MxB Disrupts Hepatitis C Virus NS5A–CypA Complex: Insights From a Combined Theoretical and Experimental Approach

Figures

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  FIGURE 1. The anti-HCV activity of MxB truncations. (A) Schematic representation of wild-type and mutated MxB. (B) The anti-HCV activities of MxB truncations. Huh7.5.1 cell line was transduced with plasmids expressing Flag-tagged MxB (WT) and MxB truncations. The expression levels were examined by Western blotting analysis. The Jc1 HCVcc virus, which expresses Gluc as a reporter, was used to infect the MxB Huh7.5.1 cells. HCV infection was determined by measuring Gluc activity in the supernatant. (C) Co-IP was applied to detect the interaction between MxB truncations and NS5A. HEK293T cells were transfected with plasmids expressing MxB-Myc truncations and NS5A-Flag. Anti-Flag antibody was used to pull down NS5A-Flag, and anti-Myc antibody was applied to detect MxB truncations. Data are representative of three independent experiments, and values are expressed as means ± SD. Difference between two conditions is considered statistically significant with p < 0.05 (*). n.s. indicates non-significance. HCV, hepatitis C virus; MxB, myxovirus resistance B; WT, wild-type; Co-IP, co-immunoprecipitation.

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  FIGURE 2. Predicted MxB–NS5A interaction sites. (A) Cartoon and surface representation of the most representative structure of MxB (blue) in complex with NS5A (yellow). NS5A uses its domains I (NS5A-D1) to bind to the GTPase domain of MxB. (B,C) Surface representations of interacting residues in MxB (top) and NS5A (bottom). The identified hot-spot residues in MxB and NS5A are labeled and highlighted in green and blue in the surface representations, respectively. (D,E) The per-residue contribution to the MxB:NS5A-D1 binding energy (kcal/mol). Only residues with total energies above 2 kcal/mol are labeled. NS5A, non-structural protein 5A; MxB, myxovirus resistance B.

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  FIGURE 3. Validation of the predicted MxB–NS5A interaction. (A,B) Schematic representation of NS5A and MxB truncations. Diagrams were constructed using IBS (version 1.0) (Liu et al., 2015). The interactions of MxB with NS5A truncations (C) and those of NS5A with MxB truncations (D) were detected by IP with anti-Flag antibody followed by immunoblotting (IB) with anti-Myc antibody in HEK293T. Data are representative of three independent experiments. MxB, myxovirus resistance B; NS5A, non-structural protein 5A; IP, immunoprecipitation.

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  FIGURE 4. Predicted CypA–NS5A interaction sites. (A) Cartoon and surface representation of the most representative structure of CypA (magenta) in complex with NS5A (yellow). NS5A uses its domain I and domain II (NS5A-D2D3) to bind CypA. (B,C) Surface representations of interacting residues in CypA (top) and NS5A (bottom). The identified hot-spot residues in CypA and NS5A are labeled and highlighted in blue and orange in the surface representations, respectively. (D,E) The per-residue contribution to the CyPA:NS5A binding energy (kcal/mol). Only residues with total energies above 1 kcal/mol are labeled. CypA, cyclophilin A; NS5A, non-structural protein 5A.

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  FIGURE 5. Validation of the predicted CypA–NS5A interaction. (A,B) Schematic representation of CypA and NS5A truncations. Diagrams were constructed using IBS (version 1.0) (Liu et al., 2015). The interactions of NS5A with CypA truncations (C) and that of CypA with NS5A truncations (D) were detected by IP with anti-Flag antibody followed by immunoblotting (IB) with anti-HA antibody in HEK293T. Data are representative of three independent experiments. CypA, cyclophilin A; NS5A, non-structural protein 5A; IP, immunoprecipitation.

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  FIGURE 6. Predicted ternary CyPA–NS5A–MxB complex structure. CyPA, NS5A, and MxB are shown in magenta, yellow, and blue surface, respectively. CypA, cyclophilin A; NS5A, non-structural protein 5A; MxB, myxovirus resistance B.

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  FIGURE 7. Comparison of per-residue energy decomposition for key residues of CyPA:NS5A interaction in the absence (blue) and presence (red) of MxB. Residues highlighted are displayed for CyPA (A) and NS5A (B). CypA, cyclophilin A; NS5A, non-structural protein 5A; MxB, myxovirus resistance B.