High-level expression and purification of biologically active recombinant pokeweed antiviral protein.

Pokeweed antiviral protein (PAP) from the leaves of the pokeweed plant, Phytolacca americana, is a naturally occurring single-chain ribosome-inactivating protein, which catalytically inactivates both prokaryotic and eukaryotic ribosomes. The therapeutic potential of PAP has gained considerable interest in recent years due to the clinical use of native PAP as the active moiety of immunoconjugates against cancer and AIDS. The clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of a homogenously pure and active PAP preparation with minimal batch to batch variability from its natural source. Previous methods for expression of recombinant PAP in yeast, transgenic plants and Escherichia coli have resulted in either unacceptably low yields or were too toxic to the host system. Here, we report a successful strategy which allows high level expression of PAP as inclusion bodies in E. coli. Purification of refolded recombinant protein from solubilized inclusion bodies by size-exclusion chromatography yielded biologically active recombinant PAP (final yield: 10 to 12 mg/L). The ribosome depurinating in vitro N-glycosidase activity and cellular anti-HIV activity of recombinant PAP were comparable to those of the native PAP. This expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PAP sufficient to carry out advanced clinical trials. To our knowledge, this is the first large-scale expression and purification of biologically active recombinant PAP from E. coli.     

Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC(50) of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID(50) of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.     

Pokeweed antiviral protein regulates the stability of its own mRNA by a mechanism that requires depurination but can be separated from depurination of the alpha-sarcin/ricin loop of rRNA

Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, alpha-sarcin/ricin loop in the large rRNA, resulting in inhibition of protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeast Saccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to the N-glycosidase activity of the protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs.     

Recombinant production of anti-HIV protein, griffithsin, by auto-induction in a fermentor culture

Griffithsin (GRFT) is a novel anti-HIV protein isolated from the red alga Griffithia sp. The potent anti-viral activity of GRFT against both laboratory and primary isolates of HIV at picomolar concentrations makes this protein an attractive candidate microbicide to prevent sexual transmission of HIV. Here, we describe the recombinant production and purification of a biologically active hexa-histidine-tagged GRFT (His-GRFT) from Escherichia coli. To facilitate a large-scale production of recombinant His-GRFT, we tested different expression conditions to optimize the expression in the cytoplasm of E. coli to increase the overall production of soluble His-GRFT. Attempts to express His-GRFT in shake flask cultures resulted in a modest yield of soluble His-GRFT, with a large accumulation of the protein in inclusion bodies. The use of a fermenter and of a rich, auto-inducing medium allowed the total amount of His-GRFT per liter to be increased by about 45-fold, with approximately 70% of the protein expressed in the soluble fraction. N-terminal sequencing and MALDI-TOF analyses of the recombinant His-GRFT confirmed that the initial methionine residue was cleaved off. Recombinant His-GRFT showed equivalent activity with natural GRFT, both in respect to gp120-binding characteristics as well as anti-HIV activity. Size-exclusion chromatography analysis showed that both native GRFT and recombinant His-GRFT existed as homodimers in solution. The expression system described in this work provides a basis for the mass production of GRFT to allow further studies of the protein and investigation of therapeutic and preventive strategies against HIV.     

A 13-week subchronic intravaginal toxicity study of pokeweed antiviral protein in mice.

Pokeweed antiviral protein (PAP), a 29-kDa plant-derived protein isolated from Phytolacca americana, is a broad-spectrum antiviral agent. PAP shows unique clinical potential to become the active ingredient of a non-spermicidal microbicide because of its potent in vivo anti-HIV activity, non-interference with in vivo sperm functions, and lack of cytotoxicity to genital tract epithelial cells. Over 13 weeks the subchronic and reproductive toxicity potential of an intravaginally administered gel formulation of PAP was studied in mice to support its further development as a vaginal microbicide. Female B6C3F1 and CD-1 mice in subgroups of 20, were exposed intravaginally to a gel formulation containing 0, 0.025, 0.05, or 0.1% PAP, 5 days/week for 13 consecutive weeks. On a molar basis, these concentrations are 500- to 2000-times higher than the in vitro anti-HIV IC50 value. After 13 weeks of intravaginal treatment, B6C3F1 mice were evaluated for survival, body weight gain, and absolute and relative organ weights. Blood was analyzed for hematology and clinical chemistry profiles. Microscopic examination was performed on hematoxylin and eosin-stained tissue sections from each study animal. Placebo-control and PAP-dosed female CD-1 mice were mated with untreated males in order to evaluate if PAP has any deleterious effects on reproductive performance. There were no treatment-related mortalities. Mean body weight gain was not reduced by PAP treatment during the dosing period. The hemogram and blood chemistry profiles revealed lack of systemic toxicity following daily intravaginal instillation of PAP for 13 weeks. No clinically significant changes in absolute and relative organ weights were noted in the PAP dose groups. Extensive histopathological examination of tissues showed no increase in treatment-related microscopic lesions in any of the three PAP dose groups. Repeated intravaginal exposure of CD-1 mice to increasing concentrations of PAP for 13 weeks showed no adverse effect on their subsequent reproductive capability (100% fertile), neonatal survival (>90%) or pup development. Collectively, these findings demonstrate that repetitive intravaginal administration of PAP at concentrations as high as 2000 times its in vitro anti-HIV IC50 value was not associated with local or systemic toxicity and did not adversely affect the reproductive performance of mice. PAP may be useful as an active ingredient of a safe vaginal microbicide for prevention of the sexual transmission of viruses, particularly of HIV-1.     

Production of pokeweed antiviral proteins nontoxic to cells by Mutagenesis of PAP-cDNA

Pokeweed antiviral protein (PAP), one of ribosome inactivating proteins (RIPs), has very strong toxicity both to prokaryotic and eukaryotic cells. To produce mutant PAPs nontoxic to cells, the PAP-cDNA was inserted into a yeast-E.coli shuttle vector under the control of galactose promoter, mutagenized using hydroxylamine, and transformed into yeast cells. Transformed yeast cells were selected on the uracil-deficient plate containing glucose or raffinose, and the yeast cells producing mutant PAPs nontoxic to cells were then selected on the galactose plate. Eighteen mutants were obtained by immunoblot analyses of 1,000 transformants: among them, three, ten and five mutants produced unprocessed, mature and truncated PAPs, respectively. Fourteen PAP mutants among them did not inhibit the yeast cell growth, and showed no or less inhibition of protein synthesisin vitro. Six among fourteen mutants were able to protect TMV infection in coinoculation experiment. The mutant PAPs showing an antiviral activity either without or reduced RIP activity contain neither the active site mutation nor C-terminal deletion mutation. These results suggest that both the RIP activity and the antiviral activity will require other amino acid residue(s) besides the active site and that the antiviral acitivity of PAP can be dissociated from its toxicity.     

Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris

Antifreeze proteins and antifreeze glycoproteins are structurally diverse molecules that share a common property in binding to ice crystals and inhibiting ice crystal growth. Type II fish antifreeze protein of Atlantic herring (Clupea harengus harengus) is unique in its requirement of Ca(2+) for antifreeze activity. In this study, we utilized the secretion vector pGAPZalpha A to express recombinant herring antifreeze protein (WT) and a fusion protein with a C-terminal six-histidine tag (WT-6H) in yeast Pichia pastoris wild-type strain X-33 or protease-deficient strain SMD1168H. Both recombinant proteins were secreted into the culture medium and properly folded and functioned as the native herring antifreeze protein. Furthermore, our studies demonstrated that expression at a lower temperature increased the yield of the recombinant protein dramatically, which might be due to the enhanced protein folding pathway, as well as increased cell viability at lower temperature. These data suggested that P. pastoris is a useful system for the production of soluble and biologically active herring antifreeze protein required for structural and functional studies.     

The C-terminus of pokeweed antiviral protein has distinct roles in transport to the cytosol, ribosome depurination and cytotoxicity

Pokeweed antiviral protein (PAP) produced by pokeweed plants is a single-chain (type I) ribosome-inactivating protein (RIP) that depurinates ribosomes at the alpha-sarcin/ricin loop of the large rRNA, resulting in inhibition of translation. Unlike the type II RIPs, which have an active and a binding moiety, PAP has only the active moiety. The mechanism by which toxins without a binding moiety gain access to cytosolic ribosomes is not known. We set up yeast as a simple and genetically tractable system to investigate how PAP accesses ribosomes and showed that the mature form of PAP is targeted to the cytosol from the endomembrane system in yeast. In the present study, we performed a systematic deletion analysis to identify the signal required for transport of PAP to the cytosol. We demonstrate here that processing of the C-terminal extension and sequences at the C-terminus of the mature protein are critical for its accumulation in the cytosol. Using a series of PAP mutants, we identified the C-terminal signal and demonstrated that it is distinct from the sequences required for ribosome depurination and cytotoxicity. The C-terminal motif showed sequence similarity to type II RIPs that retrotranslocate from the endoplasmic reticulum to the cytosol. These results demonstrate that a conserved sequence at the C-terminus of a type I RIP mediates its transport to the cytosol and suggest that type I and II RIPs may use a common signal to enter the cytosol.     

Inhibition of Pokeweed Antiviral Protein (PAP) by Turnip Mosaic Virus Genome-linked Protein (VPg)

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.     

Pokeweed antiviral protein isoforms PAP-I, PAP-II, and PAP-III depurinate RNA of human immunodeficiency virus (HIV)-1.

Pokeweed antiviral protein (PAP) is a naturally occurring broad-spectrum antiviral agent with potent anti-human immunodeficiency virus (HIV)-1 activity by an as yet undeciphered molecular mechanism. In the present study, we sought to determine if PAP is capable of recognizing and depurinating viral RNA. Depurination of viral RNA was monitored by directly measuring the amount of the adenine base released from the viral RNA species using quantitative high-performance liquid chromatography. Our findings presented herein provide direct evidence that three different PAP isoforms from Phytolacca americana (PAP-I from spring leaves, PAP-II from early summer leaves, and PAP-III from late summer leaves) cause concentration-dependent depurination of genomic RNA (63 to 400 pmols of adenine released per micrograms of RNA) purified from human immunodeficiency virus type-I (HIV-I), plant virus (tobacco mosaic virus (TMV), and bacteriophage (MS 2). In contrast to the three PAP isoforms, ricin A chain (RTA) failed to cause detectable depurination of viral RNA even at 5 microM, although it was as effective as PAP in inhibiting protein synthesis in cell-free translation assays. PAP-I, PAP-II, and PAP-III (but not RTA) inhibited the replication of HIV-1 in human peripheral blood mononuclear cells with IC(50) values of 17 nM, 25 nM, and 16 nM, respectively. These findings indicate that the highly conserved active site residues responsible for the depurination of rRNA by PAP or RTA are not sufficient for the recognition and depurination of viral RNA. Our study prompts the hypothesis that the potent antiviral activity of PAP may in part be due to its unique ability to extensively depurinate viral RNA, including HIV-1 RNA.     

Expression of pokeweed antiviral protein in mammalian cells activates c-Jun NH2-terminal kinase without causing apoptosis

Pokeweed antiviral protein (PAP) is a ribosome inactivating protein isolated from the pokeweed plant (Phytolacca americana L.) that exhibits broad range antiviral activity against several human viruses including HIV and influenza. This characteristic suggests that PAP may have therapeutic applications; however, it is not known whether the protein elicits a ribotoxic stress response that would result in cell death. Therefore, we expressed PAP in 293T cells and showed that the enzyme did not inhibit protein translation even though approximately 15% of the ribosomal RNA (rRNA) was depurinated. PAP expression induced the activation of c-Jun NH2-terminal kinase (JNK), which was specific to rRNA depurination, as the enzymatically inactive mutant PAPx did not affect kinase activity. Moreover, incubation of PAP-expressing cells with translation inhibitors diminished JNK activation, indicating that the signal for induction of the kinase pathway originated from ribosomes. JNK activation did not result in apoptosis as demonstrated by the absence of caspase-3 and poly(ADP-ribose) polymerase cleavage and by the lack of cell staining for morphological changes in membrane permeability. Unlike all ribosome inactivating proteins tested thus far, the stress response triggered by PAP expression did not result in cell death, which supports further investigation of the enzyme in the design of novel antiviral agents.     

Overexpression of a small medicinal peptide from ginseng in the yeast Pichia pastoris

A medicinal peptide, Gsp, which was initially extracted from the traditional medicinal herb ginseng, has potential use as a drug against diabetes. Gsp is a low molecular weight protein that we have secreted in a recombinant form from the yeast Pichia pastoris. A DNA fragment encoding four copies of the Gsp protein each separated by a basic amino acid was synthesized and inserted into the P. pastoris expression vector plasmid pPIC9. After electroporation of the resulting vector, pPIC9-Gsp, into the yeast, transformants were selected. Recombinant pre-Gsp secreted from P. pastoris had a molecular weight of 5.9 kDa and mature recombinant Gsp had a primary structure indistinguishable from native Gsp. After optimization of the culturing process, the yield of pre-Gsp reached 800 mg/L in the clarified broth. A continuous batch fermentation process was developed that allowed the same population of cells to be reutilized five times without loss of expression level. This continuous culturing process resulted in a substantial saving of both time and cost in pharmaceutical production and should be applicable to the production of other recombinant proteins in P. pastoris.     

黑曲霉葡萄糖氧化酶基因的克隆及其在酵母中的高效表达

将黑曲霉葡萄糖氧化酶(GOD)基因重组进大肠杆菌酵母穿梭质粒Ppic9,转化甲基营养酵母Pichia pastoris GS115,构建出GOD的高产酵母工程菌株。在酵母αFactor及AOX1基因启动子和终止信号的调控下,黑曲霉GOD在甲基酵母中大量表达并分泌至胞外,经甲醇诱导3～4d,发酵液中的GOD活力可达30～40u/mL。SDS-PAGE证实GOD在培养物上清中的含量显著高于其它杂蛋白,约占胞外蛋白总量的60%～70%,经Q SepharoseTMFast Flow离子交换柱一步纯化即达电泳纯。重组酵母GOD比活达426.63u/mg蛋白,是商品黑曲霉GOD的1.6倍。动力学性质分析表明,重组酵母GOD的K_m和K_cat分别为38.25mmol/L和3492.66s-1,与商品黑曲霉GOD相比,具有更高的催化效率。重组酵母GOD的高活力特性可有效提高葡萄糖传感器的线性检测范围。     

Pokeweed antiviral protein cleaves double-stranded supercoiled DNA using the same active site required to depurinate rRNA

Ribosome-inactivating proteins (RIPs) are N-glycosylases that remove a specific adenine from the sarcin/ricin loop of the large rRNA in a manner analogous to N-glycosylases that are involved in DNA repair. Some RIPs have been reported to remove adenines from single-stranded DNA and cleave double-stranded supercoiled DNA. The molecular basis for the activity of RIPs on double-stranded DNA is not known. Pokeweed antiviral protein (PAP), a single-chain RIP from Phytolacca americana, cleaves supercoiled DNA into relaxed and linear forms. Double-stranded DNA treated with PAP contains apurinic/apyrimidinic (AP) sites due to the removal of adenine. Using an active-site mutant of PAP (PAPx) which does not depurinate rRNA, we present evidence that double-stranded DNA treated with PAPx does not contain AP sites and is not cleaved. These results demonstrate for the first time that PAP cleaves supercoiled double-stranded DNA using the same active site that is required for depurination of rRNA.     

美洲商陆抗病毒蛋白的研究

美洲商陆抗病毒蛋白(pokeweed antiviral proteins.PAP)是一种具有多种生物功能活性的蛋白质,其在广谱抗病毒方面有重要的应用价值。综述美洲商陆抗病毒蛋白的分子结构与功能,抗病毒的分子机理及其在植物基因工程中的应用。     

An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. II. Cloning of the cDNA and expression.

Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP), that specifically blocks collagen-mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Condra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleotides whose sequences were derived from two short peptides from V8 digests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated containing the entire deduced amino acid sequence for LAPP. Computer analysis of the amino acid sequence predicts a peptidase cleavage site between a 21-residue pre-peptide and a mature protein of 126 amino acids. A DNA insert to express the predicted mature LAPP protein was generated by PCR amplification using phage-derived cDNA clones as a substrate. This insert encoded a fusion protein with the leader sequence of the yeast alpha mating factor and the mature LAPP cDNA. These PCR products were cloned into the yeast expression vector pKH4 alpha 2. A KEX 2 Lys-Arg endopeptidase cleavage site was placed NH2-terminal to the predicted mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its reactivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesion of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/function studies and to studies on the effects of an inhibitor of collagen-stimulated platelet aggregation in vivo.     

High‐level expression of Aspergillus niger β‐galactosidase in Ashbya gossypii

Ashbya gossypii has been recently considered as a host for the expression of recombinant proteins. The production levels achieved thus far were similar to those obtained with Saccharomyces cerevisiae for the same proteins. Here, the β‐galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2‐µm plasmids carrying the native signal sequence at higher levels than those secreted by S. cerevisiae laboratorial strains. Four different constitutive promoters were used to regulate the expression of β‐galactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant β‐galactosidase in A. gossypii, leading to 2‐ and 8‐fold higher extracellular activity than the AgGPD promoter and the heterologous promoters, respectively. In similar production conditions, the levels of active β‐galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and ～2.5 times higher than those previously reported for the β‐galactosidase‐high producing S. cerevisiae NCYC869‐A3/pVK1.1. The substitution of glucose by glycerol in the production medium led to a 1.5‐fold increase in the secretion of active β‐galactosidase by A. gossypii. Recombinant β‐galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger β‐galactosidase and recombinant β‐galactosidase produced by yeast. These results highlight the potential of A. gossypii as a recombinant protein producer and open new perspectives to further optimize recombinant protein secretion in this fungus. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:261–268, 2014     

Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli

A 2.7-kilobase fragment of DNA from Oerskovia xanthineolytica containing the gene for a beta-1,3-glucanase has been isolated and its complete nucleotide sequence determined. The sequence was found to contain two large open reading frames. Purification of the mature native enzyme and subsequent amino-terminal sequencing defined the glucanase gene in one reading frame which potentially encodes a protein of 548 amino acids. We have expressed this glucanase gene in Escherichia coli under control of the lacUV5 promoter and found the product to be secreted into the periplasm as a mature enzyme of about the same molecular weight as that of the native protein. The recombinant enzyme was purified to near homogeneity by a single step of high performance liquid chromatography. The ability of the recombinant enzyme to digest beta-glucan substrates and to lyse viable yeast cells was found to be indistinguishable from that of the native protein. Deletion of the cysteine-rich carboxyl-terminal 117 amino acids of the enzyme, which also contain two duplicated segments, abolished the lytic activity but did not significantly affect the glucanase function of the protein. The possible involvement of this domain in interaction with the yeast cell wall is discussed.     

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.