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. 2021 Jun 10;17(6):e1009616. doi: 10.1371/journal.ppat.1009616

Fig 1. Acetylation-mimicking mutation of NEDD4 promotes VP40 budding.

Fig 1

(A) HEK293T cells were transfected with Myc-VP40 for 40 hours and then treated with TSA, SAHA, and NAM for 8 hours and analyzed using the virus-like particle (VLP) assay. (B) HEK293T cells were transfected with indicated plasmids, immunoprecipitated with an anti-HA antibody and analyzed via immunoblotting with an anti-Acetyl-K antibody to detect the acetylation of NEDD4 and TSG101, where “*” indicated the specific NEDD4 and acetylation of NEDD4 and “■” indicated the location of the acetylation of TSG101. (C) NEDD4 acetylation sites were identified using mass spectrometry. Schematic diagram of the location of NEDD4 acetylation sites. (D) HEK293T cells were transfected with indicated plasmids, immunoprecipitated with an anti-Myc antibody and analyzed via immunoblotting with an anti-Acetyl-K antibody to detect the acetylation of the NEDD4 wild-type (WT) and mutants. (E)–(F) HEK293T cells were transfected with indicated plasmid combinations to detect the influence on the egress of VP40. Error bars, mean ± SD of three experiments. Student’s t test; *p < 0.05; **p < 0.01; ***p < 0.001.