Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is a cap-binding protein, and it was suggested that it inhibits translation of RNA by binding to the 5' m⁷G cap structure of eukaryotic mRNA, and depurinating the mRNA at sites downstream of the cap structure. PAP is a potent antiviral agent against many plant, animal, and human viruses. Depurination of capped viral RNA may be the primary mechanism for PAP's antiviral activity. However, the above mechanism does not clarify the inhibitory effect of PAP on the replication of uncapped viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and Turnip mosaic virus (TuMV) genome linked protein (VPg) were investigated. VPg is important in the initiation of protein synthesis, functioning as a cap analog. VPg stimulates the in vitro translation of uncapped IRES-containing RNA and inhibits capped RNA translation in wheat germ extracts. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP-VPg interactions. PAP interacts strongly with VPg, thus the effect of VPg on the PAP catalyzed depurination of several different RNA molecules was determined to investigate whether VPg binding to PAP influences selectivity of depurination. PAP binds to and depurinates both m⁷GpppG-capped and uncapped S/R oligo nucleotide and TEV RNAs, supporting previous conclusions that the cap structure is not the only determinant for PAP depurination of RNA. VPg decreases depurination of the above capped and uncapped RNAs and competes with TEV RNA for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the defense mechanisms of the plant. Depurination inhibition of PAP by VPg also suggests the possible use of this protein against cytotoxic activity of RIPs and inhibition of their biological potency.