Hortensins, Type 1 Ribosome-Inactivating Proteins from Seeds of Red Mountain Spinach: Isolation, Characterization, and Their Effect on Glioblastoma Cells.

Ribosome inactivating proteins (RIPs) are specific N-ß-glycosylases that are well-characterized in plants. Their enzymatic action is to damage ribosomes, thereby blocking protein translation. Recently, several research groups have been working on the screening for these toxins in edible plants to facilitate the use of RIPs as biotechnological tools and biopesticides and to overcome public prejudice. Here, four novel monomeric (type 1) RIPs have been isolated from the seeds of Atriplex hortensis L. var. rubra, which is commonly known as edible red mountain spinach. These enzymes, named hortensins 1, 2, 4, and 5, are able to release the ß-fragment and, like many other RIPs, adenines from salmon sperm DNA, thus, acting as polynucleotide:adenosine glycosidases. Structurally, hortensins have a different molecular weight and are purified with different yields (hortensin 1, ~29.5 kDa, 0.28 mg per 100 g; hortensin 2, ~29 kDa, 0.29 mg per 100 g; hortensin 4, ~28.5 kDa, 0.71 mg per 100 g; and hortensin 5, ~30 kDa, 0.65 mg per 100 g); only hortensins 2 and 4 are glycosylated. Furthermore, the major isoforms (hortensins 4 and 5) are cytotoxic toward human continuous glioblastoma U87MG cell line. In addition, the morphological change in U87MG cells in the presence of these toxins is indicative of cell death triggered by the apoptotic pathway, as revealed by nuclear DNA fragmentation (TUNEL assay).

Biosynthesized tumor acidity and MMP dual-responsive plant toxin gelonin for robust cancer therapy.

Among all kinds of anticancer agents, small molecule drugs produce an unsatisfactory therapeutic effect due to the lack of selectivity, notorious drug resistance and side effects. Therefore, researchers have begun to pay extensive attention to macromolecular drugs with high efficacy and specificity. As a plant toxin, gelonin exerts potent antitumor activity via inhibiting intracellular protein synthesis. However, gelonin lacks a translocation domain, and thus its poor cellular uptake leads to low outcomes of antitumor response. Here, tumor acidity and matrix metalloproteinase (MMP) dual-responsive functional gelonin (Trx-PVGLIG-pHLIP-gelonin, TPpG), composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP), an MMP-responsive motif PVGLIG hexapeptide and gelonin, was innovatively proposed and biologically synthesized by a gene recombination technique. TPpG exhibited good thermal and serum stability, showed MMP responsiveness and could enter tumor cells under weakly acidic conditions, especially for MMP2-overexpressing HT1080 cells. Compared to low MMP2-expressing MCF-7 cells, TPpG displayed enhanced in vitro antitumor efficacy to HT1080 cells at pH 6.5 as determined by different methods. Likewise, TPpG was much more effective in triggering cell apoptosis and inhibiting protein synthesis in HT1080 cells than in MCF-7 cells. Intriguingly, with enhanced stability and pH/MMP dual responsiveness, TPpG notably inhibited subcutaneous HT1080 xenograft growth in mice and no noticeable off-target side effect was observed. This ingeniously designed strategy aims at providing new perspectives for the development of a smart platform that can intelligently respond to a tumor microenvironment for efficient protein delivery.

Differential role of GABAergic and cholinergic ventral pallidal neurons in behavioral despair, conditioned fear memory and active coping.

The ventral pallidum (VP), a major component of the reward circuit, is well-associated with appetitive behaviors. Recent evidence suggests that this basal forebrain nucleus may have an overarching role in affective processing, including behavioral responses to aversive stimuli. We investigated this by utilizing selective immunotoxin lesions and a series of behavioral tests in adult male Wistar rats. We made bilateral GAT1-Saporin, 192-IgG-Saporin or PBS (vehicle) injections into the VP to respectively eliminate GABAergic and cholinergic neurons, and tested the animals in the forced swim test (FST), open field test (OFT), elevated plus maze (EPM), Morris water maze (MWM) and cued fear conditioning. Both GAT1-Saporin and 192-IgG-Saporin injections reduced behavioral despair without altering general locomotor activity. During the acquisition phase of cued fear conditioning, this antidepressant effect was accompanied by reduced freezing and increased darting in the 192-IgG-Saporin group, and increased jumping in the GAT1-Saporin group. In the extinction phase, cholinergic lesions impaired fear memory irrespective of the context, while GABAergic lesions reduced memory durability only during the early phases of extinction in a novel context. In line with this, selective cholinergic, but not GABAergic, lesions impaired spatial memory in the MWM. We observed no consistent effect in anxiety-like behavior assessed in the OFT and EPM. These findings indicate that both the GABAergic and cholinergic neuronal groups of the VP may contribute to emotion regulation through modulation of behavioral despair and acquired fear by suppressing active coping and promoting species-specific passive behaviors.

Streptavidin-Saporin: Converting Biotinylated Materials into Targeted Toxins.

Streptavidin-Saporin can be considered a type of 'secondary' targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the 'Molecular Surgery' capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.

LRP1-Mediated Endocytosis May Be the Main Reason for the Difference in Cytotoxicity of Curcin and Curcin C on U2OS Osteosarcoma Cells.

Curcin and Curcin C, both of the ribosome-inactivating proteins of Jatropha curcas, have apparent inhibitory effects on the proliferation of osteosarcoma cell line U20S. However, the inhibitory effect of the latter is 13-fold higher than that of Curcin. The mechanism responsible for the difference has not been studied. This work aimed to understand and verify whether there are differences in entry efficiency and pathway between them using specific endocytosis inhibitors, gene silencing, and labeling techniques such as fluorescein isothiocyanate (FITC) labeling. The study found that the internalization efficiency of Curcin C was twice that of Curcin for U2OS cells. More than one entering pathway was adopted by both of them. Curcin C can enter U2OS cells through clathrin-dependent endocytosis and macropinocytosis, but clathrin-dependent endocytosis was not an option for Curcin. The low-density lipoprotein receptor-related protein 1 (LRP1) was found to mediate clathrin-dependent endocytosis of Curcin C. After LRP1 silencing, there was no significant difference in the 50% inhibitory concentration (IC50) and endocytosis efficiency between Curcin and Curcin C on U2OS cells. These results indicate that LRP1-mediated endocytosis is specific to Curcin C, thus leading to higher U2OS endocytosis efficiency and cytotoxicity than Curcin.

From Immunotoxins to Suicide Toxin Delivery Approaches: Is There a Clinical Opportunity?

Suicide gene therapy is a relatively novel form of cancer therapy in which a gene coding for enzymes or protein toxins is delivered through targeting systems such as vesicles, nanoparticles, peptide or lipidic co-adjuvants. The use of toxin genes is particularly interesting since their catalytic activity can induce cell death, damaging in most cases the translation machinery (ribosomes or protein factors involved in protein synthesis) of quiescent or proliferating cells. Thus, toxin gene delivery appears to be a promising tool in fighting cancer. In this review we will give an overview, describing some of the bacterial and plant enzymes studied so far for their delivery and controlled expression in tumor models.

Analysis of the Sequence Preference of Saporin by Deep Sequencing.

Ribosome-inactivating proteins (RIPs) are RNA:adenosine glycosidases that inactivate eukaryotic ribosomes by depurinating the sarcin-ricin loop (SRL) in 28S rRNA. The GAGA sequence at the top of the SRL or at the top of a hairpin loop is assumed to be their target motif. Saporin is a RIP widely used to develop immunotoxins for research and medical applications, but its sequence specificity has not been investigated. Here, we combine the conventional aniline cleavage assay for depurinated nucleic acids with high-throughput sequencing to study sequence-specific depurination of oligonucleotides caused by saporin. Our data reveal the sequence preference of saporin for different substrates and show that the GAGA motif is not efficiently targeted by this protein, neither in RNA nor in DNA. Instead, a preference of saporin for certain hairpin DNAs was observed. The observed sequence-specific activity of saporin may be relevant to antiviral or apoptosis-inducing effects of RIPs. The developed method could also be useful for studying the sequence specificity of depurination by other RIPs or enzymes.

Transient Inhibition of Trastuzumab-Tumor Binding to Overcome the "Binding-Site Barrier" and Improve the Efficacy of a Trastuzumab-Gelonin Immunotoxin.

We have recently shown that coadministration of mAbs with anti-idiotypic distribution enhancers (AIDE) that inhibit mAb binding to tumor antigens enabled increased intratumoral mAb distribution and increased efficacy of an antibody-drug conjugate (trastuzumab emtansine, T-DM1). In this article, a pharmacokinetic/pharmacodynamic (PK/PD) model was applied to predict the impact of this optimization strategy on the within-tumor distribution and antitumor efficacy of trastuzumab-gelonin, where the released payload (gelonin) is expected to exhibit negligible bystander activity. Immunofluorescence histology was used to investigate trastuzumab-gelonin distribution in solid tumors following dosing with or without coadministration of anti-trastuzumab AIDEs. Antitumor efficacy of trastuzumab-gelonin, with or without coadministration of AIDEs, was also evaluated in tumor-bearing mice. Trastuzumab-gelonin efficiently induced cytotoxicity when applied to NCI-N87 cells in culture (IC50: 0.224 ± 0.079 nmol/L). PK/PD simulations predicted that anti-idiotypic single-domain antibodies AIDEs with dissociation rate constants between 0.03 and 0.2 per hour would provide optimal enhancement of trastuzumab-gelonin efficacy. LE8 and 1HE, anti-trastuzumab AIDEs, were selected for evaluation in vivo. Coadministration of trastuzumab-gelonin with the inhibitors increased the portion of tumor area that stained positive for trastuzumab-gelonin by 58% (P = 0.0059). In addition, LE8 or 1HE coadministration improved trastuzumab-gelonin efficacy in NCI-N87 xenograft-bearing mice by increasing the percent increase in life span (%ILS) from 27.8% (for trastuzumab-gelonin administered alone) to 62.5% when administered with LE8 (P = 0.0007) or 83.3% (P = 0.0007) when administered with 1HE. These findings support the hypothesis that transient, competitive inhibition of mAb-tumor binding can improve the intratumoral distribution and efficacy of immunotoxins when applied for treatment of solid tumors.

Plant-Derived Type I Ribosome Inactivating Protein-Based Targeted Toxins: A Review of the Clinical Experience.

Targeted toxins (TT) for cancer treatment are a class of hybrid biologic comprised of a targeting domain coupled chemically or genetically to a proteinaceous toxin payload. The targeting domain of the TT recognises and binds to a defined target molecule on the cancer cell surface, thereby delivering the toxin that is then required to internalise to an appropriate intracellular compartment in order to kill the target cancer cell. Toxins from several different sources have been investigated over the years, and the two TTs that have so far been licensed for clinical use in humans; both utilise bacterial toxins. Relatively few clinical studies have, however, been undertaken with TTs that utilise single-chain type I ribosome inactivating proteins (RIPs). This paper reviews the clinical experience that has so far been obtained for a range of TTs based on five different type I RIPs and concludes that the majority studied in early phase trials show significant clinical activity that justifies further clinical investigation. A range of practical issues relating to the further clinical development of TT's are also covered briefly together with some suggested solutions to outstanding problems.

Isolation, Characterization and Biological Action of Type-1 Ribosome-Inactivating Proteins from Tissues of Salsola soda L.

Ribosome-inactivating proteins (RIPs) are known as RNA N-glycosylases. They depurinate the major rRNA, damaging ribosomes and inhibiting protein synthesis. Here, new single-chain (type-1) RIPs named sodins were isolated from the seeds (five proteins), edible leaves (one protein) and roots (one protein) of Salsola soda L. Sodins are able to release Endo's fragment when incubated with rabbit and yeast ribosomes and inhibit protein synthesis in cell-free systems (IC50 = 4.83-79.31 pM). In addition, sodin 5, the major form isolated from seeds, as well as sodin eL and sodin R, isolated from edible leaves and roots, respectively, display polynucleotide:adenosine glycosylase activity and are cytotoxic towards the Hela and COLO 320 cell lines (IC50 = 0.41-1200 nM), inducing apoptosis. The further characterization of sodin 5 reveals that this enzyme shows a secondary structure similar to other type-1 RIPs and a higher melting temperature (Tm = 76.03 ± 0.30 °C) and is non-glycosylated, as other sodins are. Finally, we proved that sodin 5 possesses antifungal activity against Penicillium digitatum.

Sapovaccarin-S1 and -S2, Two Type I RIP Isoforms from the Seeds of Saponaria vaccaria L.

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

Saporin as a Commercial Reagent: Its Uses and Unexpected Impacts in the Biological Sciences-Tools from the Plant Kingdom.

Saporin is a ribosome-inactivating protein that can cause inhibition of protein synthesis and causes cell death when delivered inside a cell. Development of commercial Saporin results in a technology termed 'molecular surgery', with Saporin as the scalpel. Its low toxicity (it has no efficient method of cell entry) and sturdy structure make Saporin a safe and simple molecule for many purposes. The most popular applications use experimental molecules that deliver Saporin via an add-on targeting molecule. These add-ons come in several forms: peptides, protein ligands, antibodies, even DNA fragments that mimic cell-binding ligands. Cells that do not express the targeted cell surface marker will not be affected. This review will highlight some newer efforts and discuss significant and unexpected impacts on science that molecular surgery has yielded over the last almost four decades. There are remarkable changes in fields such as the Neurosciences with models for Alzheimer's Disease and epilepsy, and game-changing effects in the study of pain and itch. Many other uses are also discussed to record the wide-reaching impact of Saporin in research and drug development.

Protective Effects of a synthetic glycosaminoglycan mimetic (OTR4132) in a rat immunotoxic lesion model of septohippocampal cholinergic degeneration.

Using a partial hippocampal cholinergic denervation model, we assessed the effects of the RGTA® named OTR4132, a synthetic heparan-mimetic biopolymer with neuroprotective/neurotrophic properties. Long-Evans male rats were injected with the cholinergic immunotoxin 192 IgG-saporin into the medial septum/diagonal band of Broca (0.37 µg); vehicle injections served as controls. Immediately after surgery, OTR4132 was injected into the lateral ventricles (0.25 µg/5 µl/rat) or intramuscularly (1.5 mg/kg). To determine whether OTR4132 reached the lesion site, some rats received intracerebroventricular (ICV) or intramuscular (I.M.) injections of fluorescent OTR4132. Rats were sacrificed at 4, 10, 20, or 60 days post-lesion (DPL). Fluorescein-labeled OTR4132 injected ICV or I.M. was found in the lesion from 4 to 20 DPL. Rats with partial hippocampal cholinergic denervation showed decreases in hippocampal acetylcholinesterase reaction products and in choline acetyltransferase-positive neurons in the medial septum. These lesions were the largest at 10 DPL and then remained stable until 60 DPL. Both hippocampal acetylcholinesterase reaction products and choline acetyltransferase-positive neurons in the medial septum effects were significantly attenuated in OTR4132-treated rats. These effects were not related to competition between OTR4132 and 192 IgG-saporin for the neurotrophin receptor P75 (p75NTR), as OTR4132 treatment did not alter the internalization of Cy3-labelled 192 IgG. OTR4132 was more efficient at reducing the acetylcholinesterase reaction products and choline acetyltransferase-positive neurons than a comparable heparin dose used as a comparator. Using the slice superfusion technique, we found that the lesion-induced decrease in muscarinic autoreceptor sensitivity was abolished by intramuscular OTR4132. After partial cholinergic damage, OTR4132 was able to concentrate at the brain lesion site possibly due to the disruption of the blood-brain barrier and to exert structural and functional effects that hold promises for neuroprotection/neurotrophism.

Curcin C inhibit osteosarcoma cell line U2OS proliferation by ROS induced apoptosis, autophagy and cell cycle arrest through activating JNK signal pathway.

Osteosarcoma is a kind of primary bone malignant tumors. Its cure rate has been stagnant in the past decade years. Curcin C belongs to type I ribosome inactivating proteins, extracted from the cotyledons of post-germinated Jatropha curcas seeds. It can inhibit the proliferation of several tumor lines including U2OS cells with extraordinary efficiency. The treated U2OS cells were arrested in both S and G2/M phase, showed typical apoptosis morphological characteristic, formed autophagosomes and increase the ratio of LC3II to LC3I. Meanwhile, the level of ROS in the treated cells was found increasing significantly, with the change of mitochondrial membrane potential and decreased antioxidant enzyme activities. The application of ROS scavenger NAC not only significantly inhibited the toxicity of Curcin C but also prevented the happen of apoptosis and autophagy to some extent. These results suggested that Curcin C may function through ROS pathway. In addition, the Curcin C treatment could activate JNK and inhibit ERK signal pathway. Sp600125, an inhibitor of JNK signaling pathway, can prevent subsequent apoptosis and autophagy events, suggesting that JNK pathway was at least one of the pathways of Curcin C action. Moreover, the relevant including antagonistic among autophagy, apoptosis and cell cycle arresting induced by Curcin C also was found. In summary, it can be speculated that Curcin C may induce S, G2/M phase arrest, apoptosis and autophagy of human osteosarcoma U2OS cells through activating JNK signal pathway and blocking ERK signal pathway by promoting ROS accumulation in cell, thus finally reflected in the effect of inhibiting tumor cell proliferation.

Toxic proteins application in cancer therapy.

Ribosome inactivating proteins (RIPs) as family of anti-cancer drugs recently received much attention due to their interesting anti-cancer mechanism. In spite of small drugs, RIPs use the large-size effect (LSE) to prevent the efflux process governed by drug resistance transporters (DRTs) which prevents inside of the cells against drug transfection. There are many clinical translation obstacles that severely restrict their applications especially their delivery approach to the tumor cells. As the main goal of this review, we will focus on trichosanthin (TCS) and gelonin (Gel) and other types, especially scorpion venom-derived RIPs to clarify that they are struggling with what types of bio-barriers and these challenges could be solved in cancer therapy science. Then, we will try to highlight recent state-of-the-arts in delivery of RIPs for cancer therapy.

Quinoa as source of type 1 ribosome inactivating proteins: A novel knowledge for a revision of its consumption.

This study investigates on the presence of toxic proteins in quinoa seeds. To this aim, a plethora of biochemical approaches were adopted for the purification and characterization of quinoin, a type 1 ribosome-inactivating protein (RIP) contained in quinoa seeds. We determined its melting temperature (68.2 ± 0.6 °C) and thermostability (loss of activity after 10-min incubation at 70 °C). Considering that quinoa seeds are used as a food, we found that quinoin is cytotoxic against BJ-5ta (human fibroblasts) and HaCaT (human keratinocytes) in a dose- and time-dependent manner. Moreover, in an in vitro digestive pepsin-trypsin treatment, 30% of quinoin is resistant to enzymatic cleavage. This toxin was found in seeds (0.23 mg/g of seeds) and in sprouted seeds obtained after 24-h (0.12 mg/g of sprout) and 48-h (0.09 mg/g of sprout). We suggest a thermal treatment of quinoa seeds before consumption in order to inactivate the toxin, particularly in sprouts, generally consumed raw.

Novel Binding Mechanisms of Fusion Broad Range Anti-Infective Protein Ricin A Chain Mutant-Pokeweed Antiviral Protein 1 (RTAM-PAP1) against SARS-CoV-2 Key Proteins in Silico.

The deadly pandemic named COVID-19, caused by a new coronavirus (SARS-CoV-2), emerged in 2019 and is still spreading globally at a dangerous pace. As of today, there are no proven vaccines, therapies, or even strategies to fight off this virus. Here, we describe the in silico docking results of a novel broad range anti-infective fusion protein RTAM-PAP1 against the various key proteins of SARS-CoV-2 using the latest protein-ligand docking software. RTAM-PAP1 was compared against the SARS-CoV-2 B38 antibody, ricin A chain, a pokeweed antiviral protein from leaves, and the lectin griffithsin using the special CoDockPP COVID-19 version. These experiments revealed novel binding mechanisms of RTAM-PAP1 with a high affinity to numerous SARS-CoV-2 key proteins. RTAM-PAP1 was further characterized in a preliminary toxicity study in mice and was found to be a potential therapeutic candidate. These findings might lead to the discovery of novel SARS-CoV-2 targets and therapeutic protein structures with outstanding functions.

An unusual type I ribosome-inactivating protein from Agrostemma githago L.

Agrostemma githago L. (corn cockle) is an herbaceous plant mainly growing in Europe. The seeds of the corn cockle are toxic and poisonings were widespread in the past by consuming contaminated flour. The toxic principle of Agrostemma seeds was attributed to triterpenoid secondary metabolites. Indeed, this is in part true. However Agrostemma githago L. is also a producer of ribosome-inactivating proteins (RIPs). RIPs are N-glycosylases that inactivate the ribosomal RNA, a process leading to an irreversible inhibition of protein synthesis and subsequent cell death. A widely known RIP is ricin from Ricinus communis L., which was used as a bioweapon in the past. In this study we isolated agrostin, a 27 kDa RIP from the seeds of Agrostemma githago L., and determined its full sequence. The toxicity of native agrostin was investigated by impedance-based live cell imaging. By RNAseq we identified 7 additional RIPs (agrostins) in the transcriptome of the corn cockle. Agrostin was recombinantly expressed in E. coli and characterized by MALDI-TOF-MS and adenine releasing assay. This study provides for the first time a comprehensive analysis of ribosome-inactivating proteins in the corn cockle and complements the current knowledge about the toxic principles of the plant.

Production of Recombinant Gelonin Using an Automated Liquid Chromatography System.

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05-0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.

Antiviral Activity of PD-L1 and PD-L4, Type 1 Ribosome Inactivating Proteins from Leaves of Phytolacca dioica L. in the Pathosystem Phaseolus vulgaris-Tobacco Necrosis Virus (TNV).

Using the pathosystem Phaseolus vulgaris-tobacco necrosis virus (TNV), we demonstrated that PD-L1 and PD-L4, type-1 ribosome inactivating proteins (RIPs) from leaves of Phytolacca dioica L., possess a strong antiviral activity. This activity was exerted both when the RIPs and the virus were inoculated together in the same leaf and when they were inoculated or applied separately in the adaxial and abaxial leaf surfaces. This suggests that virus inhibition would mainly occur inside plant cells at the onset of infection. Histochemical studies showed that both PD-L1 and PD-L4 were not able to induce oxidative burst and cell death in treated leaves, which were instead elicited by inoculation of the virus alone. Furthermore, when RIPs and TNV were inoculated together, no sign of H2O2 deposits and cell death were detectable, indicating that the virus could have been inactivated in a very early stage of infection, before the elicitation of a hypersensitivity reaction. In conclusion, the strong antiviral activity is likely exerted inside host cells as soon the virus disassembles to start translation of the viral genome. This activity is likely directed towards both viral and ribosomal RNA, explaining the almost complete abolition of infection when virus and RIP enter together into the cells.