Figure 7
Figure 7. DCIR-mediated enhancing effect on HIV-1 replication requires phosphorylation of the ITIM domain. (A) Raji-CD4 cells were transduced with a retroviral vector expressing a wild-type form of DCIR, a T6F mutant of DCIR, or a Y7F mutant of DCIR. Surface expression levels of DCIR was assessed by flow cytometry using a combination of PE-labeled anti-DCIR Ab (dotted lines) and a control isotype-matched Ab (continuous lines). Data shown correspond to a single experiment representative of 3 independent experiments. (B) For the virus binding/entry assay shown in the left panel, cells were exposed to NL4-3. For the infection assay shown in the right panel, the same cells were exposed to NL4-3 for 2 hours at 37°C, and then maintained in culture for 9 days. Data shown correspond to the means of triplicate samples from 3 independent experiments. The statistical significance of differences between Raji-CD4-DCIR, Raji-CD4-DCIR T6F, and Raji-CD4-DCIR Y7F is denoted by asterisks: *P < .05; **P < .01. (C) IM-MDDCs were treated with Pro-Ject only, with a control peptide or an ITIM peptide, either not phosphorylated or phosphorylated on the tyrosine or threonine residue, during 5 minutes at 37°C. All peptides used in this study were labeled with the fluorescent dye TAMRA. The total cell uptake of peptides was determined by flow cytometry. (D) Cells were next pulsed with NL4-3balenv for 60 minutes at 37°C and washed extensively before measuring the p24 content (left panel). In some experiments, similarly treated IM-MDDCs were pulsed with NL4-3balenv for 2 hours at 37°C, washed extensively, and maintained in complete culture medium supplemented with GM-CSF and IL-4. Cell-free culture supernatants were quantified by measuring the p24 content. Data shown correspond to the means of triplicate samples from 3 independent experiments. The statistical significance of differences between cells treated with nonphosphorylated ITIM peptide, ITIM peptide phosphorylated on the tyrosine residue, and ITIM peptide phosphorylated on the tyrosine residue is denoted by asterisks: ***P < .001.

DCIR-mediated enhancing effect on HIV-1 replication requires phosphorylation of the ITIM domain. (A) Raji-CD4 cells were transduced with a retroviral vector expressing a wild-type form of DCIR, a T6F mutant of DCIR, or a Y7F mutant of DCIR. Surface expression levels of DCIR was assessed by flow cytometry using a combination of PE-labeled anti-DCIR Ab (dotted lines) and a control isotype-matched Ab (continuous lines). Data shown correspond to a single experiment representative of 3 independent experiments. (B) For the virus binding/entry assay shown in the left panel, cells were exposed to NL4-3. For the infection assay shown in the right panel, the same cells were exposed to NL4-3 for 2 hours at 37°C, and then maintained in culture for 9 days. Data shown correspond to the means of triplicate samples from 3 independent experiments. The statistical significance of differences between Raji-CD4-DCIR, Raji-CD4-DCIR T6F, and Raji-CD4-DCIR Y7F is denoted by asterisks: *P < .05; **P < .01. (C) IM-MDDCs were treated with Pro-Ject only, with a control peptide or an ITIM peptide, either not phosphorylated or phosphorylated on the tyrosine or threonine residue, during 5 minutes at 37°C. All peptides used in this study were labeled with the fluorescent dye TAMRA. The total cell uptake of peptides was determined by flow cytometry. (D) Cells were next pulsed with NL4-3balenv for 60 minutes at 37°C and washed extensively before measuring the p24 content (left panel). In some experiments, similarly treated IM-MDDCs were pulsed with NL4-3balenv for 2 hours at 37°C, washed extensively, and maintained in complete culture medium supplemented with GM-CSF and IL-4. Cell-free culture supernatants were quantified by measuring the p24 content. Data shown correspond to the means of triplicate samples from 3 independent experiments. The statistical significance of differences between cells treated with nonphosphorylated ITIM peptide, ITIM peptide phosphorylated on the tyrosine residue, and ITIM peptide phosphorylated on the tyrosine residue is denoted by asterisks: ***P < .001.

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