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

Pokeweed antiviral protein (PAP) is known to inactivate ribosomes by remova l of a specific adenine from the sarcin/ ricin (SIR) loop of the large rRNA , thereby inhibiting translation. We demonstrate here that in addition to t he previously identified adenine (A4324), PAP removes another adenine (A432 1) and a guanine (G4323) from the eukaryotic large rRNA. Recent results ind icate 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 mo saic virus (BMV) and potato virus X (PVX) RNAs without depurinating ribosom es. Furthermore, translation of only capped, but not uncapped, luciferase t ranscripts is inhibited by PAP, providing evidence that PAP and PAP mutants are able to distinguish between capped and uncapped transcripts. Translati on 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 G TP, indicating that PAP recognizes the cap structure. Incubation of BMV RNA s or the capped luciferase transcripts with PAP results in depurination of either RNA. In contrast, uncapped luciferase transcripts are not depurinate d after incubation with identical concentrations of PAP. These results demo nstrate for the first time that PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and spec ifically depurinating the capped RNAs.