Binding to DCAF1 distinguishes TASOR and SAMHD1 degradation by HIV-2 Vpx
Fig 5
Vpx R34A-R42A, which induces SAMHD1 but not TASOR degradation, is characterized by a reduced binding affinity for DCAF1.
(A) HA-Vpx WT or indicated mutants and TASOR-Flag were co-expressed in HeLa cells, then an anti-HA immunoprecipitation was performed. (B) Graph representing Vpx binding affinity to DCAF1. Co-immunoprecipitated DCAF1 and immunoprecipitated HA-Vpx (Vpx WT (n = 6), Vpx R34A (n = 6), R42A (n = 6) and RR (n = 4)) were quantified from independent experiments. Co-immunoprecipitated DCAF1 signal over immunoprecipitated Vpx signal ratios were calculated and reported to Vpx WT (ratio 1). (C) TASOR-Flag and HA-Vpx WT or indicated mutants were co-expressed in HeLa cells, then an anti-Flag immunoprecipitation was performed. Immunoblot is representative of at least 3 independent experiments. (D and E) HIV-2.Gh1 Vpx WT and mutants were tested for viral reactivation (D) and TASOR degradation (E). J-Lat A1 T cells were treated with VLPs. After overnight treatment with TNF-α, HIV-1-LTR-driven GFP expression was analyzed by flow cytometry and whole-cell extracts were analyzed by western blot. For each mutant, reactivation assay was tested at least 3 times (SEM is shown), and immunoblots are representative of at least 3 independent experiments. (F) HIV-2.Gh1 Vpx WT and mutants were tested for SAMDH1 degradation, THP-1 cells were treated with VLP overnight and then whole-cell extracts were analyzed by western blot. RR: Vpx double mutant R34A-R42A. For each mutant, immunoblots are representative of 3 independent experiments.