MDM2 negatively regulated HIV-1 replication in non-permissive cells through the degradation of Vif. (A) The overexpression of MDM2 inhibited HIV-1 replication in the presence of A3G. NL-43 Wt and ΔVif viruses were produced from HEK293T cells transfected with expression vectors for MDM2 Wt and a ΔRF mutant in the presence or absence of A3G. The viral infectivity was examined using M8166 cells. Values are presented as averages of more than 3 independent experiments. (B) MDM2 reduced cellular levels of Vif, resulting in more incorporation of A3G into HIV-1 virions. Immunoblotting for cell lysates (upper 3 panels) and precipitated virions (lower 2 panels) was performed with the indicated Abs. Lane numbers correspond to those in Fig. 4A. (C) HIV-1 replication in macrophages transfected with MDM2- and control-siRNA. MDM were transfected with MDM2- and control-siRNA and challenged with R5 HIV-1JR-FL (left panel). Cell lysates were subjected to immunoblotting with the indicated antibodies (right panels). (D) Coexpression of MDM2 reduced cellular levels of Vif and inversely increased A3G levels in a dose dependent manner. HEK293T cells were cotransfected with expression vectors for A3G, Vif, GFP, and MDM2 as indicated. Cell lysates were subjected to immunoblotting with the indicated Abs. (E) Immunoprecipitation assays revealed that the coexpression of MDM2 blocked the binding of A3G to Vif in a dose dependent manner. HEK293T cells were cotransfected with expression vectors for A3G, GFP-Vif, and MDM2 as indicated. Cell lysates were immunoprecipitated with anti-GFP mAb followed by immunoblotting with the indicated Abs.