POKEWEED ANTIVIRAL PROTEIN INACTIVATES POKEWEED RIBOSOMES - IMPLICATIONS FOR THE ANTIVIRAL MECHANISM

Pokeweed antiviral protein (PAP) and other ribosome-inactivating prote ins (RIPs) had previously been thought to be incapable of attacking co nspecific ribosomes, thus having no effect on endogenous processes. Th is assertion conflicts with a model for PAP's in vivo antiviral mechan ism in which PAP (a cell wall protein) selectively enters virus-infect ed cells and disrupts protein synthesis, thus causing local suicide an d preventing virus replication. We show here that pokeweed (Phytolacca americana) ribosomes, as well as ended (Phytolacca dodecandra) riboso mes, are indeed highly sensitive to inactivation by conspecific RIPs. Ribosomes isolated from RIP-free pokeweed and ended 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 PA P or dodecandrin) very similar to those from wheat germ ribosomes. To determine if Phytolacca cells produce a cytosolic 'anti-RIP' protectiv e element, ribosomes were combined with Phytolacca postribosomal super natant factors from culture cells, then challenged with conspecific RI Ps. Resulting IC50 values of 3-7 nM PAP, PAP-II, PAP-S or dodecandrin indicate that supernatants from these Phytolacca cells lack a ribosoma l protective element. This research demonstrates that PAP inactivates pokeweed ribosomes (and is therefore potentially toxic to pokeweed cel ls) and supports the local suicide model for PAP's in vivo antiviral m echanism. The importance of spatial separation between PAP and ribosom es of cells producing this RIP is emphasized, particularly if crop pla nts are transformed with the PAP gene to confer antiviral protection.