Pokeweed antiviral protein (PAP), a 29-kDa antiviral protein isolated from Phytolacca americana, catalytically removes a specific adenine residue from the alpha-sarcin loop 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, including influenza virus, poliovirus, herpes simplex virus, and HIV. While the mechanism underlying the catalytic activity of PAP has been determined, much less is known about how PAP gains access to its ribosomal target and the role other proteins play in this process. Research from these laboratories has focused on characterizing the mechanism of cytotoxicity and antiviral activity of PAP. Recently, it has been demonstrated that expression of PAP in S. cerevisiae leads to specific inhibition of frameshifting in the +1 direction and significantly inhibits retrotransposition of the yeast retrovirus Ty1. These results suggested that PAP can be used as a specific probe to dissect the mechanisms regulating programmed ribosomal frameshifting and as a general tool to study the elongation phase of protein synthesis. The primary objective of this project is to use recombinant PAP mutants and yeast cellular mutants to investigate the mechanism by which PAP gains access to the ribosome and inhibits +1 frameshifting in S. cerevisiae. These studies will enhance our basic understanding of the mechanism of action of PAP and elucidate the role of PAP in translational reading frame maintenance and virus propagation.
This project will use Pokeweed Antiviral Protein (PAP), a plant protein that modifies the ribosome and thereby affects cellular protein synthesis, as a tool to study a phenomenon known as ribosomal frameshifting. Ribosomal frameshifting is a process by which messenger RNA, which contains information defining the sequence of amino acids in a newly synthesized protein, slips in the protein synthesis machinery, so that different three letter words are read. The project will characterize the structure of PAP that is responsible for this activity and examine other cellular proteins that may be involved. This project will increase our understanding of ribosomal frameshifting, which is required for the replication of some viruses. It may also increase our understanding of translation elongation, the process by which the three letter words are read and amino acids are added to the newly synthesized protein. Finally, it could lead to therapeutic or antiviral strategies for animals and plants.
