SAMHD1-Deficient CD14+ Cells from Individuals with Aicardi-Goutières Syndrome Are Highly Susceptible to HIV-1 Infection
Figure 1
SAMHD1 is identified as Vpx binding protein leading to its degradation.
A) pNTAP-SIV Vpx was transfected in 293T cells. After lysis, isolation of SIV-Vpx and binding proteins was performed via Tandem Affinity Purification. The proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Protein bands in which the respective protein was identified are indicated. B) Schematic representation of the Vpx protein indicating the helical structure elements (black) and the introduced T17A mutation. C) 293T cells were transfected with pcDNA3.1 (mock) or plasmids encoding HA-Vpx or HA-Vpx T17A. After 48 hours, the cells were lysed and subjected to HA-immunoprecipitation. Isolated proteins were separated via SDS-PAGE and analyzed with antibodies specific for HA or SAMHD1. D) HeLa cells endogenously expressing SAMHD1 were transfected with pcDNA3.1 (mock) or HA-Vpx or HA-Vpx T17A encoding plasmids. 24 hours after transfection, the cells were fixed and stained for SAMHD1 and HA-Vpx with fluorescent antibodies. Nuclei were stained with DAPI before performing confocal microscopy. White arrows indicate nuclei of Vpx expressing cells, in which SAMHD1 is absent. White bar length corresponds to 10 µm. E) THP-1 cells were incubated with 2 MOIeq of empty VLPs (Ø), Vpx-VLPs or Vpx T17A-VLPs in the presence or absence of 50 µM MG132. After 6 hours the cells were analyzed by western blot analysis with the indicated antibodies.