Forsythoside A inhibits avian infectious bronchitis virus infection by binding the S1 subunit.

Abstract

OBJECTIVES: Avian infectious bronchitis virus (IBV) causes acute, highly contagious bronchitis inflammation and severe economic losses in the poultry industry. Such infections are difficult to treat as the virus has high mutation rates. Forsythiaside A (FTA) has an apparent inhibitory effect on IBV. This article describes evidence for the activity and the exact mechanism against IBV. METHODS: We observed the inhibitory effect of FTA on IBV infection lifecycle stages using plaque counts, immunofluorescence assay, and RT‒qPCR; then synthesized FTA-OVA, and expressed the recombination protein of IBV spike protein; thirdly, constructed an enzyme-linked immunosorbent assay (ELISA) based on the interaction assay between FTA and IBV particles or IBV spike protein. The interaction between FTA and the S protein was further confirmed using an isothermal titration calorimetry (ITC) system. We predicted the bond modes between FTA and S protein by molecular Docking. Moreover, changed the potential binding amino acids. Then we compared the affinity between FTA and the S1 mutant or the S1 wild-type. RESULTS: FTA inhibited IBV infection, especially on the viral attachment stage at non-cytotoxic concentrations, and FTA bound to virus particles or IBV S1 protein, rather than the S2 subunit. Molecular Docking via Autodock software revealed that FTA could form hydrogen bonds with eight amino acids in the S1 subunit, and these amino acids were then mutated. The affinity between FTA and the S1 mutant was much lower than that of the S1 wild-type. CONCLUSION: FTA prevents IBV infection by blocking viral absorption and contact with the S1 subunit of IBV but not with S2. We further identified the binding sites by comparing wild-type and mutant S1 affinities.