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, ␣-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.Pokeweed antiviral protein (PAP), 1 a single chain ribosomeinactivating protein (RIP) isolated from the leaves of pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, ␣-sarcin/ricin (S/R) loop in the large rRNA (1-3). The enzymatic removal of specific purines from the S/R loop has been reported to interfere with the binding of eEF-2 (elongation factor 2) and inhibit protein synthesis at the translocation step (4, 5). RIPs are protein toxins produced by organisms ranging from bacteria to plants. Because of their selective toxicity, they have been used as biological weapons, to protect plants against pathogens, and as therapies against cancer. Their biological function in the organisms that produce them is unknown. PAP is thought to be a defense protein because it is a potent inhibitor of animal and plant viral pathogens, including human immunodeficiency virus, poliovirus, herpes simplex virus, influenza, potato virus X, and brome mosaic virus (6 -10). Because of its cytotoxicity to dividing cells, PAP is currently und...

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP.The PAP cDNA was placed under control of the galactoseinducible GAL] promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast.The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.Pokeweed antiviral protein (PAP), a ribosome-inactivating protein (RIP) isolated from the leaves or seeds of Phytolacca americana, catalytically removes a specific adenine residue from a highly conserved stem-loop structure in the 28S rRNA of eukaryotic ribosomes (1, 2). This lesion interferes with elongation factor 2 binding and blocks protein synthesis. PAP was discovered when pokeweed extracts were found to inhibit the transmission of tobacco mosaic virus and was subsequently demonstrated to be equally effective against a number of other plant viruses (3). Studies that compare the relative antiviral properties of a number of RIPs showed that all of the RIPs tested had antiviral activity, but none was as effective as PAP (4). PAP has been shown to inhibit infection of both Vero and HeLa cells by herpes simplex virus (5) and to inhibit human immunodeficiency virus 1 (HIV-1) replication in T cells and macrophages infected in vitro at concentrations that do not inhibit cellular protein synthesis (6). A number of recent studies have shown that conjugating PAP with monoclonal antibodies dramatically increases its potency against cells infected with HIV and human cytomegalovirus (7). In addition, PAP has been used as the cytotoxic moiety of immunotoxins against acute lymphoblastic leukemia and PAPcontaining immunotoxins have shown significant antileukemic activity in clinical trials (7).Single-chain RIPs (type I RIPs), like PAP, are poorly characterized at the molecular level compared to the type II RIP, ricin, which consists of an enzymatically active...

Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives.

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