Fig. 6

HIV-1 restriction by mPML does not require a type I IFN-induced antiviral state, but efficient IFN-induced inhibition of HIV-1 in MEFs requires PML. a WB analysis of infection-induced up-regulation of PML in MEFs. WT MEF cells were infected with HIV-1NL-GFP, SIV_mac-GFP, or B-MLVGFP at an MOI of 1. Protein extracts were analyzed by WB at 6 or 24 h post infection, along with a no-infection control, using an anti-mPML monoclonal antibody (upper panel). Actin was analyzed as a loading control. b Expression of mPML was analyzed in WT MEFs left untreated (Ctrl), treated with IFN-β alone, or treated with a blocking antibody against IFNAR-1 at two different doses to block IFN-β-induced signal transduction prior to IFN-β treatment. mPML was detected using a monoclonal antibody (upper panel). Actin was analyzed as a loading control. c Blocking the IFN-I receptor does not alter HIV-1_NL-GFP restriction by PML. PML-KO and WT MEFs were treated with the anti-IFNAR-1 antibody, or with PBS as a control, and were then infected with increasing doses of HIV-1_NL-GFP. The percentage of infected cells (left panel) and GFP MFI (right panel) were assessed 2 days later by FACS. d The effects of IFN-I and PML on the antiviral state. PML-KO and WT MEFs were treated with IFN-β for 16 h prior to infection with HIV-1_NL-GFP. The percentage of infected cells (left panel) and GFP MFI (right panel) were assessed 2 days later by FACS. The values represent the means of three independent infections with standard deviations (**P < 0.01, two-tailed Student’s t test; ns non-significant). e Virus dose-dependent analysis of the role of PML in IFN-induced HIV-1 restriction. WT and PML-KO MEFs were treated with either IFN-α (500 U/ml) or IFN-β (100 U/ml) for 16 h, followed by infection with increasing doses of HIV-1_NL-GFP. The percentage of infected cells was assessed 2 days later by FACS