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. 2020 Sep 15;94(19):e00486-20. doi: 10.1128/JVI.00486-20

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FIG 4 — Effect of NCINI-3 resistance mutations on the CCD. (A) SEC analysis of CCD proteins carrying NCINI-3 resistance mutations together with F185K. The elution profile of CCD^WT is shown with two molecular weight standards (44 and 17 kDa) and estimating the size positions of CCD dimers and monomers. Those of CCD^A128T, CCD^H171Q, CCD^K173Q, CCD^P15, and CCD^{P26 del} at a 50 μM concentration was are shown compared with CCD^WT. (B) DSF analysis of CCD proteins carrying NCINI-3 resistance mutations. (Left) The fluorescence of CCD^WT, CCD^A128T, CCD^H171Q, CCD^K173Q, CCD^P15, and CCD^{P26 del} was detected and is plotted as a relative ratio from 25°C to 95°C. (Right) Expanded plots from 30°C to 50°C. (C) ΔT_m 25 or 50 values indicating the T_m 25 or 50 differences between CCD^WT and CCD proteins carrying the mutations at 12.5, 25, and 50 μM CCD concentrations. The assays were performed in triplicate, and error bars indicate SD from two independent experiments. The statistical significance of ΔT_m 25 or 50 was examined using two-tailed Student’s t test (*, P < 0.05). (D) Relative ratios of SYPRO orange fluorescence of CCD^WT and CCD proteins carrying the mutations from 30°C to 40°C at a 25 μM concentration. (E) Proportions of surface hydrophobic sites of CCD proteins at 37°C. The relative ratios were obtained from SYPRO orange binding to the surface hydrophobic sites of CCD proteins carrying the mutations compared to CCD^WT at 12.5, 25, and 50 μM concentrations. The assays were performed in triplicate, and error bars indicate SD from two independent experiments. Statistical significance was examined using Student’s t test (*, P < 0.05). (F) SPR analysis of NCINI-3 interactions with CCD^WT, CCD^A128T, CCD^H171Q, CCD^K173Q, and CCD^P15. Data shown represent results derived from two independent experiments. Shown are K_D values of NCINI-3 for CCD^WT and CCD proteins carrying the mutations, and fold changes indicate K_D ratios of CCD proteins carrying the mutations to CCD^WT. Error bars indicate SD from two independent experiments. N.D., not determined.

FIG 4 — Effect of NCINI-3 resistance mutations on the CCD. (A) SEC analysis of CCD proteins carrying NCINI-3 resistance mutations together with F185K. The elution profile of CCD^WT is shown with two molecular weight standards (44 and 17 kDa) and estimating the size positions of CCD dimers and monomers. Those of CCD^A128T, CCD^H171Q, CCD^K173Q, CCD^P15, and CCD^{P26 del} at a 50 μM concentration was are shown compared with CCD^WT. (B) DSF analysis of CCD proteins carrying NCINI-3 resistance mutations. (Left) The fluorescence of CCD^WT, CCD^A128T, CCD^H171Q, CCD^K173Q, CCD^P15, and CCD^{P26 del} was detected and is plotted as a relative ratio from 25°C to 95°C. (Right) Expanded plots from 30°C to 50°C. (C) ΔT_m 25 or 50 values indicating the T_m 25 or 50 differences between CCD^WT and CCD proteins carrying the mutations at 12.5, 25, and 50 μM CCD concentrations. The assays were performed in triplicate, and error bars indicate SD from two independent experiments. The statistical significance of ΔT_m 25 or 50 was examined using two-tailed Student’s t test (*, P < 0.05). (D) Relative ratios of SYPRO orange fluorescence of CCD^WT and CCD proteins carrying the mutations from 30°C to 40°C at a 25 μM concentration. (E) Proportions of surface hydrophobic sites of CCD proteins at 37°C. The relative ratios were obtained from SYPRO orange binding to the surface hydrophobic sites of CCD proteins carrying the mutations compared to CCD^WT at 12.5, 25, and 50 μM concentrations. The assays were performed in triplicate, and error bars indicate SD from two independent experiments. Statistical significance was examined using Student’s t test (*, P < 0.05). (F) SPR analysis of NCINI-3 interactions with CCD^WT, CCD^A128T, CCD^H171Q, CCD^K173Q, and CCD^P15. Data shown represent results derived from two independent experiments. Shown are K_D values of NCINI-3 for CCD^WT and CCD proteins carrying the mutations, and fold changes indicate K_D ratios of CCD proteins carrying the mutations to CCD^WT. Error bars indicate SD from two independent experiments. N.D., not determined.