Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5053-5056, July 1986 Biochemistry Extracellular localization of pokeweed antiviral MICHAEL P. READY*, DENNIS T. BROWNt, AND JON D. ROBERTUS** *Clayton Foundation Biochemical Institute, Department of Chemistry, and tCell Research Institute and Department of Microbiology, University of Texas, Austin, TX 78712 Communicated by Esmond E. Snell, March 24, 1986

ABSTRACT Pokeweed antiviral protein is an enzyme of molecule may well be inactive until it is processed and Mr 29,000 known to inactivate a wide variety of eukaryotic packaged in the seed. ribosomes. We have used electron microscopy to show that the However, the case is not so clear for such as the specific for the protein is bound within the wall pokeweed enzyme, which are not cytotoxic to animals. In matrix of leaf mesophyll cells from Phytolacca americana. Any addition, reports have suggested that pokeweed antiviral penetration or breakage of the cell wall and membrane could protein does not inhibit protein synthesis on pokeweed allow the enzyme to enter the cytoplasm, where it is likely to ribosomes (19, 20). If this were true, it would mean that inhibit protein synthesis in the damaged cell. We speculate that pokeweed could not shut down its own ribosomes if they pokeweed antiviral protein is a defensive agent whose principal were usurped by an invading . Recently we speculated 'function is probably antiviral. (5) that this state of affairs is unlikely; a protein that makes up as much as 0.5% of the plant's soluble protein and that Many higher plants contain proteins that enzymatically attacks ribosomes with a Kcat of 400 mol/mol per min must inhibit protein synthesis on eukaryotic ribosomes. These exist to inhibit protein synthesis and probably inhibits its own proteins fall into two main classes. One class contains protein synthesis under some conditions. In this communi- heterodimers of the form AB, where the B chain recognizes cation we show that pokeweed antiviral protein is heavily cell-surface receptors and mediates protein uptake and the A sequestered in the cell wall of pokeweed cells. The isolation chain attacks and inactivates large ribosomal subunits. Ex- of the enzyme outside the cytoplasm is consistent with the amples of these true cytotoxins are (1), abrin (2), and antiviral hypothesis for its action. A breach in the cell, as modeccin (3). required for many virus infections, could allow entrance of The second major class of ribosome-inhibiting proteins the protein into the cytoplasm. Ifpokeweed antiviral protein contains enzyme monomers ofMr -30,000. Their mechanism is capable of inhibiting pokeweed ribosomes, this inhibition ofaction appears to be very similar to that ofthe heterodimer would block . A chains; lacking a B-chain carrier, however, this second group of proteins is only slightly cytotoxic. Examples of this MATERIALS AND METHODS class are pokeweed antiviral protein (4), dodecandrin (5), and gelonin (6). Pokeweed Antiviral Protein-Sepharose 4B Afflnity Column. Recently we have shown that single-chain proteins such as Five grams of CNBr-activated Sepharose 4B (Sigma) was pokeweed antiviral protein are evolutionarily related to the A swollen on a sintered glass filter with five 200-ml aliquots of chains ofricin and modeccin (7). This comparison was based 1 mM HCL. The gel was then rinsed with 85 ml of coupling largely on amino-terminal sequence homologies but was also buffer (10 mM NaHCO3, pH 8.3/500 mM NaCl) and trans- consistent with kinetic comparisons (8). The relative degree ferred to a flask containing 100 mg of pokeweed antiviral of sequence similarity was also consistent with the presence, protein in coupling buffer. The flask was incubated overnight degree, or absence of immunological crossreactivity (5, at 40C with shaking. The gel was recovered on a sintered glass 9-11). We have also shown that the toxin B chain has had a filter and washed with 40 ml of coupling buffer and then separate evolutionary history (12) and was subsequently transferred to a flask containing 100 ml of 0.2 M glycine (pH joined to an inhibitor gene to produce the class of 8.0) and shaken overnight at 40C to block unoccupied heterodimeric cytotoxins. coupling sites. The physiological role of these proteins is still somewhat The gel was again recovered on sintered glass and washed controversial, although the heterodimeric proteins are prob- four times with alternating 75-ml aliquots of coupling buffer ably defensive. Several laboratories have shown that ricin, and 100 mM NaOAc, pH 4.0/500 mM NaCl. The gel was then although a powerful inhibitor of animal ribosomes, is a resuspended in 10 mM Hepes/NaOH, pH 7.7/150 mM NaCl, relatively weak inhibitor of plant ribosomes (13, 14). Harley packed in a Pharmacia K9/30 column, and washed with and Beevers (14) demonstrated that ricin is roughly 20,000 several changes of the same buffer. times more active against rat ribosomes than against Antibody Preparation. Whole IgG was prepared from the ribosomes from five plants, including castor bean (Ricinus serum of pokeweed antiviral protein-inoculated rabbits by communis) from which the toxin is derived. This is consistent the method of ref. 8. Two hundred fifty milligrams of with the notion that ricin is a defensive agent against animal antibody, dialyzed into 10 mM Hepes/NaOH, pH 7.7/150 seed predators. Ricin is synthesized as a single polypeptide, mM NaCl, was applied to the Sepharose 4B column equili- cleaved by posttranslational processing (15), and then pack- brated in the same buffer. The column was washed with aged in the endosperm (16, 17). Processing may play a role in buffer until the A2,0 of the effluent returned to the baseline; preventing self-inhibition. Indeed, although ricin is far less the column was then eluted with 10 mM sodium formate (pH active toward plant than animal ribosomes, physiologically 2.5). Three-milliliter fractions were collected in tubes con- reasonable levels of purified soluble enzyme do severely taining 1 ml of 0.5 M Hepes/KOH at pH 8.0. Twenty-nine inhibit growth of castor seedlings (18). Thus, the precursor milligrams of protein was recovered. IgG was precipitated from the eluate by addition of The publication costs ofthis article were defrayed in part by page charge ammonium sulfate to 37.5% saturation and pelleted by payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

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FIG. 1. Localization of pokeweed antiviral protein in thin sections of pokeweed mesophyll cells. Sections of cells were stained with rabbit anti-pokeweed antiviral protein, followed by ferritin-conjugated goat anti-rabbit IgG. V, vacuole; C, cytoplasm; Ch, chloroplast; W, cell wall; I, calcium oxalate inclusion. Note heavy ferritin binding in the wall matrix. The low contrast seen in these sections (here and in Fig. 2) is a result of the fact that these tissues were not treated with osmium tetroxide prior to embedding and are not poststained with lead or uranium salts. centrifugation. The pellet was resuspended in and exhaus- merized by UV exposure at 40C. The resulting blocks were tively dialyzed against phosphate-buffered saline/1 mM trimmed so that thin cross sections of the leaves could be cut MgOAc2 for a final concentration of 9.4 mg/ml. from areas close to the center ofthe disc. Unstained sections Immunoelectrophoresis (21) of the purified antibody were first floated on a solution of 1% bovine serum against pokeweed extract exhibited a single precipitin band. in distilled water (to reduce nonspecific binding) and all Purity was further tested by immunoblot analysis, modified subsequent treatments were done with reagents containing from the method of Barinaga et al. (22). Varying levels of 1% . Sections were next floated for 30 pokeweed extract (10-200 pug of protein) were subjected to min on a 1:100 dilution of anti-pokeweed antiviral protein NaDodSO4/PAGE and then transferred nonelectrophoreti- prepared as described above, washed with 1% bovine serum cally to nitrocellulose by using 25 mM Tris/192 mM albumin, and floated on a 1:200 dilution offerritin-conjugated glycine/20% methanol, pH 8.3, as the transfer buffer. The goat anti-rabbit IgG (Cappel Laboratories, Cochranville, PA) blot was blocked with bovine serum albumin as in ref. 22 and for 30 were then extensively washed with 1% then incubated with affinity-purified rabbit anti-pokeweed min. They antiviral protein followed by incubation with fluorescein bovine serum albumin and finally with distilled water prior to isothiocyanate-labeled goat anti-rabbit IgG. Examination of electron microscopy. Control sections were processed in an the blot under long-wave UV light showed a single band in identical manner, except that they were not exposed to each lane ofpokeweed extract in the same position as control anti-pokeweed antiviral protein. All specimens were photo- lanes of purified enzyme. graphed without heavy metal staining in a JOEL 100 CX Electron Microscopy. Four-milliliter discs were removed electron microscope. from the center of the youngest leaves of a pokeweed plant by using a cork borer and were deposited directly into cold RESULTS AND DISCUSSION phosphate-buffered saline, pH 7.2/1.5% gluteraldehyde and submerged in the fixative for 2 hr. The tissues were washed Fig. 1 is an electron micrograph of thin-sectioned pokeweed in distilled water, transferred through a graded alcohol series mesophyll cells that have been treated with rabbit anti- consisting of 20%, 40%, 60%, 80%, 90%, and 95% ethanol at pokeweed antiviral protein and ferritin-labeled goat anti- 40C, and finally dehydrated in 100% ethanol. The specimens rabbit as described in Materials and Methods. Fig. 2 is a were embedded in Lowicryl K4M by using the procedure control micrograph of cells under essentially identical con- outlined by Kellenberger et al. (23). The plastic was poly- ditions but not treated with anti-pokeweed antiviral protein. Downloaded by guest on October 1, 2021 Biochemistry: Ready et al. Proc. Natl. Acad. Sci. USA 83 (1986) 5055

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FIG. 2. Control micrograph for Fig. 1. Pokeweed mesophyll cells were stained with ferritin-conjugated goat anti-rabbit IgG without prior exposure to rabbit anti-pokeweed antiviral protein. Key, as in Fig. 1. Note the absence of ferritin binding in the cell wall.

The mesophyll cell wall has a natural granular appearance The conclusion to be drawn from these micrographs is that under the electron microscope in the absence of ferritin pokeweed antiviral protein is synthesized in the cytoplasm binding (Fig. 2). This granularity is distinct, however, from and extruded across the cellular membrane into the cell wall the clear electron density and uniform size of the ferritin matrix. It is found in high density along the entire circum- molecule; this can readily be seen by comparison with the ference of the wall and surrounds each cell in a pokeweed nonspecifically bound material in the vacuole of Fig. 1. The antiviral protein envelope. The enzyme-containing band is at greatly enhanced particulate definition and enhanced elec- least 1600 nm wide, suggesting that the protein is not attached tron density of the wall seen in Fig. 1 are typical of that to the wall but is trapped within the cell wall matrix. We have expected after large amounts of ferritin binding. Pokeweed no idea how the protein is held there, but the association is antiviral protein, as measured by ferritin binding, is densely probably weak, as pokeweed antiviral protein can readily be concentrated outside the cytoplasmic membrane in the cell obtained from water extracts of macerated leaf tissue. Prec- wall. A very light background of ferritin is also seen in the edents exist for this sort of arrangement; for example, cytoplasm and cellular vacuole. It is unclear whether. this soybean cell walls contain the enzyme 3-glucosylase I. This represents binding to the enzyme, some minor crossreaction, protein is readily extractable into acetate buffer from wall or residual nonspecific binding of the second antibody. This fragments and is not associated with the inner cell membrane nonspecific association is not seen when the ferritin-con- (24). The loose association of the antiviral protein with jugated second antibody alone is applied to the sections (Fig. pokeweed walls may account for some of the redistribution 2), indicating that this apparently random binding is not due ofthis material during preparation for electron microscopy as to an association of ferritin with the plastic. If this random suggested above. The embedding compound used in these association is due to pokeweed antiviral protein, as seems studies maintains many of the characteristics of proteins in likely, it remains unclear whether this is an alternate location their natural hydrated state. It is possible that pokeweed for the protein or represents contamination from cell walls antiviral protein is released from the wall of the thin section broken during sample preparation. It is very clear, however, as it floats on water after sectioning. A reassociation of this that the ferritin-binding sites are most dense in the cell wall, eluted material with the section may account for its appear- and it seems very likely that the knife used in preparing the ance in low concentration in vacuoles (Fig. 1) and in areas of ultrathin sections could drag material from these walls across the section not containing tissue (not shown). the section. An important question concerns the natural role of Almost no ferritin is visible in Fig. 2, even though the pokeweed antiviral protein in pokeweed leaves. It has been needle-like calcium oxalate inclusions are in focus in both known for many years that the enzyme strongly inhibits micrographs. transmission of in a variety of plants (25) and in Downloaded by guest on October 1, 2021 5056 Biochemistry: Ready et al. Proc. Natl. Acad. Sci. USA 83 (1986) animal cell cultures (26). For example, pokeweed antiviral not, however, guarantee resistance to virus that can enter the protein inhibits cucumber mosaic virus infection of Cheno- cell without need of a gap in the membrane, although we do podium quinoa; presumably it enters the cytoplasm along not know of any such specific virus. with the virus and, once inside, shuts down protein synthesis and prevents viral replication (25). There is evidence that the We thank Ms. Terrie Kolvoord and Ms. Vivian Benningfield for protein actually binds weakly to some virus proteins, perhaps their assistance in preparing this manuscript. This work was sup- by salt linkages (26), and may actually be carried into the ported by Grant GM30048 (to J.D.R.) from the National Institutes of cytoplasm by the invader. Health. To act as an antiviral agent for pokeweed, pokeweed 1. Olsnes, S & Pihl, A. (1973) Biochemistry 12, 3121-3126. antiviral protein would have to inhibit protein synthesis on its 2. Olsnes, S. & Pihl, A. (1973) Eur. J. Biochem. 35, 179-185. own ribosomes. Owens et al. (19) presented evidence that the 3. Gasperi-Campani, A., Barbieri, L., Lorenzoni, E., enzyme does not inactivate pokeweed ribosomes. More Montanaro, L., Sperti, S., Bonetti, E. & Stirpe, F. (1978) recently, Battelli et al. (20) could find no evidence for Biochem. J. 174, 491-496. inhibition ofpokeweed ribosomes by the closely related seed 4. Irvin, J. D. (1975) Arch. Biochem. Biophys. 169, 522-528. protein PAP-S (pokeweed antiviral protein from seeds). In 5. Ready, M. P., Adams, R. P. & Robertus, J. 1). (1984) both cases, however, protein synthesis as measured by 14C Biochim. Biophys. Acta 791, 314-319. incorporation was extremely low, comparable to that pro- 6. Stirpe, F., Olsnes, S. & Pihl, A. (1980) J. Biol. Chem. 225, 6947-6953. duced by wheat germ ribosomes after inhibition by pokeweed 7. Ready, M., Wilson, K., Piatak, M. & Robertus, J. D. (1984) J. antiviral protein. We have made several attempts to isolate Biol. Chem. 259, 15252-15256. active pokeweed ribosomes; in all cases, these ribosomes 8. Ready, M. P., Bird, S., Rothe, G. & Robertus, J. D. (1983) yielded only background levels of synthesis. Biochim. Biophys. Acta 740, 19-28. Attempts to isolate active pokeweed ribosomes in the 9. Irvin, J. D., Kelly, T. & Robertus, J. D. (1980) Arch. Biochem. presence of antibody have also failed, probably because we Biophys. 200, 418-425. lacked sufficient antibody to neutralize the vast amount of 10. Barbieri, L., Aron, G. M., Irvin, J. D. & Stirpe, F. (1982) enzyme in the tissue. We feel that the simplest explanation Biochem. J. 203, 55-59. for these observations is that breakage of the cells during the 11. Houston, L. L., Ramakrishnan, S. & Hermodson, M. A. (1983) J. Biol. Chem. 258, 9601-9604. isolation process could allow the protein to gain access to the 12. Robertus, J. D. & Ready, M. P. (1984) J. Biol. Chem. 259, ribosomes and inhibit them. Ribosomes attacked by 13953-13956. pokeweed antiviral protein retain some slight activity (8), so 13. Cawley, D. B., Hedblom, M. L., Hoffman, E. J. & Houston, we should expect some protein synthesis from intoxicated L. L. (1977) Arch. Biochem. Biophys. 182, 690-695. pokeweed ribosomes. However, the ribosomes thus isolated 14. Harley, S. M. & Beevers, H. (1982) Proc. Nati. Acad. Sci. would be insensitive to further inhibition by pokeweed USA 79, 5935-5938. antiviral protein or PAP-S. 15. Butterworth, A. G. & Lord, J. M. (1983) Eur. J. Biochem. 137, Ifpokeweed ribosomes are sensitive to pokeweed antiviral 57-65. protein, then clearly the cell must take steps to protect its 16. Youk, R. J. & Huang, A. H. C. (1976) Plant Physiol. 58, 703-709. ribosomes during synthesis of this enzyme. We have shown 17. Tully, R. E. & Beevers, H. (1976) Plant Physiol. 58, 710-716. that the enzyme is an exported protein. Proteins destined for 18. Harley, S. M. & Beevers, H. (1984) Plant Sci. Lett. 36, 1-5. export typically possess an N-terminal leader peptide that is 19. Owens, R. A., Bruening, G. & Shepherd, R. J. (1973) Virology inserted into the membrane of the . 56, 390-393. Protein synthesis occurs through the membrane into the 20. Battelli, M. G., Lorenzoni, E. & Stirpe, F. (1984) J. Exp. Bot. lumen. Folding of the nascent polypeptide and cleavage of 35, 882-889. the leader thus occur outside the cytosol (27). We have no 21. Graber, P. & Williams, C. A. (1953) Biochim. Biophys. Acta reason to believe that synthesis ofpokeweed antiviral protein 10, 193-194. does not follow the same general pathway. Indeed, the 22. Barinaga, M., Franco, R., Mienkroth, J., Ong, E. & Wahl, nucleotide sequence has been determined for cDNA coding G. E. (1981) Methods for the Transfer of DNA, RNA and the related protein ricin (28). Preproricin, the precursor Protein to Nitrocellulose and Diazotized Paper Solid Supports molecule, carries a signal peptide at its N terminus and (Schleicher & Schuell, Keene, NH). never exists as an active in 23. Kellenberger, E., Carlemalm, E., Villiger, W., Roth, J. & presumably enzyme the cytosol. Garavito, R. M. (1980) Low Denaturation Embedding for In conclusion, we feel that the evidence presented here, Electron Microscopy ofThin Sections (Chemiscle Werke Lowi although indirect, is consistent with the notion that pokeweed GmBH, Waldkraiburg, F.R.G.). antiviral protein functions as an antiviral agent. It is synthe- 24. Cline, K. & Albersheim, P. (1981) Plant Physiol. 68, 207-220. sized in an inactive form, sequestered in the cell wall matrix, 25. Thomlinson, J. A., Walker, V. M., Flewett, T. H. & Barclay, and reenters the cytoplasm, perhaps being carried in by virus, G. R. (1974) J. Gen. Virol. 22, 225-232. when the wall and membrane are breached, for example, by 26. Ussery, M. A., Irvin, J. D. & Hardesty, B. (1977) Ann. N. Y. an insect wound. Ifthe antiviral hypothesis is true, then once Acad. Sci. 284, 431-440. in the cytoplasm the enzyme would inactivate ribosomes, 27. Krell, G. (1981) Annu. Rev. Biochem. 50, 317-348. preventing viral replication and further infection. The exist- 28. Lamb, F. I., Roberts, L. M. & Lord, J. M. (1985) Eur. J. ence of pokeweed antiviral protein in the wall matrix would Biochem. 148, 265-270. Downloaded by guest on October 1, 2021