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. 2019 Aug 5;15(8):e1007962. doi: 10.1371/journal.ppat.1007962

Fig 4. Analysis of the ACBD3 mutations at the GOLD: EVD68 3A interface.

Fig 4

a, List of the ACBD3 mutants designed for further experiments. b, Mammalian-two-hybrid assay with the ACBD3 mutants and wild-type 3A. HeLa cells were transfected as indicated and the firefly luciferase activity normalized to the Renilla luciferase activity was determined using a dual-luciferase reporter assay system. c, Co-immunoprecipitation of the ACBD3 mutants and wild-type 3A. EGFP-fused wild-type 3A and GST-fused wild-type ACBD3 and its mutants were overexpressed in HEK293T cells. The 3A and ACBD3 complexes were affinity captured by the GFP-Trap nanobody or glutathione sepharose, respectively, and resolved by immunoblotting as indicated. d, Localization of the ACBD3 mutants. EGFP-fused wild-type ACBD3 and its mutants were overexpressed in HeLa ACBD3 KO cells. Cells were fixed and immunostained with the anti-giantin antibody. Scale bars represent 10 μm. e, Rescue of enterovirus replication by the ACBD3 mutants. HeLa ACBD3 KO cells were transfected with wild-type ACBD3 or its mutants, and enterovirus replication was determined using the Renilla luciferase-expressing CVB3 virus by the Renilla luciferase assay system. GalT and ACBD3 F258A/Q259A were used as controls.