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Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants

Fig 1

UbVs inhibit activity of MERS-CoV PLpro and CCHFV OTU in vitro.

(A) Sequences of UbVs that bind MERS-CoV or CCHFV vDUBs. Only regions subjected to diversification relative to Ub.wt in the phage-displayed library are shown. Amino acids discussed in the text are highlighted. (B) The binding specificities of phage-displayed UbVs (y-axis) are shown across a group of 12 DUBs (x-axis), as assessed by phage ELISA. Sub-saturating concentrations of phage were added to immobilized proteins as indicated. Bound phages were detected by the addition of anti-M13-HRP and colorimetric development of TMB peroxidase substrate. The mean value of absorbance at 450 nm is shaded in a black-red-yellow gradient. (C) Inhibition of MERS-CoV PLpro (solid lines) or CCHFV OTU (dashed lines) by the cognate UbVs shown as dose-response curves using Ub-AMC (left) or ISG15-AMC (right) as a substrate. The IC50 value was determined as the concentration of UbV that reduced proteolytic activity by 50% (S1 Table). The Ub.wt data obtained in the deISGylation assay cannot be fitted by GraphPad Prism so no lines are shown. (D) Effects of UbV inhibitors on vDUB activity against K48/K63 tetra-Ub substrates. Purified MERS-CoV PLpro (top panels) or CCHFV OTU (bottom panels) was incubated with the indicated UbV or Ub.wt (negative control) and biotinylated tetra-Ub at 37°C for a time course of 30 minutes. Western blots were probed with ExtrAvidin-HRP (EA-HRP) to detect biotin-Ub. Inhibition of proteolysis was shown by a delay of appearance of the digestion products tri-Ub (Ub3), di-Ub (Ub2) and mono-Ub (Ub1).

Fig 1