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 2.
Structural basis for UbV inhibition of MERS-CoV PLpro.
(A-C) Crystal structure of (A) the MERS-CoV PLpro-ME.4 complex, (B) the MERS-CoV PLpro-ME.2 complex, and (C) the MERS-CoV PLpro-Ub.wt complex (PDB ID: 4RF0). PLpro domains are shown as surface representations, and coloured in wheat, gray and chartreuse for the PLpro-ME.4, -ME.2 and–Ub.wt complexes, respectively. ME.4, ME.2 and Ub.wt are shown as tubes and coloured in marine, red and orange, respectively. (D) Close up of a superposition of the MERS-CoV PLpro-ME.4 and -ME.2 complexes (left panel) showing detailed interactions between PLpro and residue Ile70 of ME.4 or ME.2, and a comparison (right panel) of the same region in the PLpro-Ub.wt complex. PLpro residues are shown as sticks and labeled with in italics with asterisks. (E) Close up of the MERS-CoV PLpro-ME.4 and -ME.2 complexes (left panel) showing detailed interactions between PLpro and residue Phe46 of ME.4 or ME.2, and a comparison (right panel) of the same region in the PLpro-Ub.wt complex. (F) Close up of the MERS-CoV PLpro-ME.4 and -ME.2 complexes (left panel) showing detailed interactions between PLpro and residue Tyr64 of ME.4 or ME.2, and a comparison (right panel) of the same region in the PLpro-Ub.wt complex. (G) Close up of ME.4 residue Asn74 bound near the active site of PLpro. Hydrogen bonds are represented by dashed black lines. (H) Close up of the C-terminus of Ub.wt covalently bound in the active site of PLpro. (I) Close up of ME.2 residue Pro74 bound near the active site of PLpro. Figures were generated using PyMOL [61].
Table 1.
Crystallographic and refinement statistics for MERS-CoV PLpro and CCHFV OTU bound to UbVs.
Fig 3.
Structural basis for UbV inhibition of CCHFV OTU.
(A-C) Crystal structure of (A) the CCHFV OTU-CC.2 complex, (B) the CCHFV OTU-CC.4 complex, and (C) the CCHFV OTU-Ub.wt complex (PDB ID: 3PT2). OTU domains are shown as surface representations, and coloured in cyan, light cyan and slate for the OTU-CC.2, -CC.4 and–Ub.wt complexes, respectively. CC.2, CC.4 and Ub.wt are shown as tubes and coloured in yellow, magenta and orange, respectively. (D) Overlay of the CCHFV OTU-CC.2, CC.4 and–Ub.wt structures showing interactions between CCHFV OTU and CC.2 or CC.4 residue Tyr68 or Ub.wt residue His68. UbV and Ub.wt residues are shown as sticks and labeled in regular font. CCHFV OTU residues are shown as sticks and labeled in italics with asterisks. (E) Close up of interactions between the C-terminus of CC.2 and CCHFV OTU. (F) Close up of interactions between the C-terminus of CC.4 and CCHFV OTU. (G) Close up of interactions between the C-terminus of Ub.wt and the active site of CCHFV OTU. Figures were generated using PyMOL [61].
Fig 4.
UbVs inhibit proteolytic activity of MERS-CoV PLpro in cell culture and affect MERS-CoV replication.
(A) The effects of UbVs on the DUB activity of MERS-CoV PLpro was determined by co-transfecting HEK293T cells with plasmids encoding HA-Ub, MERS-CoV PLpro-V5 (wild type or the active site mutant C1592A annotated as “C” throughout the rest of the figure), FLAG-ME-UbV as indicated (in increasing dose) and GFP (as a transfection control). Cells were lysed 18 hours post transfection and expressed proteins were analyzed by western blotting. DUB activity of MERS-CoV PLpro was visualized by the deconjugation of HA-Ub from cellular proteins. (B) Assessment of the inhibitory effects of UbVs on the suppression of the IFN-β promoter by MERS-CoV PLpro. HEK293T cells were transfected with plasmids encoding firefly luciferase reporter gene under control of the IFN-β promoter, Renilla luciferase, innate immune response inducer mitochondrial antiviral signaling protein (MAVS), MERS-CoV PLpro-V5 (wild type or the active site mutant C) and FLAG-tagged UbVs (in increasing dose). Cells were lysed 16 hours post transfection and both firefly and Renilla luciferase activities were measured. Results represent at least three independent experiments. Significance relative to wild-type without expression of a UbV was calculated using an unpaired two-tailed Student’s t test and significant values were indicated: ** p < 0.01. Bars represent mean and error bars represent S.D. (C) Proteolytic cleavage capability of MERS-CoV PLpro was assessed in the presence of the UbVs. N-terminally HA-tagged and C-terminally V5-tagged nsp3C-4 (excluding the PLpro domain) was co-expressed with V5-tagged MERS-CoV PLpro-V5 (wild type or the active site mutant C), FLAG-ME-UbV (with increasing doses) and GFP (as a transfection control). Cells were lysed 18 hours post transfection and proteolytic cleavage activity was assessed by western blotting to detect generation of N-terminal HA-tagged nsp3C and C-terminal V5-tagged nsp4 cleavage products. (D) MERS-CoV titers of collected supernatants from lentivirus transduced and, subsequently, MERS-CoV infected MRC5 cells. MRC-5 cells were transduced with lentiviruses encoding FLAG-UbVs, FLAG-Ub.AA or GFP (latter two as controls) and, either 32 hours or 48 hours post-transduction, the cells were infected with MERS-CoV at a multiplicity of infection of 0.01. After another 32 hours, culture supernatants were harvested and MERS-CoV titers were determined by plaque assays on Vero cells. Significant difference relative to MERS-CoV titers from lentivirus transduced MRC5 cells expressing Ub.AA is indicated: * p<0.05. Bars represent mean and error bars represent S.D.