*Review* **Engineering of Ribosome-inactivating Proteins for Improving Pharmacological Properties**

#### **Jia-Qi Lu 1,**†**, Zhen-Ning Zhu 1,**†**, Yong-Tang Zheng <sup>2</sup> and Pang-Chui Shaw 1,\***


Received: 19 February 2020; Accepted: 6 March 2020; Published: 9 March 2020

**Abstract:** Ribosome-inactivating proteins (RIPs) are N-glycosidases, which depurinate a specific adenine residue in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. This loop is important for anchoring elongation factor (EF-G for prokaryote or eEF2 for eukaryote) in mRNA translocation. Translation is inhibited after the attack. RIPs therefore may have been applied for anti-cancer, and anti-virus and other therapeutic applications. The main obstacles of treatment with RIPs include short plasma half-life, non-selective cytotoxicity and antigenicity. This review focuses on the strategies used to improve the pharmacological properties of RIPs on human immunodeficiency virus (HIV) and cancers. Coupling with polyethylene glycol (PEG) increases plasma time and reduces antigenicity. RIPs conjugated with antibodies to form immunotoxins increase the selective toxicity to target cells. The prospects for future development on the engineering of RIPs for improving their pharmacological properties are also discussed.

**Keywords:** ribosome inactivating protein; therapeutic applications; immunotoxin; anti-HIV; anti-cancer

**Key Contribution:** This review summarizes an update of knowledge of the anti-HIV and anti-cancer activities of representative RIPs and the engineering methods to improve their pharmacological properties.

#### **1. Introduction**

Ribosome inactivating proteins (RIPs) are a group of cytotoxic N-glycosidases. They are mostly found from plants and a few from bacteria [1]. RIPs are classified into three types according to the number of subunits and the organization of the precursor sequences [2]. Type 1 RIPs such as trichosanthin (TCS) and momorcharin (MMC) have a single polypeptide chain with catalytic activity. Type 2 RIPs such as ricin and abrin are heterodimeric, with an active A chain linked to a lectin-binding B chain by a disulfide bond. Type 3 RIPs such as maize ribosome-inactivating protein and barley jasmonate-induced RIP (JIP60) contain a region within the protein that is removed for activation [3,4].

In general, RIPs remove a specific adenine in the α-sarcin/ricin loop (α-SRL) of rRNA, resulting in blocking the binding of elongation factor [5]. The depurination of α-SRL loop causes the GTP binding site to lose the ability to activate GTP hydrolysis. Protein synthesis is thus impeded and the cell dies [6]. Because of lectin-binding properties, most of type 2 RIPs have higher rate of cell entry and hence cytotoxicity [7]. RIPs have also been found to exhibit other enzymatic activities, including superoxide dismutase (SOD) [8], phospholipase [9] and depurination of DNA, RNA and

poly (A) [10]. Several RIPs such as Momordica anti-HIV protein (MAP30) and gelonium anti-HIV protein (GAP31) exhibit a topological activity on plasmid and viral DNA, for example HIV-1 long terminal repeats (LTRs) [11]. RIPs can also induce cell apoptosis by a mechanism independent from the depurination of the SRL loop [12]. Because of their cytotoxicity, several RIPs have been tested for anti-tumor, anti-viral, anti-bacterial and anti-fungal properties. Clinical trials, such as gelonin for treating myeloid malignancies [13], ricin for treating leukemia [14] and TCS and pokeweed antiviral protein (PAP) for treating human immunodeficiency virus (HIV) [15,16] have been carried out.

Despite RIPs showing great potential in clinical applications, side effects such as inducing immune responses, short plasma half-life and non-specificity have limited their uses. Over the years, a number of works have been carried out to modify RIPs to reduce these problems. Antibodies can be conjugated with RIPs to form immunotoxins (ITs). Because of the selective function of the antibodies for targeting, ITs can achieve higher efficacy and lower side effects. Polyethylene glycol (PEG) has been used to couple with RIPs. The complex has increased molecular size. As a result, renal clearance, proteolytic degradation, immunogenicity and antigenicity are reduced.

#### **2. Anti-HIV activity of RIPs**

#### *2.1. Anti-HIV Activity of Representative RIPs*

RIPs including MAP30, saporin, TCS, gelonium anti-HIV protein (GAP31) and α-momorcharin (α-MMC) possess anti-HIV activity and other anti-viral activities. It has been found that RIPs affect the life cycle of human immunodeficiency viruses (HIV) including reverse transcription, integration, replication and assembly (Figure 1), not only due to their N-glycosidase activity.

**Figure 1.** The cell cycle of HIV and the anti-HIV mechanism of representative RIPs. RIPs including TCS, GAP31, MAP30, PAP, marmorin and saporin can attack different steps of the life cycle of HIV and inhibit its growth. The mechanisms are not just due to rRNA depurination. TCS: trichosanthin; MAP30: momordica anti-HIV protein. GAP31: gelonium anti-HIV protein; PAP: pokeweed antiviral protein; MOD: maize ribosome-inactivating protein; DAP30: dianthus anti-HIV protein; MAPK: mitogen-activated protein kinase; HIV: human immunodeficiency virus.

Integration of the viral DNA plays a significant role in the replicative cycle of retroviruses. Saporin, TCS, GAP31 and luffin have all been reported possessing HIV-1 integrase inhibitory activities.

TCS is one of the most studied RIPs, regarding its anti-HIV activity. HIV-1 integration was also inhibited by TCS, which was attributed to the temporary interaction between TCS and HIV-1 long-terminal repeats (LTRs) [17]. Some TCS mutants without anti-HIV-1 activity still had depurination activity, which meant that N-glycosidase activity may be dissociated from the anti-HIV mechanism [18]. The anti-HIV activity of TCS was eliminated by c-Jun N-terminal kinase (JNK) inhibitor CEP-11004. Jun amino-terminal kinase (JNK) is a member of stress-activated protein kinases (SAPK), which belong to the mitogen-activated protein kinase (MAPK) family. TCS exhibited anti-HIV activity and it may through the MAPK signal pathway [19]. A similar result was found when HSV-1 infected cells were treated with TCS. The p38 MAPK and B-cell lymphoma 2 (Bcl-2) induced by HSV-1 were inhibited by TCS [20]. A singular TCS hijacking HIV-1 strategy was revealed. HIV-1 scaffold protein Gag and lipid raft membrane facilitated the formation of TCS-enriched virions. The infection ability of HIV-1 with TCS was reduced dramatically [21]. Chemokine (C-C motif) ligand 5 (RANTES) and alpha-stimulated chemotaxis by stromal cell-derived factor (SDF)-1 were significantly increased by TCS, while pertussis toxin-sensitive G proteins were activated simultaneously. The activation of chemokine receptors provoked by chemokine was strengthened by TCS [22]. The relaxed circular DNA can be broken into a linear DNA by TCS, which indicated that TCS has DNase-like activity [23], which may also be a possible mechanism. Trichobitacin isolated from the root tubers of *Trichosanthes kirilowii* transiently decreased the expression of HIV-1 p24 antigen [24].

Saporin was found to inhibit HIV integrase 3' end processing activity (anti-HIV-1 integrase), induce viral apoptosis and suppress HIV propagation, which are unrelated to N-glycosidase activity [25]. Saporin-6 and saporin L3 exhibit classical depurination activity targeting the GAGA conserved sequence of RNA [26]. Saporin-6 was also reported to have DNA nuclease activity [27]. Isoform saporin-L1 can inhibit viral replication which may be related to the adenosine glycosidase activity on DNA, genomic RNA and mRNA [28]. However, the anti-HIV activity of saporin-6 is found independent of its RNA N-glycosidase activity, and may be related to apoptosis [25]. Like saporin, luffin also showed HIV integrase inhibitory activities on 3 end processing and strand-transferring, which leads to anti-HIV-1 replication [29].

MAP30 (Momordica anti-HIV protein) displays DNA glycosylase activity contributing to HIV-1 integrase inhibition. Besides, MAP30, alpha- and beta-momorcharins depress HIV replication [30]. MAP30 is also able to relax supercoiled DNA [31]. MAP30 was found suppressing the expression of HIV core protein p24 and viral-related reverse transcriptase (RT) activity without cytotoxicity and cytostaticity [32]. MAP30 can assist other anti-HIV drugs including dexamethasone and indomethacin in achieving higher efficiency [30].

GAP31 (gelonium anti-HIV protein of 31 kDa) and MAP30 block the infection of HIV-1 in T lymphocytes and monocytes and viral replication [11]. They also show both anti-HIV and anti-HSV activity [33]. They manifest inhibitory activity on HIV-1 integrase attributed to the topological activity toward HIV-1 long-terminal repeats (HIV-1 LTRs) [11]. GAP31 interacts with 5 overhanging adenosine ends, but not with blunt ends, which revealed that it acts like DNA adenosine glycosidase towards the accessible adenosine [34]. A 33-aa segment (KGATYITYVNFLNELRVKTKPEGNSHGIPSLRK) of GAP31, K10-K42, was shown to be the shortest peptide that elicits anti-HIV effect [35].

PAP (pokeweed antiviral protein) inhibits viral protein synthesis in HIV-1 infected cluster of differentiation 4 (CD4) + T cells [36]. Engineered nontoxic PAPs, FLP-102((151)AA (152)) and FLP-105((191)AA(192)), have the potency of nucleoside reverse transcriptase inhibition toward inhibitor-resistant HIV-1 with less cytotoxicity than native PAP [37]. PAP, MAP30 and GAP31 were nontoxic to human sperm, thereby they could be applied to inactivate infective viruses and virus-infected cells in semen [38].

DAPs 30 and 32 (dianthus anti-HIV proteins, 30 and 32 kDa), as well as GAP31, are able to relax supercoiled DNA and cleave double-stranded DNA into a linear one [39].

Balsamin, purified from *Momordica balsamina*, inhibits HIV-1 replication in both human T lymphocyte cell lines and human primary CD4+ T cells [40]. Balsamin is also capable of relaxing super-coiled DNA into the linear form [41].

#### *2.2. Engineering of RIPs for Improving the Anti-HIV E*ffi*cacy*

Acquired immune deficiency syndrome (AIDS) patients treated with TCS showed non-dose-related reversible mental status changes including dementia, and even coma [15]. GLQ223, an inhibitor of HIV replication, is a highly purified TCS formula to treat HIV patients with higher safety than TCS. A flu-like syndrome was the major adverse effect associated with GLQ223 [42]. Different strategies were used to remit the side-effect of RIPs, such as competitive binders and steric hindrance [43]. However, immunotoxins (ITs) are more effective because of their high specificity and selectivity. RIPs, especially PAP and ricin A chain (RTA), have been utilized to make ITs for therapeutic use [43].

Immunotoxins (ITs) are chimeric proteins that consist of RIPs or RIP fragments and moiety for targeting [43]. Targeting moiety includes antibodies, cytokines, growth factors, hormones and lectins. ITs were first designed with whole RIP linking to full length monoclonal antibody (mAb) by disulfide bond. Type 2 RIP has lectin-binding domain (B-chain) with multiple binding sites appearance maintained at a high level of non-specific internalization. To improve the performance of the IT, B-chain of type 2 RIP was removed, or its binding sites were blocked in the second generation of ITs. The third generation ITs are recombinant immunotoxins. Recombinant RIPs genetically fused to the targeting portion of mAb by a peptide linker increases homogeneity. A single chain Fv fragment (scFv) with retained targeting ability leads to smaller size of ITs, which may affect both cell penetration and clearance. Two major drawbacks of ITs are immunogenicity and vascular leak syndrome (VLS). Human or humanized antibody formats are applied to reduce immunogenicity in the fourth generation. Antigenic epitopes modification of RIPs is also applied. ITs can be used combining with other therapeutic agents to achieve synergic effect [44,45].

Most anti-HIV ITs were designed targeting the HIV envelope glycoprotein and surface antigens. Several RTA based ITs with different ligands targeting to an external envelope glycoprotein (gp120) of HIV and CD4 were tested for the anti-HIV efficacy; ligands included 0.5beta, anti-gp120 and mAb 924 [46–48]. It was later found not much improvement was observed, while anti-gp41 (mAb 7B2) together with soluble CD4 showed anti-HIV activity [46]. Pulchellin, was conjugated to mAb 924 and mAb 7B2 for recognizing gp120 and gp41; it showed similar characteristic with RTA ITs [49]. mAb 924 and 7B2 were conjugated to RTA and pulchellin by succinimidyl 6-[3(2-pyridyldithio) propionamido] hexanoate. The lysine and N-terminus on antibody and cysteine on RTA and pulchellin were involved in this conjugation [49].

RTA and Maize RIP variants were linked with HIV-1 protease recognition sequences to the C-terminal or internal inactivation region (Figure 2), which could be activated by HIV-1 protease [50,51]. Maize RIP has a 25-amino acid internal inactivation region, which is able to sterically block the interaction with ribosome. This provides a switching mechanism resulting in specific targeting to HIV-infected cells with low cytotoxicity to normal cells. The internal inactivation region was replaced by HIV-1 protease recognition site. Transcriptional activator Tat protein (TAT) sequence was fused to the N-terminus. The scheme of engineering is shown in Figure 2. Recombinant active maize RIP also exerted better anti-viral activity in vivo, with the decrease of plasma virial burden transiently in chimeric simian-human immunodeficiency virus (SHIV) 89.6-infected macaque model [52]. RTA linked with HIV-1 protease recognition sequences also exhibited a more specific activity towards HIV-1 infected cells [51].

**Figure 2.** Schematic diagram of the RTA and maize RIP variants. The recombinant maize RIP precursor pro-RIP contains a 25 amino acids internal inactivation region. RTA: ricin A chain; HIV: human immunodeficiency virus; TAT: transcriptional activator Tat protein. TAT-Pro-HIV-P2/NC and TAT-Pro-HIV-MA/CA: N-termini of pro-RIP were fused with a TAT sequence. First and last 10 aa in internal inactivation region were replaced by the HIV-1 recognition p2/NC site (TATIM/MQRGN) and the MA/CA site (VSQNY/PIVQN), respectively. RTA-C10: MA/CA site fused to C-terminal of RTA. RTA-C25: two MA/CA sites were fused and separated by MQMPE (middle five residues of pro-RIP).

PAP was linked to mAbs targeting CD4, CD5 or CD7 antigens. The variants exerted anti-viral activity through inhibition of HIV-1 replication in HIV-1 infected CD4+ T cells and activating T cells from two asymptomatic HIV-1-seropositive patients [53].

TXU (anti-CD7)-PAP increased plasma half-life to 12.4 +/− 1.4 h and decreased systemic clearance to 2.7 +/− 0.7 mL/h/kg in adult HIV-infected patients. TXU-PAP had low toxicity. All six patients treated by 5 microg/kg dose level showed no adverse effects with viral burden reduction [16].

#### **3. Anti-tumor activity of RIPs**

#### *3.1. Anti-Tumor Activity of Representative RIPs*

TCS exerts anti-tumor activity to a wide spectrum of cancers by multiple mechanisms. The invasion, migration and epithelial-mesenchymal transition (EMT) of cervical cancer cells were inhibited, which might be relevant to the restriction of signal transducer and activator of transcription (STAT5))/C-myc signaling pathway activation by TCS. The level of B-cell lymphoma 2 (Bcl-2) and expression of antigen ki-67 associated with cellular proliferation and ribosomal RNA transcription and Phospho-c-Myc (P-C-myc) were decreased while the activation of caspase-3 was increased [54]. The apoptosis-inducing activity of TCS was attributed to the promotion of caspase-8 and caspase-9 pathways, along with the activation of caspase-3 and PARP cleavage in breast cancer cells [55]. TCS mediated Phosphoinositide 3-kinase (PI3K)/Protein kinase B (AKT) pathway and thus enhanced cytotoxicity and apoptosis-inducing activity of an anti-cancer therapy Gemcitabine against non-small cell lung cancer [56]. TCS also enhanced the cell penetration of Granzyme B leading to apoptosis of tumor cells [57]. It was shown that TCS incited autophagy in gastric cancer cells via increasing the level of autophagy protein 5 (ATG5), altering microtubule-associated protein 1A/1B-light chain 3 (LC3) I to LC3 II, inducing reactive oxygen species (ROS) and stimulating nuclear factor kappa-B (NF-κB)/Tumor protein p53 (p53) pathway [58]. ROS induction might be related to extracellular Ca2+, which is involved in the apoptosis of human choriocarcinoma (JAR) cells [59]. TCS was able to inhibit angiogenesis in JAR cells through the reduction of vascular endothelial growth factor and inhibition of angiogenic signal, which contributed to the anti-cancer effect [60]. Smac (a mitochondrial protein) pathway was regulated by TCS in CaSki cervical cancer cells [61]. Low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a receptor facilitating TCS to enter JAR cells, while no significant endocytosis of TCS was found in Hela cells [62]. Notch signal was downregulated by TCS in the nasopharyngeal carcinoma (NPC) cell line CNE2 [63]. Besides tumor cell apoptosis induction and antiproliferation ability, the host immune system mediated by TCS might be another pathway for inhibition. T cells such as interferon-gamma (IFN-γ) producing CD4(+) and CD8(+) T cells, were increased by TCS in the 3LL Lewis lung carcinoma tumor model. TCS had upregulatory activity towards the expression of tumor suppressor in lung cancer 1 (TSLC1) and class I-restricted T cell-associated molecule (CRTAM) [64]. TCS exhibited antiproliferative activity on leukemia and lymphoma, which attributed to the induction of T-lymphocyte cell apoptosis and inhibition of B-lymphocyte cell growth by S-phase cell cycle arrest [65].

Ricin also exhibits an anti-tumor property. Ricin inhibited the growth of sarcomas in rats [66], and it increased the survival rate of Ehrlich ascites tumor-bearing mice [67]. It also shows promising effect on nude mice with human xenograft [68]. A phase I clinical study on 54 cancer patients with different kinds of tumors was taken and thus confirmed its properties [69]. The inhibition of protein synthesis was first considered attributing its anti-tumor activity. Cell apoptosis and the secretion of cytokine inflammatory mediators were shown to be the related mechanisms [70,71] (Figure 3). The treatment of ricin caused the activation of p38 and jun-N-terminal kinases (JNKs) [72]. Phosphoinositide 3-kinase (PI3K) and Janus kinase 2 (JAK2) were also involved in the activation of RAW264.7 mouse macrophage cells treated by ricin toxin-binding subunit B [73]. Ricin caused proinflammatory responses on human airway cells, which was related to stress-activated protein kinases and nuclear factor kappa-B (NF-kappaB) [74]. Both two main pathways, extrinsic (receptor mediated) and intrinsic (mitochondrial pathway), were involved in the ricin mediated cell apoptosis, following the activation of poly (ADP-ribose) polymerase (PARP) [75,76]. In addition, rapid release of cytochrome c was observed in ricin treated cells [75]. Ricin has been shown to induce the secretion of proinflammatory cytokines mediator such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) [77,78].

**Figure 3.** Ricin-induced cell death in the anti-tumor process. Arrows represent the activation of receptors and blunt arrows represent inhibition of receptors. Pathways involved are stated in blue boxes. The inhibition of protein synthesis, cell apoptosis and the release of cytokine inflammatory mediators are considered as the possible mechanism. p38: p38 mitogen-activated protein kinases; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; PI3K: phosphoinositide 3-kinases; JAK2: Janus kinase 2; STAT: signal transducer and activator of transcription; PARP: poly ADP-ribose polymerase; iNOS: inducible nitric oxide synthase; TNF-α: tumor necrosis factor-α; IL: Interleukin; Bcl-xl: B-cell lymphoma-extra large; Bax: Bcl-2-associated X protein.

Riproximin is a type 2 RIP that up-regulated the anti-cancer cytokine IL24/MDA-7 and ER-stress-related GADD genes; it also down-regulated the genes relating to migration (RHO GTPases), anti-apoptotic activities (BCL family), and cell cycle (cyclins) in selected human breast cancer cells MDA-MB-231 and MCF-7 [79]. Similar results were confirmed by using selected human and rat colorectal cancer (CRC) cell lines [80].

The anti-cancer effect of α-MMC was tested in human breast cancer cells MDA-MB-231 and MCF-7, but the relatively high cytotoxicity limited its therapeutic use [81]. After treated with α-MMC, cytochrome c was released, and intracellular free calcium concentration was increased, and calcium overloading led to cell death [82]. c-Jun N-terminal kinases (JNKs) signal pathway, which relates to cell apoptosis, was also triggered by α-MMC [83]. Many studies demonstrated that α-MMC involved in similar pathways with TCS, such as caspase-3 and 9 activations and interaction with low density lipoprotein receptor-related protein 1 (LRP1) [83,84]. LRP1 plays a vital role in the cytotoxicity mechanism of α-MMC because α-MMC mediated cytokine expression and MAPK pathway, which would be hindered by LRP1 silencing. α-MMC inhibited immune response through the inhibition of IL-1β, IL-2, IL-8, IL-9, IL-12, MIP-1α/β, MCP-1 and elevated the expression of IL-1ra and RANTES in human monocyte THP-1 cells. The regulation of cytokine release by α-MMC revealed that α-MMC might be applied to treat tumor-associated macrophages (M2 subtypes) [85].

Curcin, a type 1 RIP, could inhibit the growth of several tumor cell lines at 5 μg/mL, such as NCL-H446, SGC-7901 and S180 [86]. Curcin C, which shares highly conserved sequence with curcin, elicited inhibitory activity against the osteosarcoma cell line U20S with the half maximal inhibitory concentration (IC50) value 0.019 μM when IC50 of curcin is 0.27 μM [81].

Viscumarticulatum RIP (Articulatin-D), one of the mistletoe RIPs, could selectively inhibit acute T-cell leukemia. Caspase-8 and -3 were also involved. Early signals of apoptosis induction of Articulatin-D are exposure of phosphatidylserine and increased level of mitochondrial membrane potential [87]. Aviscumine and its native form mistletoe lectin-I increased the amount of cancer cell-specific T-cells resulting in more T-cell-mediated tumor cell lysis in a mouse glioma model. The level of the proteins associated with immune response was increased [88].

#### *3.2. Engineering of RIPs for Improving the Anti-Cancer E*ffi*cacy*

Most RIPs ITs have shown anti-cancer potential, particularly for hematological malignancies, which are easier accessed than solid tumors [45]. The presence of clusters of differentiation (CD) on hematological cells surface are considered as ideal targets for better design of ITs [89].

Sap-SO6 (the main isoform of saporin) was linked to CD2, CD7, CD19 and CD22 antigens found on human leukemia and lymphoma plasma membrane surface to make ITs [90]. The saporin ITs generated increased selective cytotoxicity at least 100-fold more than saporin alone [91]. Anti CD30-Saporin was reported reducing 60% tumor mass when used to treated refractory Hodgkin lymphoma patients [86]. However, it had transient hepatotoxicity when a single dose up to 0.8 mg/kg was applied [86].

RTA was also used in constructing ITs; some recent studies are reviewed below. A preliminary clinical study found that BCMab1-Ra, an IT consisting of RTA and BCMab1 (a novel monoclonal antibody that specifically recognized the aberrantly glycosylated Integrin a3b1 in bladder cancer), cured a patient with multiple tumors on the bladder and achieved no tumor recurrence in 3 years [92]. RTA conjugated with anti-HER2 scFv 4D5 and the endoplasmic reticulum-targeting peptide KDEL had 440-fold increase in anti-ovarian cancer cell activity compared to RTA alone. The specificity of this IT RTA-4D5-KDEL to HER2 was high so the toxicity to normal cells was low [93]. Combotox, a 1:1 combination of anti-CD19 and anti-CD22 immunotoxins, was conjugated to deglycosylated RTA, which showed higher efficacy than either IT in pediatric precursor B-lineage acute lymphoblastic leukemia (pre-B ALL) [94].

Besides plant RIPs, bacterial-originated toxins such as Pseudomonas exotoxin (PE) and diphtheria toxin (DT) were also used in ITs. Denileukin diftitox (Ontak) was the first immunotoxin approved by the Food and Drug Administration (FDA), which consists of Interleukin-2 ligand and DT [95]. A number of PE-based ITs have been under clinical trials. A recent example is the antimesothelin immunotoxin SS1(dsFv)PE38 (SS1P), which is the combination of PE38 (a modified Pseudomonas exotoxin A) and a murine antimesothelin variable antibody fragment. SS1P displayed high activity against malignant pleural mesothelioma in phase I clinical trial [96]. To achieve higher efficacy, researchers applied a tumor-seeking bacterial system by engineering *Salmonella typhimurium* to make it selectively expressed and released TGFα-PE38 (transforming growth factor alpha-PE38). The released TGFα-PE38 was then tested in mice with implanted colon or breast tumor cells, which expressed high levels of EGFR (epidermal growth factor receptor). Lower solid tumor growth rate was shown comparing to just intracerebral infusion of TGFα-PE38 [97].

Vascular leak syndrome is a major side effect of many RIP-based ITs. Ricin and T22, a ligand of the cell surface marker C-X-C motif chemokine receptor type 4 (CXCR-4), were assembled to form nanostructures, which exhibited specific anti-tumor activity and avoided VLS [98].

Besides targeting CD antigen, cell penetrate peptide (CPP) was adopted to improve specificity towards cancer cells. TCS fused with heparin-binding domain (HBD), a human derived cell-penetrating peptide CPP, could increase the apoptosis rate of HeLa cells when compared with treated TCS alone [99]. It offered an efficient delivery to cancer cells.

A co-delivery system of TCS and albendazole (ABZ) inhibited drug-resistant tumor cells (A549/T and HCT8/ADR) proliferation and tumor metastasis [100]. ABZ was covered by albumin-coated silver nanoparticles linked with low-molecular-weight protamine (a CPP) modified TCS; it could impair cytoskeleton.

#### **4. Challenges in Therapeutic Applications**

The immunogenicity of RIPs is an obstacle in usage. Although RIPs exhibit immunosuppressive activity [101], plant-originated RIPs readily stimulated immune system of patients, even causing allergic symptom [102]. Furthermore, the short plasma half-life of RIPs reduced drug exposure to targets, thus limiting clinical application. The plasma half-life of wild-type TCS was 9 min [103]. Repeated administration is needed to preserve the adequate level owing to renal insufficiency of small molecular weight RIPs [103]. However, repeating administration caused strong immune reaction [102]. Another side effect is neurotoxicity. TCS was examined without direct toxicity to the central nervous system (CNS). However, HIV-infected patients treated by TCS were reported to have adverse CNS reactions [104]. The HIV-infected macrophages might be altered by TCS treatment, which aggravated neurological symptoms [104]. Solid tumor mass is hard to access, leading to reduced efficacy. Intracavitary therapy with ITs might solve this problem [105]. Several studies have been carried out to enhance the pharmacological properties of RIPs.

#### **5. Coupling with Polymer Polyethylene glycerol (PEG) and Dextran**

PEGylation is a common strategy used to improve drug performance. PEG is biocompatible with high hydrophilicity, low toxicity and non-immunogenicity [106]. After coupling, the size and molecular weight are increased to avoid rapid renal clearance and proteolytic degradation for longer half-life. Antigenicity and immunogenicity can be decreased, while pharmacokinetics and pharmacodynamics can be improved. Moreover, permeation retention effect was increased by PEGylation, which helped to target tumoral tissues [107]. Non-specific PEGylation was applied at first. Although reactive amino acids such as cysteine, arginine and serine can be chosen [108], site-directed conjugation has provided better achievements. The antigenic sites are mapped and then antigenicity can be alleviated through PEGylation, while the original enzymatic activity could be least affected. For successful conjugation, a site extending from the protein surface is preferred [109]. Many RIPs PEGylations were attempted to advance their pharmacological properties (Table 1).


#### **Table 1.** Representative PEGylated RIPs.

Gelonin (GAP31) was covalently coupled to methoxypoly (ethylene glycol) (mPEG) 2000, mPEG 5000 and mPEG succinimidyl succinate 20K (SS-20PEG). mPEG does not affect the positive charge of protein, while charges alteration may result in lowering biological activity. The plasma half-life time of all conjugations above was increased. PEGylation also decreased organ uptake. Coupled of mPEG retained immunogenicity, while SS-20PEG conjugated decreased cytotoxicity [110].

Site-directed PEGylation of TCS showed that PEG 20,000 is better than PEG 5000. Coupled to PEG 20000, the plasma half-life extended due to the enlarged size and resistance to proteolysis. Immunoglobulin G (IgG) level was also reduced because of decrease in immunogenicity [118]. The IgE level was reduced but the IgG level was maintained when conjugated to PEG 5000 [109,118]. Because tremendous liver uptake is through carbohydrate-mediated recognition, carbohydrate-directed PEGylation can improve the pharmacokinetics of RTA. Meanwhile, the circulation time was increased and antigenicity was reduced by masking the epitopes. This makes carbohydrate-directed PEG-RTA conjugate a potential anti-tumor drug [120]. Coupling PEG to maize RIP (MOD) gave similar results, including pharmacokinetics improvement and antigenicity reduction [109]. Mono-, di-, tri-PEGlyatedα-MMC and MAP30 dramatically reduced the immunogenicity and maintained biological activities [112,113]. The enzymatic activity assay showed that mono-PEGylated α-MMC strongly inhibited the growth of human cervix adenocarcinoma cells [116]. Sun et al. provided a method to isolate high purity mono-mPEGylated MAP30 and α-MMC, which can be useful for further RIP drug examination [111]. In vivo study implied that the hepatic toxicity of α-MMC was reduced after PEGylation [115]. The plasma half-life of α-MMC was sharply increased from 6.2–7.5 h to 52–87 h [114]. The homogeneity of PEGlyated α-MMC was further improved by site-specific conjugation

of mPEG-ALD [116]. Due to steric hindrance of active sites, PEGylation may lead to biological activity reduction, which can be compensated by longer plasma half-life [119].

Coupling dextran on RIPs was also studied to improve their performance on anti-cancer and anti-HIV. Similar to PEGylation, dextran can increase circulation time and reduce IgG or IgE responses [103,121,122]. Dextran may be used as a linker to connect monoclonal antibodies and RTA, and to improve selectivity toward target cells [123]. A lower rate of plasma clearance prolongs the plasma half-life of TCS after coupling to dextran T40 by a dialdehyde method [103]. The toxicity and potency were decreased [124]. TCS coupled with dextran had the IgE level reduced eight times compared to wild-type [121] To reduce the antigenicity, TCS was also coupled by bromodextran T20 [125]. To obtain better conjugate, potential antigenic site K173 of TCS was mutated to cysteine and coupled with dextran. Site-directed conjugate dextran-K173C had the hypersensitivity reaction and the level of IgG andIgE decreased [126].

#### **6. Perspectives**

The therapeutic applications of RIPs, in particular anti-HIV and anti-tumor potential were exploited in the last several decades [43]. The main obstacles of utilization include short plasma half-life, non-selective cytotoxicity and antigenicity. Most PEGylation increases plasma time and reduces antigenicity. RIPs conjugated with antibodies to form immunotoxins increase the selective toxicity to target cells.

Immunotherapy can assist chemotherapy to improve efficacy [127]. An IT is co-applied with a small molecular drug or another IT to achieve high efficacy and reduce side effects. Anti-CD3 and anti-CD7 were conjugated separately to RTA and mixed to treat steroid-refractory acute graft-versus-host-disease. The mixture exerted higher efficacy than one alone with low side effects in clinical trial I/II [128]. Saponin, which is generally classified as steroidal or triterpenoidal, can act as an endo/lysosomal escape enhancer. It was commonly used together with type I RIP to facilitate escape of RIP from endo/lysosomal degradation in order to increase efficacy of RIP or ITs [43].

"Cocktail therapy" is an effective strategy to increase the effectiveness of ITs [129]. The synergistic effect can be achieved when two or even more suitable ITs corporate. Lung cancer cells can easily generate resistance against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [130]. TCS was found inducing TRAIL sensitivity of non-small cell lung cancer (NSCLC) by regulating death receptors and proteins involved in invasion and cell cycle [130].

A number of RIP-derived drugs reached clinical trial, but then failed due to severe side effects and little efficacy (Table 2). At present, an RIP diphtheria toxin (DT) derived drug denileukin diftitox (Ontak) has been approved by FDA for treating cutaneous T-cell lymphoma (CTCL).


**Table 2.** Recent (after 2000) representative ricin & RTA immunotoxins tested.

With the rapid development of immunotoxin engineering technology, it is possible to acquire RIP-based drugs with high efficacy and low side effects. A list of endosomal escape enhancers such as saponin, TAT (transcriptional activator Tat protein), perforin and ricin B-chain were observed to facilitate RIP [43]. mAbs help increase the specificity; adequate combination of ITs with different mAbs can achieve better curative effect [96]. Novel technology has been tested, such as photochemical internalization (PCI). PCI is a light-based method and is employed to trigger the endosomal escape of RIP. Saporin linked with a photosensitizer functionalized CPP showed cytotoxicity augmentation in MC28 fibrosarcoma cells [135]. Another study also showed that the cytotoxicity of IT 225.28-saporin was strengthened by using PCI with a photosensitizer disulfonated tetraphenyl chlorin (TPCS2a) [136]. TCS was conjugated with an albumin-binding domain and a legumain-substrate peptide as a modified IT for better delivery efficiency, which can make use of the nutrient transporter pathway of albumin-binding proteins. Protease legumain at the tumor sites can dissociate TCS from an albumin-binding domain, which gives a new strategy for tumor therapy [137]. These new research findings provide RIP therapy a promising future.

**Author Contributions:** Conceptualization, P.-C.S.; writing, Z.-N.Z. and J.-Q.L.; review and editing: P.-C.S. and Y.-T.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by The Chinese University of Hong Kong: 3110130, The Research Grants Council of Hong Kong SAR: 14176617.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Antiviral Activity of Ribosome-Inactivating Proteins**

**Lucía Citores †, Rosario Iglesias † and José M. Ferreras \***

Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, E-47011 Valladolid, Spain; luciac@bio.uva.es (L.C.); riglesia@bio.uva.es (R.I.)

**\*** Correspondence: josemiguel.ferreras@uva.es

† Authors contributed equally to this work.

**Abstract:** Ribosome-inactivating proteins (RIPs) are rRNA N-glycosylases from plants (EC 3.2.2.22) that inactivate ribosomes thus inhibiting protein synthesis. The antiviral properties of RIPs have been investigated for more than four decades. However, interest in these proteins is rising due to the emergence of infectious diseases caused by new viruses and the difficulty in treating viral infections. On the other hand, there is a growing need to control crop diseases without resorting to the use of phytosanitary products which are very harmful to the environment and in this respect, RIPs have been shown as a promising tool that can be used to obtain transgenic plants resistant to viruses. The way in which RIPs exert their antiviral effect continues to be the subject of intense research and several mechanisms of action have been proposed. The purpose of this review is to examine the research studies that deal with this matter, placing special emphasis on the most recent findings.

**Keywords:** adenine polynucleotide glycosylase; antiviral therapy; human virus; immunotoxin; ribosome-inactivating protein (RIP); rRNA glycosylase (EC 3.2.2.22); virus-resistant transgenic plant (VRTP)

**Key Contribution:** Ribosome-inactivating proteins might help in the fight against human and plant viruses.

#### **1. Introduction**

One of the main efforts of virologists and molecular biologists is the search for antivirals that can help in the fight against viruses causing diseases in animals and especially in humans. Strategies are also being searched to tackle the challenge of plant viruses causing significant crop losses. This has led to the discovery of a number of antivirals with different chemical nature or proteins with different enzymatic activities [1,2]. The search for more effective and safer antivirals continues to be a field of intense investigation and plants are one of the most used sources, since they have evolved a variety of protein-based defense mechanisms to tackle viral infections [3]. Regarding ribosome-inactivating proteins (RIPs), it is worth noting the fact that one of the first RIPs to be purified was PAP (pokeweed antiviral protein) and although many RIPs have been purified as protein synthesis inhibitors, many others have been isolated as powerful antivirals. For many years, RIPs have been studied as potent inhibitors of protein synthesis that can be used for the construction of immunotoxins [4]. Since linked to a monoclonal antibody or a protein that specifically binds to a receptor, they can be used to specifically kill tumor cells [4,5]. RIPs have initially been studied as a family of proteins widely distributed among angiosperms although they have also been found in other taxons [6,7]. They irreversibly inactivate ribosomes inhibiting protein synthesis and thus causing cell death [6,7]. The first RIPs to be isolated, the extremely potent toxins ricin and abrin, were purified at the end of the nineteenth century and it was believed that their red cell agglutinating activity was the reason for the toxic effect [8,9]. In the early 1970s, it was reported that abrin, ricin, and PAP strongly inhibited protein synthesis in a cell-free rabbit reticulocyte system [8–10]; and Barbieri

**Citation:** Citores, L.; Iglesias, R.; Ferreras, J.M. Antiviral Activity of Ribosome-Inactivating Proteins. *Toxins* **2021**, *13*, 80. https://doi.org/ 10.3390/toxins13020080

Received: 22 December 2020 Accepted: 20 January 2021 Published: 22 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and Stirpe classified these and other related proteins as type 1 RIPs (a single polypeptide chain, such as PAP) and type 2 RIPs (two chains, an A chain similar to type 1 RIPs, and a B chain that possesses lectin activity, such as abrin and ricin) [4]. The enzymatic activity of ricin was discovered by Endo and colleagues, that is, RIPs are considered as 28S rRNA N-glycosylases (EC 3.2.2.22) that cleave the N-glycosidic bond between the adenine No. 4324 and its ribose in the 60S subunit of rat ribosomes [11] or the equivalent one in sensitive ribosomes from other organisms [12]. This adenine is located in the sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factors EFG and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. This loop is also the target of ribotoxins such as α-sarcin, enzymes with rRNA endonuclease activity (EC 3.1.27.10) [13]. However, some RIPs are also able to remove more than an adenine from the rRNA [14] and many of them are able to deadenylate not only rRNA but also other polynucleotide substrates such as DNA, poly(A), mRNA, tRNA, and viral RNA [15], and because of this, the name of adenine polynucleotide glycosylase (or polynucleotide: adenosine glycosidase) was proposed for RIPs [15]. Additionally, other activities have been reported for RIPs, just as shown in Table 1.

**Table 1.** Proposed activities and other biological properties of ribosome-inactivating proteins (RIPs).


A convincing picture of the role played by these proteins in plants is not yet available. They seem to play different roles in different species, so antiviral, antifungal, plant defense, storage, programmed senescence, antifeedant, stress protection, and development regulation roles have been proposed for RIPs [7].

The need to find new antivirals has encouraged researchers to study the antiviral activity of RIPs. On the other hand, much research is underway, focused on the use of these proteins to obtain crops with resistance to viral pathogens. The aim of this review is to compile the advances that have been made within this field, placing special emphasis on the most recent findings.

#### **2. Activity on Animal (Human) Viruses**

Global health threats such as the emergence of human viruses resistant to commonly used antiviral drugs, has prompted the study of RIPs as possible tools for fighting these agents. Antiviral activity of RIPs against different animal viruses has been reported (Table 2).


**Table 2.** RIPs active against animal viruses. RIPs with antiviral activity, the families and species from which they have been obtained and the viruses in which this activity has been demonstrated are shown.

Virus name abbreviations: CHIKV (chikungunya virus), DENV (dengue virus), FLUV (human influenza virus), HBV (hepatitis B virus), HHV (human gammaherpesvirus), HIV (human immunodeficiency virus), HPV (human poliovirus), HSV (herpes simplex virus), HTLV (human T-cell leukemia virus), JEV (Japanese encephalitis virus), LCMV (lymphocytic choriomeningitis virus), PICV (Pichinde virus), SHIV (simian–human immunodeficiency virus).

> RIPs with antiviral activity belong to the main types of RIPs found in angiosperms [7]: monocot type 1 RIPs (Poaceae), dicot type 1 RIPs (Euphorbiaceae, Caryophyllaceae, Phytolaccaceae), type 2 RIPs (ricin, Euphorbiaceae), and type 1 RIPs derived from type 2 RIPs (Cucurbitaceae); which suggests that all these proteins could have, to a greater or lesser extent, antiviral activity and that their main biological role could be precisely the defense of the plant against viruses. However, researchers have focused on the study of proteins obtained from species of the families Phytolaccaceae, Cucurbitaceae, Caryophyllaceae, and Euphorbiaceae; and the most studied RIPs are pokeweed antiviral protein (PAP), trichosanthin (TCS) and Momordica antiviral protein (MAP30), which have been the subject of recent reviews [10,35,36,38,58]. It is noteworthy that RIPs have shown to be active against viruses of very different nature: double-stranded (ds) DNA viruses (hepatitis B virus,

HBV; human gammaherpesvirus, HHV; human poliovirus, HPV; herpes simplex virus, HSV), retroviruses (human immunodeficiency virus, HIV; human T-cell leukemia virus, HTLV; simian–human immunodeficiency virus, SHIV), positive-sense single-stranded (ss) RNA viruses (Japanese encephalitis virus, JEV; dengue virus, DENV; chikungunya virus, CHIKV), and negative-sense (ss) RNA viruses (human influenza virus, FLUV; lymphocytic choriomeningitis virus, LCMV; Pichinde virus, PICV). Most of the viruses studied are enveloped viruses that infect humans, with the exceptions of the simian–human immunodeficiency virus (SHIV), the Pichinde virus (PICV), and the non-enveloped human poliovirus. This virus was the first in which activity against an animal virus was reported [59]. Results obtained with HEp-2 cells infected with human poliovirus or herpes simplex virus (HSV) showed that gelonin, momordin, dianthin 32, and PAP-S impaired viral replication by inhibiting protein synthesis in virus-infected cells, in which presumably they enter more easily than in uninfected cells [30], suggesting that antiviral activity could be a general property of RIPs.

#### *2.1. Activity on Human Immunodeficiency Virus*

The most studied virus is the human immunodeficiency virus (HIV). The lack of effective antivirals against this virus and its rapid spread around the world prompted studies on the activity of RIPs against this virus since 1989 [60]. At least 20 RIPs have shown activity against HIV (Table 2). Thus, several RIPs obtained from Euphorbiaceae and Caryophylaceae, but mostly from Cucurbitaceae and Phytolocaceae, inhibit the replication of HIV in vitro [35]. It has also been reported that maize RIP transiently reduces viral load in SHIV infected Chinese rhesus macaques [27]. The results obtained with RIPs promoted their use in clinical trials [61]. Although the development of specific HIV antivirals such as reverse-transcriptase and protease inhibitors have directed AIDS therapy to other treatments, these studies demonstrated the potential of RIPs for the treatment of virus-related diseases.

#### *2.2. Activity on Herpes Simplex Virus*

Another virus that has been targeted by RIPs is the herpes simplex virus (HSV). Currently, there is no treatment that completely eliminates HSV infection from the body, because once the virus enters an organism, it remains dormant until reactivated. This has encouraged researchers to study RIPs as candidates for HSV therapy. Gelonin, trichosanthin, dianthin 32, PAP, PAP-S, and several RIPs obtained from *Momordica charantia* have shown anti-HSV activity in vitro (Table 2).

#### *2.3. Activity on Other Animal Viruses*

Exposure of HepG2.2.15 cells to MAP30 [44], PAP-S [56], α-momorcharin [41], and an eukaryotic expression plasmid encoding PAP [56] inhibits the production of hepatitis B virus (HBV). Additionally, an extract from Radix Trichosanthis had a stronger inhibitive effect on expression of HBsAg and HBeAg in HepG2.2.15, and trichosanthin has been proposed as the main component of the aqueous extract responsible for the anti-hepatitis B viral effect [62].

On the other hand, it has also been reported that PAP inhibits replication of human T-cell leukemia (HTLV), human influenza, chikungunya (CHIKV), Japanese encephalitis (JEV), and lymphocytic choriomeningitis (LCMV) viruses, gelonin inhibits Pichinde virus replication, and MAP30 inhibits human gammaherpesvirus 8 (HHV8) and dengue virus [10,31,35,42,52–55].

#### *2.4. Citotoxicity of RIPs*

An important aspect to consider when working with antivirals is their cytotoxicity. In this sense, type 1 RIPs and type 2 RIPs can be distinguished. Type 1 RIPs consist of a polypeptide chain with rRNA N-glycosylase activity, while type 2 RIPs are constituted by two chains linked by a disulfide bond: The A chain (active) is equivalent to a type

1 RIP and the B chain (binding) is a lectin able to bind to membrane glycoproteins and glycolipids allowing endocytosis of RIP by cells. This is why RIPs such as ricin and abrin are extremely toxic showing IC50 (concentration that inhibits protein synthesis by 50%) values of 0.67–8 pM in cell cultures [63]. There are type 2 RIPs such as those from *Sambucus* which are much less toxic to cultured cells with IC50 values of 27–64 nM [64]. Type 1 RIPs are much less toxic and have highly variable IC50 values (0.2–10 μM) [63]. Due to the high cytotoxicity of type 2 RIPs, only type 1 RIPs or the ricin A-chain (which has a cytotoxicity similar to that of type 1 RIPs) [63] have been used as antiviral agents.

A good antiviral should display a substantial difference between the antiviral concentration and the cytotoxic concentration. Due to the diverse toxicities of type 1 RIPs, there are also differences in this regard, but the most commonly used proteins such as PAP, MAP30, or trichosanthin always show a substantial difference between toxic concentrations for cells (3–30 μM) [63,65,66] and concentrations that have antiviral activity (around 30 nM) [35].

Finally, it should be noted that some bacterial and fungal enzymes targeting the sarcin-ricin loop have also been reported to possess antiviral activity [2,67–73].

Therefore, RIPs have awakened over many years, and continue to do so, a keen interest as tools to fight viruses that cause diseases in humans. In fact, recently saporin and RTAM-PAP1 (a chimera constructed with ricin A-chain and PAP) have been proposed as candidates for therapy of COVID-19 [74,75].

#### **3. Activity against Plant Viruses**

To date, 39 RIPs have been described that display some type of activity against plant viruses (Table 3).

These RIPs have been found in 26 plant species belonging to one family of monocotyledons and ten families of dicotyledons, that are distributed throughout the phylogenetic tree of angiosperms in a similar way to the RIP-containing plants [7], thus suggesting that most RIPs could be active against plant viruses. As a matter of fact, only two type 2 RIPs from *Sambucus nigra* (SNAI and SNLRP) have been reported to fail to protect transgenic plants against viral infection [76].

Despite the fact that these antiviral proteins are distributed in a great variety of families, most of them (thirty one) belong to the orders Caryophyllales and Lamiales (families Caryophyllaceae, Amaranthaceae, Phytolaccaceae, Nyctaginaceae, Basellaceae, Lamiaceae), which are RIPs with well-defined structural and phylogenetic characteristics [7].

RIPs seem to be active against a wide range of viruses (Table 3), all of them belonging to different families of positive-sense single-stranded (ss) RNA viruses. The exception is the geminivirus ACMV (African cassava mosaic virus), which contains a single-stranded circular DNA genome. They seem to protect all kinds of plants and, although the most commonly used plant for testing has been *Nicotiana tabacum* L., RIPs have also shown ability to protect other species of the genus *Nicotiana* (*N. benthamiana* Domin and *N. glutinosa* L.) as well as other species commonly used in research or crops such as *Brassica rapa* L. (=*B. parachinensis* L.H.Bailey) (choy sum), *Cyamopsis tetragonoloba* (L.) Taub. (guar), *Crotalaria juncea* L. (sunn hemp). *Phaseolus vulgaris* L. (common bean), *Momordica charantia* L. (bitter melon), *Beta vulgaris* L. (sugar beet), *Cucurbita pepo* L. (squash), *Solanum tuberosum* L. (potato), *Carica papaya* L. (papaya), *Chenopodium quinoa* Willd. (quinoa), or *Lycopersicon esculentum* Mill. (tomato).


**Table 3.** RIPs active against plant viruses. RIPs with antiviral activity, the families and species from which they have been obtained and the viruses in which this activity has been demonstrated are shown.


**Table 3.** *Cont.*

Virus name abbreviations: ACMV (African cassava mosaic virus), AMCV (artichoke mottled crinkle virus), AMV (alfalfa mosaic virus), BMV (brome mosaic virus), BYMV (bean yellow mosaic virus), ChiVMV (Chilli veinal mottle virus), CMV (cucumber mosaic virus), ICRSV (Indian citrus ringspot virus = citrus ringspot virus, CRSV), PLRV (potato leafroll virus), PMV (pokeweed mosaic virus), PRSV (papaya ringspot virus), PVX (potato virus X), PVY (potato virus Y), SBMV (southern bean mosaic virus), SHMV (sunn-hemp mosaic virus = sunn-hemp rosette virus, SRV), SPMV (satellite panicum mosaic virus), TBSV (tomato bushy stunt virus), TEV (tobacco etch virus), TMV (tobacco mosaic virus), TNV (tobacco necrosis virus), TuMV (turnip mosaic virus), ZYMV (zucchini yellow mosaic virus).

> It is difficult to compare the antiviral activity of the different RIPs because different criteria have been used to evaluate their antiviral capacity. In some cases, the putative antiviral character is based on their N-glycosylase activity on the virus genome [105]; all RIPs are able to release adenines from any kind of RNA or DNA, including viral genomes [4]. This adenine polynucleotide glycosylase activity has been detected by electrophoresis [87], or HPLC [103,105]. In many cases, the test has involved applying a RIP solution on the leaf surface of the plant together with the virus and comparing the result with the control that does not contain RIP. In some cases, the virus is applied simultaneously [86,92,113] and in others, sometime after the application of the RIP [90,115]. The evaluation of antiviral activity has been done by counting the number of lesions [88,93], the time of onset of symptoms [77,79], the number of infected plants [105], or the severity of the infection symptoms [78,115]. Virus levels have also been estimated by ELISA [99], Western blotting analysis [81], RT-PCR analysis [101], quantitative real-time PCR analysis [81,82],

electron microscopy [92], or by determining the infection capacity of an extract from the infected plant [92]. Another approach has been the construction of virus-resistant transgenic plants [80,102]. The virus has been inoculated mechanically or by aphids [102] and the resistance has been determined by one of the methods listed above.

Other studies link RIPs to the defense of plants against viruses, especially studies of induction of RIPs through signaling compounds such as salicylic acid, hydrogen peroxide, or jasmonic acid, which are involved in the systemic acquired resistance (SAR) of plants against viruses and other pathogens. Thus, it has been reported that artichoke mottled crinkle virus (AMCV), salicylic acid, and hydrogen peroxide induce the expression of BE27 in both treated and untreated leaves of sugar beet plant [86,117]. On the other hand, it has been reported that alpha-momorcharin induces the generation of salicylic acid, jasmonic acid, and reactive oxygen species, which improve tobacco mosaic virus (TMV) tolerance [118]. Additionally, alpha-momorcharin induces the expression of the N gene [118], which encodes the N protein that recognizes the TMV replicase fragment and triggers signal transduction cascades, initiating a hypersensitive response (HR) and inhibiting the spread of TMV [118]. Other RIPs in which some type of elicitor activity has been reported are pokeweed antiviral protein II (PAPII) [104], CIP-29 [111], and CA-SRI [113,115]. By contrast, the antiviral activity of SNAI' [116], IRIP and IRAb [77], and nigrin b [76] is not accompanied by an induction of pathogenesis-related proteins. All this suggests that some, but not all RIPs, could be part of the SAR or/and HR to defend the plant against viral infections.

#### **4. Antiviral Mechanisms of RIPs**

RIPs have long been recognized as antiviral proteins in both plants and animals, but the mechanism responsible for this activity continues to be the subject of intense research today. The mechanism that triggers protection against viruses could have both common and different elements in plants and animals (Figure 1).

#### *4.1. Antiviral Mechanisms of RIPs in Plants*

#### 4.1.1. Protein Synthesis Inhibition (rRNA N-glycosylase)

It has long been known that RIPs can inhibit protein synthesis in plants [119–122]. The mechanism is the same as that described for inhibition of protein synthesis in animals, i.e., RIPs act as N-glycosylases of the major rRNA by removing a specific adenine from the sarcin-ricin loop (SRL), which is highly conserved in animals and plants [120]. Moreover, it has been shown that some RIPs can inhibit protein synthesis carried out by ribosomes of the same plants that produce them [123] and in addition, in the case of some RIPs, a positive correlation between rRNA N-glycosylase activity on tobacco ribosomes and antiviral activity against TMV has been reported [124].

The fact that RIPs do not cause cell death in the absence of the virus and allow plant growth is due to the fact that, at least for type 1 RIPs from dicots, they are synthesized as preproteins with a leader peptide that directs them into the apoplastic space [125]. Viral infection is supposed to facilitate the entry of the RIP, which inactivates cell ribosomes, causing cell death and preventing the virus from using the cellular machinery to replicate and spread [125]. So far, the mechanism by which the virus facilitates the entry of RIPs has not been shown, although the ability of viruses to modify plasma membrane permeability is well-known [126].

**Figure 1.** Proposed mechanisms for the antiviral activity of RIPs against plant viruses (upper panel), animal viruses (lower panel), and retroviruses (lower panel including dashed square). (**upper panel**) In plants, viral infection promotes the passage of the RIP from the apoplast to the cytosol. In the cytosol, it can inactivate ribosomes (rRNA glycosylase activity), causing the death of infected cells and thus preventing the spread of the virus. The RIP can also depurinate the viral RNA (adenine polynucleotide glycosylase, APG, activity), inhibiting its replication, transcription, translation, and assembly. It can also trigger antiviral defense signaling pathways, causing an increase in the levels of salicylic acid, jasmonic acid, pathogenesis-related (PR) proteins, and both reactive oxygen species (ROS) and ROS scavenging enzymes. (**lower panel**) In animal cells, the RIP can enter by pinocytosis or receptor-mediated endocytosis. RIP can inactivate ribosomes (rRNA glycosylase activity), causing the death of infected cells or inactivate the viral genome, DNA, or RNA (APG activity), preventing their replication, transcription, and translation. Some RIPs depurinate specific sequences (APG activity), blocking critical functions for the virus life cycle. In the case of retroviruses, the RIP can also depurinate the long terminal repeats (LTRs) (APG activity) or cleave the circular DNA (APG activity) preventing its integration into the cell genome. It can also be introduced into virions during budding (viral membrane association), making them less infective. Ribotoxic stress (rRNA glycosylase activity or APG activity on mRNA) and DNA damage (APG activity) caused by RIPs can trigger the activation of signaling pathways that cause infected-cell death preventing virus spreading.

#### 4.1.2. Adenine Polynucleotide Glycosylase Activity

However, although some type 1 RIPs can inactivate ribosomes of some plants, they do not do so with those of others and usually act at much higher concentrations than in animal ribosomes [127]. In addition, mutants have been obtained from PAP that do not depurinate tobacco or reticulocyte lysate ribosomes but inhibit translation of brome mosaic virus (BMV) and potato virus X (PVX) [128].

The specificity of RIPs is highly variable, therefore some RIPs can act on other adenines in both animal [14] and plant [120,129] ribosomes. In addition, all RIPs release adenines from eukaryotic DNA and many of them also release adenines from other RNAs, including viral RNAs [15,22,87]. It has also been reported that some RIPs may have DNA nicking, DNase or RNase activities (Table 1). This can alter the life cycle of the virus, both its replication and transcription [130], translation [91], and assembly [131].

The adenine polynucleotide glycosylase activity on viral RNAs might be more specific. Thus, it has been reported that some RIPs can inhibit the translation of capped RNA by binding to the cap of viral RNAs and depurinating these RNAs downstream of the cap structure. For these RIPs, viral RNA depurination could be the main mechanism of their antiviral activity [51]. On the other hand, one of them (PAP) can also bind to translation initiation factors, allowing it to depurinate preferentially uncapped viral RNAs [103]. Viral capped RNA sequestration has also been proposed as an antiviral mechanism for MbRIP-1, a RIP from *Momordica balsamina* [132]. All this suggests that the antiviral mechanism of RIPs could be more complex than a simple and direct depurination of viral RNA.

#### 4.1.3. Antiviral Protection through Signaling Pathways

The other proposed mechanism involves signaling molecules that defend the plant from viral infection. However, different results have been obtained depending on the RIP studied and the approach used. Thus, it has been reported that α-momorcharin (α-MMC), in *N. benthamiana* plants sprayed with a solution of the RIP, up-regulates the expression of reactive oxygen species (ROS) scavenging-related genes, modulating ROS homeostasis and conferring resistance to TMV, ChiVMV, and CMV infection [81,133]. Additionally, this RIP also up-regulates some salicylic acid-responsive defence-related genes [81]. By contrast, the same RIP sprayed in *M. charantia* plants increases plant resistance to CMV but by increasing jasmonic acid biosynthesis and inducing ROS without a relevant increase in salicylic acid [82]. It has also been reported that α-momorcharin induces an increase of both jasmonic acid and salicylic acid in tobacco plants, enhancing TMV resistance [118]. On the other hand, it has been postulated that PAP generates a signal that leads to the overexpression of pathogenesis-related proteins rendering transgenic tobacco plants resistant to virus infection in the absence of an increase in the salicylic acid levels [129,134,135]. Finally, it has been reported that the expression of IRAb and IRIP in transgenic tobacco plants provides a strong local protection against TMV and TEV but without induction of pathogenesis-related proteins [77]. The relationship between the enzymatic activity of RIPs and their ability to induce production of signaling molecules in plants has not been studied. In animals, the enzymes that exert their cytotoxic function through modification of the sarcin-ricin loop (SRL), such as ricin, α-sarcin, or Shiga toxin, strongly activate signaling pathways through the mitogen-activated protein kinases (MAPKs) p38 and JNK [136]. The trichothecenes deoxynivalenol (DON) and T-2 toxin inhibit protein synthesis and have been shown to induce activation of ERK1/2 and p38 MAP kinase in several animal and human cell lines followed by increased cytokine production [137]. This ribosome mediated activation of MAPKs is termed 'ribotoxic stress response' [137]. In Arabidopsis, DON and T-2 toxin led to the expression of MPK3 and MPK6 MAP kinases, implicated as positive regulators of the hypersensitive response via ethylene signaling and ROS [137]. Therefore, it would be possible that the generation of signaling compounds by plants was a response to ribotoxic stress produced by RIPs.

#### *4.2. Antiviral Mechanisms of RIPs in Animals*

#### 4.2.1. Protein Synthesis Inhibition (rRNA N-glycosylase)

Early studies on the mechanism of antiviral action of RIPs in animal cells focused on their ability to inhibit protein synthesis [30]. Several type 1 RIPs (gelonin, *Momordica charantia* inhibitor, dianthin 32, and PAP-S) reduced viral production and plaque formation in HEp-2 cells infected with Herpes simplex virus-1 (HSV-1) or poliovirus I. In addition, the four RIPs inhibited protein synthesis more efficiently in cells infected with one of the two viruses than in uninfected cells, suggesting that RIPs inhibited viral replication by inhibiting protein synthesis of infected cells, presumably because they entered infected cells more easily than uninfected cells [30]. Although the mechanism by which viruses can facilitate the entry of RIPs is not established, it is known that type 1 RIPs can enter cells through pinocytosis or receptor-mediated endocytosis [138,139] and that both processes are stimulated by viruses [140,141].

#### 4.2.2. Adenine Polynucleotide Glycosylase Activity

However, RIPs can inhibit virus replication without apparently inactivating ribosomes [34,52,142,143]. The adenine polynucleotide glycosylase activity on viral RNA [57] or DNA [33] is able to inactivate the viral genome and explains inhibition of virus replication [37,142,143]. In addition, RIPs can also depurinate viral mRNAs, thus avoiding the synthesis of proteins that are vital for its functions [52,144,145]. In the case of HIV, a strong inhibition of the integration of viral DNA into the host genome [32,45,50], caused by the adenine polynucleotide glycosylase activity on LTRs (long-terminal repeats) [33,146,147] and the nicking activity on the supercoiled DNA [148,149] of the virus, has been reported. Trichosanthin is also able to enter viral particles during budding, resulting in virions unable to infect other cells [150,151].

#### 4.2.3. Antiviral Protection through Signaling Pathways

Finally, it has also been proposed that the antiviral activity of RIPs can be carried out through signaling pathways. Thus, it has been reported that RIPs promote p53 and c-Jun N-terminal kinase (JNK) activity [152,153] and block the activation of KF-κB, p38MAPK, and Bcl-2 [152,154,155] during viral infection. The modulation of these pathways would lead to the death of infected cells, thus preventing the spread of the virus. Cell DNA damage [152] or ribotoxic stress [153] caused by RIPs could trigger some of these signaling pathways. Ribotoxic stress response (RSR) is a response of cells to a variety of agents that affect the functions of ribosome, such as some antibiotics, alkaloids, mycotoxins, RIPs, ribotoxins, or ultraviolet radiation [136]. Ribotoxic stress is sensed by the MAP3K ZAKα that transduces the signal from ribosomes to activate MAP2K that in turn activates SAPKs. There are two SAPKs (stress-activated protein kinases) families in mammals: p38 and c-Jun N-terminal kinase (JNK). Activation of p38 induces cell-cycle arrest whereas activation of JNK promotes apoptosis [156], inducing both pro-survival and pro-apoptotic signaling. Additionally, mRNA damage by the adenine polynucleotide glycosylase activity of RIPs could trigger RSR as has been reported for ultraviolet radiation [156]. However, much research is still required to clarify how RIPs protect cells from viral infection through these pathways.

Therefore, RIPs can exert their antiviral effect through different mechanisms that could originate from their activity on the different nucleic acids from both the virus and the infected cell. Depending on the type of RIP, virus and infected cell, some mechanisms could predominate over others and more research is required to determine in each case which are the predominant ones.

#### **5. Experimental Therapy**

Because of its strong antiviral activity, RIPs have been used in experimental therapy, especially to treat the acquired immune deficiency syndrome (AIDS), but also against hepatitis, chikungunya, dengue, and lymphomas caused by the Epstein–Barr virus. Additionally, they have also been tested in vivo against viruses that infect animals, such as the murine cytomegalovirus, the Pichinde virus, or the simian–human immunodeficiency virus (Table 4).

#### *5.1. RIPs and PEGylated RIPs*

Trichosanthin (GLQ223) was used alone [61,157] or in combination with zidovudine (azidothymidine, AZT) [158] in clinical trials with AIDS patients. Trichosanthin infusions were safe and relatively well tolerated [157]. In patients, a decrease in serum p24 antigen [61] and an increase in CD4<sup>+</sup> and CD8<sup>+</sup> T cells [157,158] were observed. Recently, it has also been reported that maize RIP reduces the viral load of an HIV-related virus, the simian–human immunodeficiency virus in Chinese rhesus macaques [27].

Despite its potential as therapeutic agents, the strong immunogenicity, allergic reaction, and short half-life are the biggest barriers to their application as therapeutic agents. Polyethylene glycol (PEG) conjugation (PEGylation) can confer on these proteins, increasing plasma half-life, decreasing toxicity, and reducing immunogenicity and antigenicity. PEGylated alpha-momorcharin and MAP30 showed about 60%–70% antivirus activities against HSV-1, and at the same time decreased 50%–70% immunogenicity when compared with the non-PEGylated proteins [40].

#### *5.2. Immunotoxins and Other Conjugates*

RIPs have been used in medicine mainly as the toxic part of immunotoxins, that is, chimeric proteins consisting of an antibody specifically directed against a target, linked to a toxin of plant or bacterial origin. The design of immunotoxins has been improved over the past 40 years to minimize the off-target toxicity and immunogenicity [159,160]. Several types of antiviral immunotoxins have been constructed using either bacterial toxins (or their fragments) such as pseudomonas exotoxin A or diphteria toxin [161], and RIPs from plants (Table 4). The most commonly used RIP has been the ricin A-chain and the most studied virus the HIV. Viral proteins (gp41, gp120, or gp 160) or proteins from infected cells (CD4, CD25, or CD45RO) have been selected as targets. Despite the success of highly active antiretroviral therapy (HAART), antiviral immunotoxins continue to be developed in order to deplete persisting HIV-infected cell reservoirs [162]. Immunotoxins have also shown to be active in vitro against Epstein–Barr [163,164] and Pichinde [31] viruses and in vivo (in combination with the synthetic analogue of 2- -deoxy-guanosine ganciclovir) against the murine cytomegalovirus [165].

Targeting can also be carried out by conjugating RIPs with other proteins or peptides that specifically bound to viral proteins or proteins present only in infected cells [49,166].

#### *5.3. Designed Antiviral Proteins and Nanocapsules*

RIPs have also been used to design antiviral proteins. One of these engineered proteins contains an internal sequence that is recognized by the HIV protease and that is blocking the N-glycosylase activity of the RIP. This protein is activated in infected cells and has shown antiviral activity [28]. Similarly, variants of the ricin A-chain with the sequence recognized by the HIV protease in the C-terminus are activated in infected cells and show antiviral activity [29].

Another approach is to fuse the sequences of RIPs with antimicrobial peptides such as latarcin, thanatin, protegrin-1, and plectasin that are able to inhibit viral replication inside the infected cells, viral entry and replication, dengue NS2B-NS3 serine protease, and virus replication, respectively [42,53]. The aim is to target different stages of the viral life cycle. Thus, the peptide-fusion proteins Latarcin-PAP1-Thanatin and Protegrin1-MAP30-Plectasin inhibit virus replication in vitro and protect the virus-infected mice from chikungunya and dengue viruses, respectively [42,53]. Another fusion protein containing ricin A-chain and PAP-S displays antiviral activity in vitro against hepatitis B virus suggesting a synergistic activity of both proteins [167]. This has encouraged its authors to propose it as an anti-SARS-CoV-2 agent [75].


**Table 4.** Ribosome-inactivating proteins used in experimental antiviral therapy. RIPs have been used alone, PEGylated, or as part of immunotoxins, conjugates, engineered proteins, or nanocapsules.

Virus name abbreviations: CHIKV (chikungunya virus), DENV (dengue virus), EBV (Epstein–Barr virus), HBV (hepatitis B virus), HIV (human immunodeficiency virus), HSV (herpes simplex virus), MCMV (murine cytomegalovirus), PICV (Pichinde virus), SHIV (simian– human immunodeficiency virus). RIP name abbreviations: MAP (Momordica antiviral protein), α-MMC (alpha-momorcharin), PAC (Pulchellin A-chain), PAP (pokeweed antiviral protein), RAC (ricin A-chain), TCS (trichosanthin).

> The latest approach is the use of nanocapsules to deliver RIPs to virus-infected cells. Nanocapsules are vesicular objects in which the encapsulated compound is confined in an internal cavity surrounded by an outer membrane [182,183]. Nanocapsules containing MAP30 [180] or ricin A-chain [181] have shown antiviral activity in vitro against HIV. In the latter case, targeting has been achieved by using peptide crosslinkers that are sensitive to cleavage by HIV-1 protease [181].

#### *5.4. Side Effects of RIP Therapy*

Although trichosanthin was, in general, well tolerated in clinical trials when used in AIDS patients [157], some side effects were reported [61,157,158]. Clinical trials using RIPs as antivirals are scarce, but there are many clinical trials that have used RIPs as part of immunotoxins for the treatment of malignancies [9,64,184]. Side effects that may be mild or moderate like fever, nausea, vomiting, diarrhea, myalgia, edema, and hypoalbuminemia have been reported in these trials. Other effects are severe, such as immunogenicity and vascular leak syndrome (VLS), and could limit the therapeutic use of immunotoxins [64,184]. Immunogenicity may be the result of the formation of human anti-mouse antibodies (HAMA) or human anti-toxin antibodies (HATA). These antibodies can prevent repeated treatment cycles. The development of immunotoxins containing humanized antibodies or the use of part of antibodies containing only the variable domains can solve this problem [64,184]. To address the problem of the immunogenicity of RIPs, PEGylation [40,184] and elimination of epitopes through genetic manipulation have been used [184]. Vascular leak syndrome, characterized by increased vascular permeability, is caused by the nonspecific binding of RIP to vascular endothelial cells. The identification and elimination of some peptides present in RIPs, nonessentials for RIP activity and responsible for this unspecific binding, have allowed the obtaining of less toxic recombinant RIPs [184].

#### **6. Genetically Engineered Virus-Resistant Plants**

Viruses cause epidemics in all major crops, representing a significant restriction on the yield and quality of agricultural production. As strict intracellular pathogens, they cannot be chemically controlled and prophylactic measures consist mainly in the destruction of infected plants and biocide applications to limit the population of vector organisms (arthropods, nematodes, and plasmodiophorids). A powerful alternative often used in agriculture is based on the use of crop genetic resistances, an approach that depends on mechanisms governing plant-virus interactions [185]. Several transgenic plants carrying virus resistance genes have been obtained by transferring virus-derived genes, including viral coat proteins, replicases, movement proteins, defective interfering RNAs, non-coding RNA sequences and proteases into susceptible plants, or non-viral genes including R genes, microRNAs, RIPs, protease inhibitors, dsRNAses, RNA modifying enzymes, and scFvs [186]. In recent years, transgenic plants carrying RIP genes that are resistant to fungi, insects and, above all, viruses have been reported. Thus, transgenic plants bearing RIP genes have been obtained that are resistant to a wide variety of viruses (Table 5).

**Table 5.** Transgenic plants bearing RIP genes. The degree of protection achieved is indicated as the percentage reduction of lesions, infected plants or detected virus levels, or as the delay in the onset of symptoms.


Virus name abbreviations: ACMV (African cassava mosaic virus), CMV (cucumber mosaic virus), PLRV (potato leafroll virus), PVX (potato virus X), PVY (potato virus Y), TEV (tobacco etch virus), TMV (tobacco mosaic virus), TuMV (turnip mosaic virus). Protection abbreviations: L.L. (less lesions), D.D. (days of delay), L.I.P. (less infected plants), L.V.L. (less virus level).

> Most of the times, tobacco has been transformed (*Nicotiana tabacum* L. and *N. benthamiana* Domin) but also potato (*Solanum tuberosum* L.) and tomato (*Lycopersicon esculentum* Mill.). *Agrobacterium tumefaciens* containing the plant transformation vectors has been used to transform either tobacco by the leaf disc co-cultivation method or potato (*S. tuberosum*) by the stem or tuber section co-cultivation method. The CaMV 35S promoter has always been used to express the RIPs, except in the case of dianthin 30 [84]. In the case of trichosanthin, tissue-specific promoters have also been used [80]. The CaMV 35S promoter is the most studied and most widely used plant promoter for transgenic expression [189], it is a very strong constitutive promoter that facilitates a high level of RNA transcription in a wide variety of plant species. For effective protection against viruses, it is preferable to achieve high levels of RIP expression since there is a direct correlation between expression level and

resistance to viruses [78]. So, for example, in lines expressing small amounts of curcin 2, symptoms of TMV infection begin to appear after about 7 days, while lines that accumulate the highest level of curcin 2 (about 1.45 μg/mg) begin to develop symptoms after about 18 days.

Using the promoter CaMV 35S, plants with a RIP content of up to 2.7% of the total soluble protein have been obtained [80]. However, a high expression of RIP results in plants with an aberrant phenotype, which usually includes leaf mottling, extreme leaf discoloration, stunted leaf growth and/or excessive curvature, slow rooting and growth rates, and high plant mortality rates [80,188]. This could be because some RIPs can kill plant cells by inactivating their ribosomes [120–122]. Several approaches have been used to overcome this problem. One strategy might be to introduce the gene encoding for the preprotein [80], this allows the RIP to accumulate in the apoplasma instead of the cytosol, thus preventing access to the ribosomes. Transgenic tobacco plants expressing the preprotein of trichosanthin exhibited resistance to cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) but did not show an abnormal phenotype [80]. In the case of PAP, despite being the most widely used, it inhibits protein synthesis and is toxic to plant cells, but transgenic plants have been obtained with mutants that are not toxic to the plant maintaining the antiviral activity [188]. The lack of toxicity of these mutants has been attributed to a change in the location of the protein preventing contact with ribosomes [188]. PAP (PAPI) has also been replaced by PAPII in order to obtain virusresistant plants [104]. The protein sequence of PAPII shows only 41% identity to PAPI. PAPII expressed in transgenic tobacco was correctly processed to the mature form and accumulated to at least 10-fold higher levels than wild-type PAP (up to 250 ng/mg PAPII). PAPII is less toxic than PAP and symptomless transgenic lines expressing PAPII were resistant to TMV and PVX [104]. Another approach is to use a promoter that is induced by viral infection, thus, the gene that encodes for dianthin 30 was introduced into *N. benthamiana* and expressed from the promoter ACMV virion-sense [84]. This promoter is induced specifically by the ACMV infection and transgenic plants displayed a normal phenotype and were resistant to ACMV [84].

Finally, it should be noted that some virus-resistant transgenic plants have been reported to be also resistant to fungi [78,104], which adds interest to this type of approach to improve crop resistance.

#### **7. Conclusions**

After decades of research, RIPs continue to be a topic of interest and a useful tool in many research fields. The new advances in plant molecular biology, virology, immunotherapy, and nanotechnology open new possibilities in the use of RIPs in medicine and agriculture in order to find solutions to the continuous challenge posed by viruses to human health and crop yields.

**Author Contributions:** Conceptualization, L.C. and J.M.F.; writing—original draft preparation, R.I. and J.M.F.; writing—review and editing, L.C. and R.I.; funding acquisition, J.M.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Consejería de Educación (Junta de Castilla y León) to the GIR ProtIBio, grant number VA033G19.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are available upon request. Please, contact the contributing authors.

**Conflicts of Interest:** The authors declare no conflict of interest.

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