Expression of novel fusion antiviral proteins Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro

Ricin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with 50% protein synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and protein synthesis inhibition activity assayed. Results showed that the optimized protein RTA mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on protein synthesis inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infection.