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.
Highlights
Of the large rRNA [3, 4]
We show that Pokeweed antiviral protein (PAP) depurinated the RNA, which resulted in decreased viral protein synthesis and increased viral RNA degradation
The enzymatic activity of PAPwt was required for the decrease in viral RNA, as the active-site mutant PAPx did not cause a similar decrease in the level of Brome mosaic virus (BMV) RNA3
Summary
Plasmids and Transformation into Yeast Strains—cDNAs of PAPwt (wild-type) or PAPx (active-site mutant E176V [31]) under the control of the GAL1 promoter, were transformed into Saccharomyces cerevisiae strains W303, referred to as PSY (MATa ade trp ura leu 112 his can1–100) and RP1674 (MATa his3⌬1 leu2⌬ met15⌬ ura3⌬), both used as wild-type strains. Cells were switched to medium containing 50 g/ml tetracycline (to suppress further RNA3 transcription) and 2% galactose (to induce PAPwt or PAPx transcription) This switch represented the zero time point of a 6-h time course during which 15-ml aliquots were removed for total RNA isolation. Primer Extension of BMV RNA3—For analysis of depurination, PSY yeast cells were induced to transcribe BMV RNA3 and express PAPwt or PAPx as described above. Polysome Profiles—Wild-type PSY and deletion mutant (dom34⌬xrn1⌬ and hbs1⌬xrn1⌬) yeast cells were induced to transcribe BMV RNA3 and PAPwt or PAPx as described above. BMV RNA3 was detected with a probe complementary to the 3Ј-end of the transcript, as described above for Northern blot analysis. Cells were lysed and total protein (10 g) was loaded onto 12% SDS-PAGE, transferred to nitrocellulose and probed with a polyclonal antibody for PAP (1:5000) (A). Products of the reaction were denatured in formamide buffer and separated in a 7 M urea/6% polyacrylamide gel
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