Characterization of Pokeweed Antiviral Protein (PAP) Isoforms and Comparison of Their Enzymatic Activities toward the Tobacco Etch Virus RNA

Abstract

Ribosome‐inactivating proteins (RIPs) are found in a wide range of plants. Their antiviral, antibacterial and antifungal biological activities point toward RIPs playing important roles in plant's defense mechanisms. RIPs are RNA N‐glycosidases. They specifically recognize and depurinate universally conserved sarcin/ricin (S/R) loop of the large rRNA. This leads to ribosomal inactivation and termination of protein synthesis. Phytolacca americana, the common pokeweed plant, produces pokeweed antiviral protein (PAP), a type 1 RIP. PAP possesses antiviral properties and inhibits translation of viral RNAs, including HIV‐1, influenza, poliovirus, brome mosaic virus (BMV) RNAs. PAP inhibits translation of capped RNAs by directly binding to the 5'‐cap of the RNA, and depurinating it downstream of the cap structure. The mechanism of uncapped viral RNA translation inhibition is not known. Pokeweed plant produces nine seasonal tissue‐distinct PAP isoforms. Some isoforms are known to exhibit stronger antiviral potential than others: PAP‐S, isolated from seeds. The exact mechanism of how PAP selects its viral RNA substrates for depurination is not well understood, and is of interest research. This study will provide a deeper understanding of the strategies used by plants to battle infectious pathogens, and may lead to the development of efficient plant antiviral agents, and to improvement of the applied solutions for the regulation of plant and animal diseases. Here, we isolate and characterize PAP isoforms and employ fluorescence spectroscopy and HPLC techniques to compare of their enzymatic activities toward tobacco etch virus (TEV) RNA.