Inhibition of Pokeweed Antiviral Protein (PAP) by Turnip Mosaic Virus Genome-linked Protein (VPg)

Abstract

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and an RNA N‐glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap‐binding protein, and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and Turnip mosaic virus genome linked protein (VPg) were investigated. VPg can function as a cap analog in cap‐independent translation, and potentially target PAP to uncapped IRES‐containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP‐VPg interactions. PAP binds to VPg with high affinity (29.5 nM); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate, and competes with structured RNA derived from tobacco etch virus (TEV) for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms, and also suggests the possible use of this protein against the cytotoxic activity of RIPs.

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

“…This viral peptide serves as an analog of the 5ʹ-m 7 G cap of viral RNA, and has been shown to play an important role in mRNA translation since it interacts with the cap-binding proteins (e.g., eIF4E, eIFiso4E, eIF4F, eIFiso4F) [64,65]. Our laboratory has shown that PAP interacts with VPg (K d is 29.5 ± 1.8 nM) and inhibits PAP's activity in a dose-dependent manner [57], perhaps providing an evolutionary advantage for the virus to…”

“…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]. This viral peptide serves as an analog of the 5ʹ-m 7 G cap of viral RNA, and has been shown to play an important role in mRNA translation since it interacts with the cap-binding proteins (e.g., eIF4E, eIFiso4E, eIF4F, eIFiso4F) [64,65].…”

“…Recent work performed in our laboratory showed that PAP binds to and depurinates the 5ʹ-leader sequence from tobacco etch virus (TEV) RNA [57]. The TEV 5ʹ-leader is sufficient to confer cap-independent translation, even in the absence of the 5ʹ-terminal VPg (a viral genomelinked protein) [58,59].…”

Pokeweed (Phytolacca americana L.) antiviral protein inhibits Zucchini yellow mosaic virus infection in a dose-dependent manner in squash plants

“…the genus Potyvirus, whose members are characterized by having a VPg protein at the 5' end of their genome. Recently it was shown that the VPg of a potyvirus binds PAP with high affinity, being a potent inhibitor of PAP depurination (Domashevskiy et al, 2012), which can explain the lack of antiviral effects observed at the local level. However, pretreatment with PAP before inoculation could prepare the plant to avoid viral infection as observed in our experiments where the virus was inoculated 4 days after PAP treatment.…”

Potyviral Genome-Linked Protein and its Interaction with Plant Defense Ribosome Inactivating Protein from Phytolacca Americana

“…Recent studies examined the interactions between PAP (cap-binding protein) and VPg from TuMV [70], and revealed that VPg competes with TEV RNA for PAP binding [71]. These PAP-VPg interactions are enthalpically-driven and entropically favorable [70], exhibiting a similarity to those of eIFiso4E-and eIFiso4F-VPg binding [63].…”