Pokeweed antiviral protein: Ribosome inactivation and therapeutic applications

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

“…The direct effect of PAP on RNAs, namely capped BMV RNAs and capped and uncapped luciferase transcripts, was determined by incubating the PAP-treated RNA with acidic aniline, which causes chain cleavage at the depurination site (Irvin & Uckun, 1992)+ This assay has been widely used for detection of RIPmediated depurination of the large rRNA )+ Depurination results in increased susceptibility of the sugar-phosphate backbone to hydrolysis at the depurination site+ Hence, treatment of depurinated rRNA with aniline breaks the sugar-phosphate bond of the RNA+ Therefore, degradation of the RNA following aniline treatment will occur only if that RNA has been depurinated+ BMV RNAs and luciferase transcripts were incubated with wild-type PAP and one half of the treated RNAs was incubated with aniline, and the other half was incubated with buffer+ After precipitation, RNAs were visualized by urea-polyacrylamide gel analysis and staining with ethidium bromide+ As shown in Figure 9, 10 pg (3+33 pM) of PAP did not affect the integrity of the capped or uncapped luciferase transcripts or the BMV RNAs+ However 5 ng (1+65 nM) of PAP were capable of directly depurinating the capped luciferase transcripts and the BMV RNAs, as shown by degradation of the RNA after treatment with aniline ( Fig+ 9A,B)+ In contrast, 5 ng of PAP did not depurinate the uncapped luciferase transcripts (Fig+ 9C)+ At greater concentration (100 ng, 33+3 nM), PAP was capable of degrading the capped luciferase transcripts or the BMV RNAs even in the absence of aniline treatment ( Fig+ 9A,B), whereas 100 ng of PAP had no effect on the uncapped luciferase RNA (Fig+ 9C)+ These results demonstrate that PAP specifically depurinates the capped RNAs and does not affect the uncapped RNA+…”

Modeling and Alanine Scanning Mutagenesis Studies of Recombinant Pokeweed Antiviral Protein

“…1 is a 29-kDa naturally occurring protein isolated from the leaves of the pokeweed plant, Phytolacca americana (1)(2)(3). PAP belongs to a family of plant ribosome-inactivating proteins (RIPs) that catalytically depurinate ribosomal RNA (3,4).…”

“…PAP belongs to a family of plant ribosome-inactivating proteins (RIPs) that catalytically depurinate ribosomal RNA (3,4). The enzymatic activity of PAP has been shown to be the specific cleavage of the glycosidic bond of a single adenine residue (A 4324 of the tetraloop sequence GAGA) that is located in the highly conserved sarcin/ ricin stem loop (4 -7) and is found in both eukaryotic rRNA (28 S) and in prokaryotic rRNA (23 S).…”

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

“…In the instances where the 5ʹ-cap is absent, PAP directly binds to either the 5ʹ-or 3ʹ-UTRs (untranslated regions), containing either translational enhancer sequences or an internal ribosome entry site (K d for PAPuncapped full length TEV 5ʹ-leader RNA is 28.5 ± 3.7 nM; K d for PAP-m 7 GpppG-capped full length TEV 5ʹ-leader RNA is 87.5 ± 4.8 nM) [62]; binding of the eIFs increases PAP-RNA affinity, promoting depurination of RNA (presence of eIFiso4G increases 2.4-fold PAP-cap interactions) [53]. Additionally, PAP isoforms selectivity for different ribosomes and RNAs varies (e.g., PAP-I, found in spring leaves of the pokeweed plant, exhibits RC 50 of 1.5 nM towards rat liver ribosomes and 4.7 nM towards E. coli ribosomes [38]; PAP-S1, an isoform found in seeds of the plant, exhibits IC 50 of 3.2 nM towards rat liver ribosomes and 280 nM towards E. coli ribosomes [41,42]; whereas α-PAP, expressed in all organs of the plant, exhibits IC 50 of 1.3 nM towards rat liver ribosomes and 25 nM towards E. coli ribosomes [13,42]. In recent years, a viral protein (VPg), linked to genome of turnip mosaic virus (TuMV) was shown to inhibit PAP activity in vitro [13,57].…”

“…It has been postulated that a direct interaction of PAP with viral RNA (or DNA) is an alternative antiviral mechanism in play. The pokeweed plant produces several isozymes of PAP, all exerting potent antiviral properties [11,13,[36][37][38][39][40][41][42]. PAP isozymes evoke depurination of genomic HIV-1 RNA [43][44][45], TMV RNA [46], poliovirus [47], herpes simplex virus (HSV) [48], influenza virus [49], and brome mosaic virus (BMV) [50], among many others, showing a broad spectrum of antiviral activity [13].…”