggregation and could act synergistically in the local tissue damage induced by HF3.

Two novel flavi-like viruses shed light on the plant-infecting koshoviruses.

The family Flaviviridae is composed of viruses with a positive-sense single-stranded RNA genome and includes viruses that are important veterinary and human pathogens. Most members of the family are arthropod- and vertebrate-infecting viruses, but more recently, divergent flavi-like viruses have been identified in marine invertebrate and vertebrate hosts. The striking discovery of gentian Kobu-sho-associated virus (GKaV), along with a recent report of a related virus from carrot, has expanded the known host range of flavi-like viruses to plants, suggesting they could be grouped in a proposed genus tentatively named "Koshovirus". Here, we report the identification and characterization of two novel RNA viruses that show a genetic and evolutionary relationship to the previously identified "koshoviruses". Their genome sequences were obtained from transcriptomic datasets of the flowering plants Coptis teeta and Sonchus asper. These two new viruses, which we have named "coptis flavi-like virus 1" (CopFLV1) and "sonchus flavi-like virus 1" (SonFLV1), are members of novel species characterized by the longest monopartite RNA genome observed so far among plant-associated RNA viruses, which is ca. 24 kb in size. Structural and functional annotations of the polyproteins of all koshoviruses resulted in the detection not only of the expected helicase and RNA-dependent RNA polymerase but also of several additional divergent domains, including AlkB oxygenase, trypsin-like serine protease, methyltransferase, and envelope E1 flavi-like domains. Phylogenetic analysis showed that CopFLV1, SonFLV1, GKaV, and the carrot flavi-like virus were grouped together in a monophyletic clade, strongly supporting the recent proposal for creation of the genus "Koshovirus" for the group of related plant-infecting flavi-like viruses.