Figure 3
Figure 3. The HTLV-1 SU/NRP-1 interaction depends on the b domain of NRP-1 and on HSPGs. (A) Effect of the deletion of the b domain of NRP-1 on NRP-1 synthesis, binding to heparin, and cell-surface expression. COS cells were transfected with a control plasmid (Cont.) or a plasmid encoding either wild-type (wt) HA-NRP-1 or HA-NRP-1Δb. (Left panels) Total cell proteins and proteins pulled-down on heparin-coated Sepharose beads were analyzed by immunoblot using an anti-HA mAb. (Right panel) Transfected COS cells were stained with the anti-HA mAb and analyzed by flow cytometry. (B) Effect of deletion of the b domain of NRP-1 on HTLV-1 virus binding. COS cells transfected as in panel A were incubated with culture medium (gray lines) or concentrated supernatant from HTLV-1–infected T cells (black lines), and the level of binding of HTLV-1 virions was determined. (C) Quantification of the level of HTLV-1 virus binding to COS cells overexpressing wt HA-NRP-1 or HA-NRP-1Δb. Results correspond to the ratio between the mean fluorescence intensity (MFI) of cells overexpressing either WT HA-NRP-1 or HA-NRP-1Δb and the MFI of control cells (×100) and are the mean ± SD of 3 independent experiments. *Statistically significant (unpaired t test analysis, 2-tailed P < .05). ns indicates not significant. (D) Effect of reducing HSPG synthesis on the formation of complexes between the HTLV-1 Env proteins and the ectodomain of NRP-1. COS cells were cotransfected with either the CMV-Env-LTR construct or a control plasmid and a plasmid encoding the soluble version of HA-NRP-1 (HA-sNRP-1). After 4 hours, cells were either left untreated (−) or treated with 30 mM sodium chlorate for 24 hours (+). Total proteins precipitated with an anti–HTLV-1 serum (IP Env) were examined by immunoblot using the anti-HA or the anti-SU mAb, as indicated. The levels of HA-NRP-1 and Env in cell lysates are also shown. The positions of HA-sNRP-1 and of the HTLV-1 Env precursor (gp61) and SU (gp46) are indicated. (Bottom panel) Quantification of the effect of sodium chlorate on the Env/HA-sNRP-1 coprecipitation. Results represent the normalized amount of HA-sNRP-1 coprecipitated with Env and are the mean ± SD of 2 independent experiments.

The HTLV-1 SU/NRP-1 interaction depends on the b domain of NRP-1 and on HSPGs. (A) Effect of the deletion of the b domain of NRP-1 on NRP-1 synthesis, binding to heparin, and cell-surface expression. COS cells were transfected with a control plasmid (Cont.) or a plasmid encoding either wild-type (wt) HA-NRP-1 or HA-NRP-1Δb. (Left panels) Total cell proteins and proteins pulled-down on heparin-coated Sepharose beads were analyzed by immunoblot using an anti-HA mAb. (Right panel) Transfected COS cells were stained with the anti-HA mAb and analyzed by flow cytometry. (B) Effect of deletion of the b domain of NRP-1 on HTLV-1 virus binding. COS cells transfected as in panel A were incubated with culture medium (gray lines) or concentrated supernatant from HTLV-1–infected T cells (black lines), and the level of binding of HTLV-1 virions was determined. (C) Quantification of the level of HTLV-1 virus binding to COS cells overexpressing wt HA-NRP-1 or HA-NRP-1Δb. Results correspond to the ratio between the mean fluorescence intensity (MFI) of cells overexpressing either WT HA-NRP-1 or HA-NRP-1Δb and the MFI of control cells (×100) and are the mean ± SD of 3 independent experiments. *Statistically significant (unpaired t test analysis, 2-tailed P < .05). ns indicates not significant. (D) Effect of reducing HSPG synthesis on the formation of complexes between the HTLV-1 Env proteins and the ectodomain of NRP-1. COS cells were cotransfected with either the CMV-Env-LTR construct or a control plasmid and a plasmid encoding the soluble version of HA-NRP-1 (HA-sNRP-1). After 4 hours, cells were either left untreated (−) or treated with 30 mM sodium chlorate for 24 hours (+). Total proteins precipitated with an anti–HTLV-1 serum (IP Env) were examined by immunoblot using the anti-HA or the anti-SU mAb, as indicated. The levels of HA-NRP-1 and Env in cell lysates are also shown. The positions of HA-sNRP-1 and of the HTLV-1 Env precursor (gp61) and SU (gp46) are indicated. (Bottom panel) Quantification of the effect of sodium chlorate on the Env/HA-sNRP-1 coprecipitation. Results represent the normalized amount of HA-sNRP-1 coprecipitated with Env and are the mean ± SD of 2 independent experiments.

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