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

Abstract

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 dose-response 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.