Quantification of SAMHD1 mRNA expression.

<p>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.</p

Effects of SAMHD1 siRNA on HIV-1 infection.

<p>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.</p

Western blot analysis of SAMHD1 in undifferentiated monocytes and macrophages.

<p>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.</p

Effects of VSV-G-pseudotyped HIV-2 particles on macrophage-tropic and T-cell line-tropic HIV-1 strains in M-CSF-induced macrophages.

<p>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.</p

Effects of VSV-G-pseudotyped HIV-2 particles on macrophage-tropic and T-cell line-tropic HIV-1 strains in undifferentiated monocytes.

<p>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.</p

Effects of macrophage-tropic HIV-1 strain and VSV-G-pseudotyped HIV-2 particles on lentivirus vector infection.

<p>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.</p

Phosphorylation state of SAMHD1.

<p>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.</p

HIV-1 Vpr Abrogates the Effect of TSG101 Overexpression to Support Virus Release

<div><p>HIV-1 budding requires interaction between Gag and cellular TSG101 to initiate viral particle assembly and release via the endosomal sorting complexes required for transport (ESCRT) pathway. However, some reports show that overexpression of TSG101 inhibits virus release by disruption of Gag targeting process. Since a HIV-1 accessory protein, Vpr binds to Gag p6 domain at the position close to the binding site for TSG101, whether Vpr implicates TSG101 overexpression effect has not been investigated. Here, we found that Vpr abrogates TSG101 overexpression effect to rescue viral production. Co-transfection of TSG101 and Gag with Vpr prevented TSG101-induced Gag accumulation in endosomes and lysosomes. In addition, Vpr rescued virus-like particle (VLP) production in a similar manner as a lysosomal inhibitor, Bafilomycin A1 indicating that Vpr inhibits TSG101-induced Gag downregulation via lysosomal pathway. Vpr and Gag interaction is required to counteract TSG101 overexpression effect since Vpr A30F mutant which is unable to interact with Gag and incorporate into virions, reduced ability to prevent Gag accumulation and to rescue VLP production. In addition, GST pull-down assays and Biacore analysis revealed that Vpr competed with TSG101 for Gag binding. These results indicate that Vpr overcomes the effects of TSG101 overexpression to support viral production by competing with TSG101 to bind Gag.</p></div

Vpr and Gag interaction is required to abrogate the effect of TSG101 overexpression.

<p>(A) HeLa cells were co-transfected with 0.8 μg of pCAGGS/Gag and 1.5 μg of pCAGGS/eCFP-TSG101 either without/with 0.5 μg of pCAGGS/HA-Vpr or pCAGGS/HA-Vpr A30F for 48 h prior to immunofluorescence staining with an anti-Gag and anti-HA antibodies, followed by an Alexa Fluor 594 goat anti-rabbit antibody and an Alexa Fluor 633 goat anti-mouse antibody. Co-localization of Gag/TSG101 was analyzed by Pearson’s correlation coefficients (B). Data represent the means ± SD of the result of two independent experiments. *, P < 0.05 (unpaired t-test). (C) HEK293T cells were transfected with 0.8 μg of pCAGGS/Gag, or with 0.8 μg of pCAGGS/Gag plus 1.5 μg of pCAGGS/FLAG-TSG101 either without/with different amounts of pCAGGS/Vpr A30F or 0.08 μg of pCAGGS/Vpr (positive control). After 48 h, VLPs in the cultured medium were collected by a 20% sucrose cushion. Whole cell lysates were prepared and samples were subjected to western blot analysis with anti-Gag, anti-Vpr, anti-FLAG, and anti-β-actin antibodies. The bottom panel represents intensity of VLP Gag from western blot analysis. Data represent the result of one representative experiment from two independent performs.</p

Vpr prevents TSG101-induced Gag accumulation at perinuclear region.

<p>(A) Schematic showing the binding motifs for TSG101 (PTAP; green) and Vpr (FRFG, ELY, and LXSLFG; red) within the Gag p6 domain. (B) HeLa cells were co-transfected with 0.8 μg of pCAGGS/Gag and 1.5 μg of pCAGGS/eCFP-TSG101 either without/with 0.5 μg of pCAGGS/HA-Vpr or 0.5 μg of pCAGGS/Vif-HA for 48 h prior to immunofluorescence staining with an anti-Gag and anti-HA antibodies, followed by an Alexa Fluor 594 goat anti-rabbit antibody and an Alexa Fluor 633 goat anti-mouse antibody. Image acquisition was performed under a confocal laser-scanning microscope. (C) Co-localization of Gag/TSG101 was analyzed by Pearson’s correlation coefficients. Data represent the means ± SD of the result of two independent experiments. *, P < 0.05 (unpaired t-test). (D-E) HeLa cells were co-transfected with 1.5 μg of pCAGGS/eCFP-TSG101 or 0.8 μg of pCAGGS/Gag-Venus (D) or 1.5 μg of pCAGGS/eCFP-TSG101 and 0.8 μg of pCAGGS/Gag-Venus either without/with 0.5 μg of pCAGGS/mRFP-Vpr (E) for 48 h before fixation and image acquisition. The precision FRET (PFRET) signal was analyzed using the sensitized emission method. The FRET (Ex.eCFP/Em.Venus) and PFRET image colors were converted by Hi/Lo function of FV10-ASW v.2.1 software (Olympus) to facilitate visualization of Gag/TSG101 co-localization. (F) FRET ratio (PFRET signal divided by the donor eCFP-TSG101 signal) of Gag/TSG101 co-localization in the absence or presence of Vpr. Data represent means ± SD of the result of one representative experiment from two independent performs. *, P < 0.05 (unpaired t-test).</p