Pokeweed antiviral protein inactivates pokeweed ribosomes; implications for the antiviral mechanism

• Published in: The Plant journal : for cell and molecular biology 1994-02, Vol.5 (2), p.173-183
• Main Authors: Bonness, M.S, Ready, M.P, Irvin, J.D, Mabry, T.J
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Antiviral Agents - toxicity; antiviral properties; Biological and medical sciences; Blotting, Western; cell wall components; Cells, Cultured; Fundamental and applied biological sciences. Psychology; Generalities. Techniques. Transmission, epidemiology, ecology. Antiviral substances, control; inactivation; N-Glycosyl Hydrolases; Phytolacca americana; Phytolacca dodecandra; Phytopathology. Animal pests. Plant and forest protection; Plant Proteins - isolation & purification; Plant Proteins - toxicity; Plant viruses and viroids; Plants - drug effects; Plants - metabolism; Protein Biosynthesis - drug effects; proteins; Ribosome Inactivating Proteins, Type 1; ribosome-inactivating proteins; ribosomes; Ribosomes - drug effects; Ribosomes - metabolism; Triticum - drug effects; Triticum - metabolism
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313X.1994.05020173.x

Pokeweed antiviral protein (PAP) and other ribosome‐inactivating proteins (RIPs) had previously been thought to be incapable of attacking conspecific ribosomes, thus having no effect on endogenous processes. This assertion conflicts with a model for PAP’s in vivo antiviral mechanism in which PAP (a cell wall protein) selectively enters virus‐infected cells and disrupts protein synthesis, thus causing local suicide and preventing virus replication. We show here that pokeweed (Phytolacca americana) ribosomes, as well as endod (Phytolacca dodecandra) ribosomes, are indeed highly sensitive to inactivation by conspecific RIPs. Ribosomes isolated from RIP‐free pokeweed and endod suspension culture cells were found to be highly active in vitro, as measured by poly(U)‐directed polyphenylalanine synthesis. Phytolacca ribosomes challenged with conspecific RIPs generated doseresponse curves (IC50 of 1 nM PAP or dodecandrin) very similar to those from wheat germ ribosomes. To determine if Phytolacca cells produce a cytosolic ‘anti‐RIP’ protective element, ribosomes were combined with Phytolacca postribosomal supernatant factors from culture cells, then challenged with conspecific RIPs. Resulting IC50 values of 3–7 nM PAP, PAP‐II, PAP‐S or dodecandrin indicate that supernatants from these Phytolacca cells lack a ribosomal protective element. This research demonstrates that PAP inactivates pokeweed ribosomes (and is therefore potentially toxic to pokeweed cells) and supports the local suicide model for PAP’s in vivo antiviral mechanism. The importance of spatial separation between PAP and ribosomes of cells producing this RIP is emphasized, particularly if crop plants are transformed with the PAP gene to confer antiviral protection.