FIG 4.
A single point mutation in the active site (ISG20D94G) abolishes the antibunyaviral activity of ISG20. (A) Schematic representation of the three exonuclease active motifs (Exo I, II, and III) of ISG20 (shown in gray boxes). The conserved amino acid residues (from the DEDDh family) are highlighted in green. The Asp94 residue (shown in green) of wild-type ISG20 was replaced with Gly to generate the catalytically inactive mutant ISG20D94G. The four amino acid differences between the human (H.s) and macaque (M.m) ISG20 proteins are highlighted with yellow boxes and red letters. (B) Plaque phenotypes of wt BUNV on Vero-EMPTY and Vero-ISG20/ISG20D94G cells. (C and D) Quantification of plaque areas (C) and virus titers (D) of BUNV on Vero-EMPTY and Vero-ISG20/ISG20D94G cells. (E) Western blot showing ISG20/ISG20D94G protein expression. Cell lysates were probed with an anti-ISG20 polyclonal antibody. Tubulin served as a sample loading control. (F) Same as panel E, but using bulk populations of Vero cells expressing human and macaque ISG20. (G) Titration curves for the bulk populations from panel F infected with BUNV-EGFP (46) or single-cycle RVFV (57). Means and standard deviations (SD) for triplicate titrations are shown.