Depurination of Brome mosaic virus RNA3 in vivo results in translation-dependent accelerated degradation of the viral RNA

Abstract

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that exhibits antiviral activity against several plant and animal viruses. We have shown previously that PAP depurinates Brome mosaic virus (BMV) RNAs in vitro and that prior incubation of these RNAs with PAP reduced their synthesis in barley protoplasts. To investigate the post-transcriptional effect of PAP on viral RNA in vivo, we transcribed BMV RNA3 and expressed PAP in the yeast, Saccharomyces cerevisiae, which is a surrogate host for BMV. With an inducible transcription system, we show that the half-life of RNA3 in PAP-expressing cells was significantly less than in cells expressing PAPx, its enzymatically inactive form. PAP bound to RNA3 and depurinated the RNA within open reading frames 3 and 4 and within untranslated regions of the RNA. The depurinated RNA was associated with polysomes, caused ribosomes to stall at the point of depurination, and was targeted for accelerated degradation by components of the No-go decay pathway. As a consequence of translation elongation arrest and increased RNA degradation, expression of PAP in yeast also decreased the level of protein 3a, encoded by the 5'-proximal open reading frame 3 of BMV RNA3. These data provide the first evidence of viral RNA depurination in vivo by any ribosome-inactivating protein and support our hypothesis that depurination contributes to the antiviral activity of PAP, by enhancing viral RNA degradation and reducing translation of viral protein product.