Figure 1.
Effects of macrophage-tropic HIV-1 strain and VSV-G-pseudotyped HIV-2 particles on lentivirus vector infection.
Monocytes were differentiated into macrophages for 11 days in the presence of GM-CSF. Macrophages or monocytes were treated with macrophage-tropic HIV-1 strain (SF162) or VSV-G pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G) and then infected with lentivirus vector NL43-Luci/VSV-G. Luciferase activity was measured 4 days after infection. Data are plotted as the mean ± SD of triplicate samples; presented data are representative of three independent experiments using two donors. A: Results of samples obtained from a donor 1. B: Results of samples obtained from a donor 2.
Figure 2.
Effects of VSV-G-pseudotyped HIV-2 particles on macrophage-tropic and T-cell line-tropic HIV-1 strains in GM-CSF-induced macrophages.
Monocytes were differentiated into macrophages for 6 days in the presence of GM-CSF. Macrophages were treated with VSV-G pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G) and then infected with HIV-1 strain SF162 or NL43. HIV-1 replication was quantified by ELISA measurement of p24 antigen in the supernatant after infection. Data are plotted as the mean ± SD of triplicate samples; presented data are representative of three independent experiments using two donors. A: Results of samples obtained from a donor 1. B: Results of samples obtained from a donor 2.
Figure 3.
Effects of VSV-G-pseudotyped HIV-2 particles on macrophage-tropic and T-cell line-tropic HIV-1 strains in M-CSF-induced macrophages.
Monocytes were differentiated into macrophages for 6 days in the presence of M-CSF. Macrophages were treated with VSV-G pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G) and then infected with HIV-1 strain SF162 or NL43. HIV-1 replication was quantified by ELISA measurement of p24 antigen in the supernatant after infection. Data are plotted as the mean ± SD of triplicate samples; presented data are representative of two independent experiments using two donors. A: Results of samples obtained from a donor 1. B: Results of samples obtained from a donor 2.
Figure 4.
Effects of VSV-G-pseudotyped HIV-2 particles on macrophage-tropic and T-cell line-tropic HIV-1 strains in undifferentiated monocytes.
Monocytes were treated with VSV-G pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G) and then infected with HIV-1 strain SF162 or NL43. HIV-1 replication was quantified by ELISA measurement of p24 antigen in the supernatant after infection. Data are plotted as the mean ± SD of triplicate samples obtained from a single blood donor; presented data are representative of three independent experiments using two donors. A: Results of samples obtained from a donor 1. B: Results of samples obtained from a donor 2.
Figure 5.
Syncytium formation in undifferentiated monocytes pretreated with HIV-2 particles and then infected with macrophage-tropic HIV-1.
Monocytes were treated with VSV-G pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G) and then infected with HIV-1 strain SF162 or NL43. Presented data are representative of two independent experiments.
Figure 6.
Western blot analysis of SAMHD1 in undifferentiated monocytes and macrophages.
Monocytes were differentiated into macrophages for 6 days in the presence of GM-CSF or M-CSF. Macrophages or monocytes were treated with or without VSV-G-pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G), and harvested. Whole-cell extracts were separated on SDS-PAGE and analyzed by western blot using the indicated antibodies. Presented data are representative of two independent experiments.
Figure 7.
Quantification of SAMHD1 mRNA expression.
Monocytes were differentiated into macrophages for 6 days in the presence of GM-CSF or M-CSF. Macrophages or monocytes were treated with or without VSV-G-pseudotyped and Env-defective HIV-2 particles (GH123/VSV-G), and harvested. SAMHD1 mRNA expression levels were detected by real-time RT-PCR and normalized against GAPDH. Data are shown as mean ± SD.
Figure 8.
Phosphorylation state of SAMHD1.
Monocytes (Monocyte), M-CSF-differentiated macrophages (Macrophage (M-CSF)), and GM-CSF-differentiated macrophages (Macrophage (GM-CSF)) were treated with GH123-Nhe/VSV-G for 2 h and incubated at 37°C for 4 h (4 h) or 24 h (24 h), or mock-treated (−). Cells were lyzed and subjected for SDS-PAGE containing Phos-tag to separate phosphorylated proteins from nonphosphorylated ones. SAMHD1 proteins were detected by anti-SAMHD1 antibody. Upper bands shown by a vertical bar and a lower band shown by an arrow represent phosphorylated and nonphosphorylated SAMHD1, respectively. Ratios of phosphorylated SAMHD1 levels to total SAMHD1 levels (ratio) are shown in vertical numbers.
Figure 9.
Effects of SAMHD1 siRNA on HIV-1 infection.
Monocytes (Monocyte) or macrophages (Macrophage) were treated with SAMHD1- (siSAMHD1) or GFP- (siGFP) targeting siRNAs. Three days after transfection, cells were harvested or infected with NL43-Luci/VSV-G virus. SAMHD1 mRNA expression levels in harvested cells were detected by real-time RT-PCR and normalized against GAPDH. Data are shown as mean ± SD. Luciferase activity in infected cells was measured 4 days after infection. Data are plotted as the mean ± SD of triplicate samples; presented data are representative of two independent experiments using two donors. A: Results of samples obtained from a donor 1. B: Results of samples obtained from a donor 2.