A novel mechanism for inhibition of translation by pokeweed antiviral protein: Depurination of the capped RNA template

Abstract

Pokeweed antiviral protein (PAP) is known to inactivate ribosomes by removal of a specific adenine from the sarcin/ ricin (S/R) loop of the large rRNA, thereby inhibiting translation. We demonstrate here that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA. Recent results indicate that the antiviral activity of PAP may not be due to depurination of host ribosomes. Using PAP mutants that do not depurinate either tobacco or reticulocyte lysate rRNA, we show that PAP inhibits translation of brome mosaic virus (BMV) and potato virus X (PVX) RNAs without depurinating ribosomes. Furthermore, translation of only capped, but not uncapped, luciferase transcripts is inhibited by PAP, providing evidence that PAP and PAP mutants are able to distinguish between capped and uncapped transcripts. Translation inhibition of BMV RNAs is overcome by treatment with PAP in the presence of increasing concentrations of the cap analog m7GpppG, but not GpppG or GTP, indicating that PAP recognizes the cap structure. Incubation of BMV RNAs or the capped luciferase transcripts with PAP results in depurination of either RNA. In contrast, uncapped luciferase transcripts are not depurinated after incubation with identical concentrations of PAP. These results demonstrate for the first time that PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and specifically depurinating the capped RNAs.

The N-Glycosidase Activity of the Ribosome-inactivating Protein ME1 Targets Single-stranded Regions of Nucleic Acids Independent of Sequence or Structural Motifs

“…Hudak et al (21,22) have proposed recently that recognition of luciferase mRNA by PAP requires the presence of a cap structure. Recognition is followed by depurination of downstream adenine residues.…”

“…Recent reports propose that PAP recognizes and binds to the cap structure of mRNAs, specifically depurinating downstream adenine residues (21,22). Based on these results, PAP may bind to capped viral RNA, subsequently depurinating viral RNAs rather than host ribosomes during the infection process (21). However, this hypothesis does not explain the activity of PAP with substrates such as rRNA or DNA that lack a cap structure (8).…”

“…In contrast, pokeweed antiviral protein (PAP) does not exhibit an absolute requirement for the tetraloop structure to exhibit enzymatic activity (18). Recent reports propose that PAP recognizes and binds to the cap structure of mRNAs, specifically depurinating downstream adenine residues (21,22). Based on these results, PAP may bind to capped viral RNA, subsequently depurinating viral RNAs rather than host ribosomes during the infection process (21).…”

Insights into the Molecular Antiviral Mechanism of Pokeweed Protein from Phytolacca americana

“…Recent findings have put forward an interesting mechanism for the translation inhibition by PAP [51], where PAP specifically targets and depurinates capped mRNA directly. Hudak et al [51] generated several PAP mutations (PAP x , an active site mutant (E176V); PAP n , a mutant with a substitution (G75D) in the N-terminal sequence; PAP c , a mutant lacking the C-terminal 25 amino acid residues) and showed that these PAP mutants do not depurinate rabbit or tobacco ribosomes, yet inhibit the in vitro translation of potato virus X and BMV with no notable depurination of ribosomes.…”

“…Hudak et al [51] generated several PAP mutations (PAP x , an active site mutant (E176V); PAP n , a mutant with a substitution (G75D) in the N-terminal sequence; PAP c , a mutant lacking the C-terminal 25 amino acid residues) and showed that these PAP mutants do not depurinate rabbit or tobacco ribosomes, yet inhibit the in vitro translation of potato virus X and BMV with no notable depurination of ribosomes. These studies showed that PAP is proficient in differentiating capped from uncapped mRNAs, since wild type (WT) PAP, and several of its mutants, prompted inhibition of only capped (but not uncapped) luciferase transcripts.…”