Mechanism of Toxicity and Antiviral Activity of Pokeweed Antiviral Protein

Tumer 9982498 Pokeweed antiviral protein (PAP), a 29-kDa ribosome inactivating protein (RIP) isolated from Phytolacca americana, catalytically removes a specific adenine residue from the highly conserved, surface-exposed a-sarcin/ricin loop (S/R) in the large rRNA of eukaryotic and prokaryotic ribosomes. Ribosomes depurinated in this manner are unable to bind the EF-2/GTP complex and protein synthesis is blocked at the translocation step. PAP displays broad-spectrum antiviral activity against plant and animal viruses. This laboratory recently demonstrated that PAP accesses ribosomes by binding to the ribosomal protein L3. Yeast cells expressing a mutant form of L3 are resistant to the cytostatic effects of PAP because PAP cannot interact with the mutant L3 in vivo. These results showed for the first time that a ribosomal protein provides a receptor site for a RIP and allows optimal binding to the target adenine. Recent evidence from this laboratory indicates that PAP mutants which do not depurinate rRNA inhibit translation of viral RNAs, indicating that the antiviral activity of PAP is not solely due to rRNA depurination. Translation of capped, but not uncapped RNAs are inhibited by PAP and translation inhibition is overcome by increasing concentrations of the cap analog, m7GpppG, indicating that PAP recognizes the cap structure. The main objective of this project is to investigate the mechanism of the interaction between PAP and the cap structure to determine if PAP binds and depurinates capped RNAs in vitro and in vivo and whether PAP inhibits viral infection by depurinating capped RNAs. While ribosome inactivating proteins (RIPs) have been studied for many years and their enzymatic activity has been extensively investigated, very little is known about their substrate specificity and how they recognize their substrates in vivo. This laboratory recently demonstrated that PAP accesses yeast ribosomes by binding to ribosomal protein L3. These studies provided new insights towards understanding the roles of ribosomal proteins in translation elongation and ribosome structure and function. Preliminary studies indicate that PAP recognizes the cap structure on viral and cellular RNAs, providing the first evidence for interaction between PAP and substrates other than rRNA. This research will investigate the mechanism of the interaction between PAP and the cap structure and the role of this interaction in virus resistance. Analysis of the interaction between PAP and the cap structure will bring new insights into this unusual mechanism of translation inhibition and virus resistance. These studies may pave the way for the development of more effective agents against viral infection in agriculture and medicine.