Abstract
Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human 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 genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.
Highlights
Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that depurinates RNA and inhibits protein synthesis
VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding
To examine the extent to which PAP discriminates between capped and uncapped RNA transcripts, a synthetic S/R oligonucleotide RNA, tobacco etch virus (TEV) RNA, and luciferase mRNA were used as substrates for PAP enzymatic activity
Summary
PAP is a ribosome-inactivating protein that depurinates RNA and inhibits protein synthesis. PAP inhibits the in vitro translation of brome mosaic virus and potato virus X RNAs without ribosomal depurination [19] by binding to the cap structure and depurinating the RNA This may be the primary mechanism for PAP antiviral activity [20]; it does not clarify the inhibitory effects of PAP on the replication of uncapped viruses, such as influenza [17] and poliovirus [15]. The effect of VPg on the depurination of selected RNA molecules, including structured RNA derived from TEV [33,34,35], showed that VPg decreases depurination of RNAs and competes with IRES containing TEV RNA for PAP binding These findings correlate with the inhibition of PAP enzymatic activity by VPg in wheat germ lysate. Depurination inhibition by VPg may confer an advantage for viral replication, and may be a novel mechanism to overcome plant defenses
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