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. Author manuscript; available in PMC: 2017 Sep 27.
Published in final edited form as: Cell Rep. 2017 Sep 5;20(10):2396–2407. doi: 10.1016/j.celrep.2017.08.040

Figure 1. Design of OTU mutations and in vitro assessment of OTU activity.

Figure 1

(A) CCHF virus-like particles (VLPs) were generated by transfecting Huh7 cells with plasmids encoding CCHFV polymerase (L), nucleoprotein (NP), and glycoprotein (GPC), as well as a minigenome encoding luciferase and T7 polymerase. After 3 days, supernatants containing the VLPs were harvested and transferred to new Huh7 cells, and the luciferase signal was determined as a measure of VLP activity. Data are presented as mean ± SD of 3 biological replicates and * indicate p < 0.05 (B) A model of the CCHFV OTU in complex with ubiquitin (PDB entry 3PRP) was used to assist with the design of mutations that disrupt the OTU-Ub and OTU-ISG15 binding. The protease surface that forms the binding interface with ubiquitin is shaded in brown, with the residues targeted for mutation in this study indicated. The residues forming the shared consensus sequence of ubiquitin and ISG15, LRLRGG, are shown in purple. (C) OTU mutant activity profiles on 7-amino-4-methylcoumarin (AMC) fluorogenic substrates of monoubiquitin (Ub-AMC) and ISG15 (ISG-AMC) was assessed in vitro. Data are presented as mean of two biological replicates ± SD and * indicate p < 0.05 relative to WT (D) Overview of selected OTU mutations and their predicted effect on deubiquitinase and deISGylase activities. between the wild-type OTU or L protein and the individual mutants.