Purification and partial characterization of another form of the antiviral protein from the seeds of Phytolacca americana L. (pokeweed)

Abstract

1. The pokeweed antiviral protein, previously identified in two forms (PAP and PAP II) in the leaves of Phytolacca americana (pokeweed) [Obrig. Irvin & Hardesty (1973) Arch. Biochem. Biophys. 155, 278-289; Irvin, Kelly & Robertus (1980) Arch. Biochem. Biophys. 200, 418-425] is a protein that prevents replication of several viruses and inactivates ribosomes, thus inhibiting protein synthesis. 2. PAP is present in several forms in the seeds of pokeweed. One of them, which we propose to call 'pokeweed antiviral protein from seeds' (PAP-S) was purified in high yield (180 mg per 100 g of seeds) by chromatography on CM-cellulose, has mol.wt. 30 000, and is similar to, but not identical with. PAP and PAP II. 3. PAP-S inhibits protein synthesis in a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of 1.1 ng/ml (3.6 x 10(-11) M), but has much less effect on protein synthesis by whole cells, with an ID50 of 1 mg/ml (3.3 x 10(-5) M), and inhibits replication of herpes simplex virus type 1.

Isolation and characterization of inhibitors of animal cell-free protein synthesis from barley seeds

“…Furthermore, the difference in the chromatographic behavior of the three inhibitors on the cation exchange resins, CM-cellulose and Mono S, as described in section 3.1. may be due to different extents of deamidation of asparagine and glutamine in the three inhibitors. Comparison of the amino acid compositions of translation inhibitors from plants, including the A chain of toxic proteins, reveals large differences (2,9,11,18,33). The amino acid compositions of translation inhibitors from barley were also distinctly different from those of other translation inhibitors.…”

“…The A chain inhibits the translation, but requires the B chain to enter the intact cells (26). On the other hand, the non-toxic inhibitors such as pokeweed antiviral protein (PAP) from Phytolacca americana (2,18) and tritin from wheat (Triticum aestirum) (28,29) are both single chain polypeptides analogous to the A chain of the toxic proteins. The non-toxic inhibitors isolated from different plants so far are all basic proteins with molecular weights of about 30,000.…”

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].…”

Expression of Pokeweed Antiviral Protein in Transgenic Plants Induces Virus Resistance in Grafted Wild-Type Plants Independently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis

“…Three different kinds of RIPs, PAP, PAPII, and PAP-S, have been purified from pokeweed (Phytolacca americana) plants. These proteins are similar in molecular mass (29, 30, and 29.5 kD, respectively) but are expressed at different developmental stages and in different tissues of pokeweed (Wyatt and Shepherd, 1969;Irvin et al, 1980;Barbieri et al, 1982). PAP depurinates ribosomes from pokeweed and other plants (Bonness et al, 19941, as well as mammalian, yeast, and bacterial ribosomes.…”