Figures & data
Figure 1. Expression of GPI-scFvs in transduced TZM-bl cells and their effects on CD4, CCR5, and CXCR4. (A) Schematic diagram of lentiviral transfer vector expressing a GPI-scFv or bifunctional inhibitor. The encoding sequence of scFv or a bifunctional inhibitor was genetically linked with the sequences encoding a His tag and the GPI attachment signal of DAF. LTR, long terminal repeat; RRE, Rev response element; cPPT, central polypurine track; PGK, human PGK promoter; DAF, delay-accelerating factor; WPRE, woodchuck hepatitits virus posttranscription regulatory element. (B) The expression of GPI-scFvs on the surface of transduced TZM-bl cells with or without PI-PLC treatment was determined by FACS analysis using an anti-His tag antibody. (C) Effects of GPI-scFvs on the expression of HIV receptors. The expression levels of CD4, CCR5, and CXCR4 on the surface of GPI-scFv-transduced TZM-bl cells were detected by FACS analysis using a PE-conjugated anti-human CD4, CCR5, or CXCR4 antibody and judged by the fluorescence intensity.
![Figure 1. Expression of GPI-scFvs in transduced TZM-bl cells and their effects on CD4, CCR5, and CXCR4. (A) Schematic diagram of lentiviral transfer vector expressing a GPI-scFv or bifunctional inhibitor. The encoding sequence of scFv or a bifunctional inhibitor was genetically linked with the sequences encoding a His tag and the GPI attachment signal of DAF. LTR, long terminal repeat; RRE, Rev response element; cPPT, central polypurine track; PGK, human PGK promoter; DAF, delay-accelerating factor; WPRE, woodchuck hepatitits virus posttranscription regulatory element. (B) The expression of GPI-scFvs on the surface of transduced TZM-bl cells with or without PI-PLC treatment was determined by FACS analysis using an anti-His tag antibody. (C) Effects of GPI-scFvs on the expression of HIV receptors. The expression levels of CD4, CCR5, and CXCR4 on the surface of GPI-scFv-transduced TZM-bl cells were detected by FACS analysis using a PE-conjugated anti-human CD4, CCR5, or CXCR4 antibody and judged by the fluorescence intensity.](/cms/asset/03e74f54-1af8-4962-917d-2b3b05be4c8f/temi_a_2011616_f0001_oc.jpg)
Figure 2. Confocal analyses of GPI-scFvs in transduced TZM-bl cells. Alexa555-CtxB stands for the lipid raft marker GM1 stained with Alexa 555-conjugated cholera toxin B subunit; Alexa488-Anti-His stands for the transduced cells stained with mouse anti-His tag antibody followed by Alexa 488-conjugated goat anti-mouse IgG antibody. Scale bar: 20 μm.
![Figure 2. Confocal analyses of GPI-scFvs in transduced TZM-bl cells. Alexa555-CtxB stands for the lipid raft marker GM1 stained with Alexa 555-conjugated cholera toxin B subunit; Alexa488-Anti-His stands for the transduced cells stained with mouse anti-His tag antibody followed by Alexa 488-conjugated goat anti-mouse IgG antibody. Scale bar: 20 μm.](/cms/asset/57fd3085-79ce-4875-9efc-81dc659f22b8/temi_a_2011616_f0002_oc.jpg)
Table 1. Inhibitory activities of GPI-scFvs against HIV-1 infection and Env-mediated cell-cell fusion.
Figure 3. Anti-HIV activities of GPI-scFvs in transduced cells. The inhibitory activities of GPI-scFvs against a panel of replication-competent HIV-1 isolates (A), the “global panel” of HIV-1 pseudoviruses (B), and viral Env-mediated cell-cell fusion (C) in were summarized for convenient overall view. Error bars indicate the means ± standard deviations (SD). (D-F) The inhibitory activities of GPI-scFvs on cell-cell HIV-1 transmission. TZM-bl cells expressing GPI-scFvs were used as a target and cocultured with CEMss-CCR5 donor cells that were preinfected with the HIV-1 isolates RHPA.c/2635 (D), THRO.c/2626 (E) and MJ4 (F). % cell-cell transmission was monitored by quantifying the production of the reporter luciferase in TZM-bl cells. Error bars indicate the means ± standard deviations (SD) from three independent experiments with triplicate samples, and statistical comparisons relative to the GPI-FluIgG03 control were conducted by ANOVA (ns, not significant; *, P< 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
![Figure 3. Anti-HIV activities of GPI-scFvs in transduced cells. The inhibitory activities of GPI-scFvs against a panel of replication-competent HIV-1 isolates (A), the “global panel” of HIV-1 pseudoviruses (B), and viral Env-mediated cell-cell fusion (C) in Table 1 were summarized for convenient overall view. Error bars indicate the means ± standard deviations (SD). (D-F) The inhibitory activities of GPI-scFvs on cell-cell HIV-1 transmission. TZM-bl cells expressing GPI-scFvs were used as a target and cocultured with CEMss-CCR5 donor cells that were preinfected with the HIV-1 isolates RHPA.c/2635 (D), THRO.c/2626 (E) and MJ4 (F). % cell-cell transmission was monitored by quantifying the production of the reporter luciferase in TZM-bl cells. Error bars indicate the means ± standard deviations (SD) from three independent experiments with triplicate samples, and statistical comparisons relative to the GPI-FluIgG03 control were conducted by ANOVA (ns, not significant; *, P< 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).](/cms/asset/a81c5ee4-221a-4a31-8176-b78804f54807/temi_a_2011616_f0003_oc.jpg)
Figure 4. Inhibitory activity of GPI-10E8 in transduced human CD4+ T cells against CXCR4-tropic and CCR5-tropic HIV-1 isolates. CEMss-CCR5 cells transduced with GPI-10E8/GFP or GPI-FluIgG03/GFP were infected with 1,000 TCID50 of NL4-3 (A), SG3.1 (B), MJ4 (C), or RHPA.c/2635 (D) and intracellular HIV-1 p24 antigen and GFP expression were monitored over time by flow cytometry. (E) Infection curves of the transduced CEMss-CCR5 cells. Data from a representative experiment of at least two independent experiments are shown.
![Figure 4. Inhibitory activity of GPI-10E8 in transduced human CD4+ T cells against CXCR4-tropic and CCR5-tropic HIV-1 isolates. CEMss-CCR5 cells transduced with GPI-10E8/GFP or GPI-FluIgG03/GFP were infected with 1,000 TCID50 of NL4-3 (A), SG3.1 (B), MJ4 (C), or RHPA.c/2635 (D) and intracellular HIV-1 p24 antigen and GFP expression were monitored over time by flow cytometry. (E) Infection curves of the transduced CEMss-CCR5 cells. Data from a representative experiment of at least two independent experiments are shown.](/cms/asset/b5be39ef-078c-4970-bba3-b3609a757956/temi_a_2011616_f0004_oc.jpg)
Figure 5. Selective survival of human CD4+ T cells expressing GPI-10E8 during CXCR4-tropic or CCR5-tropic HIV-1 infection. CEMss-CCR5 cells were transduced with GPI-10E8/GFP and mixed with untransduced cells at a ratio of approximately 20% GFP-positive cells. The mixed population was challenged with 1,000 TCID50 of CXCR4-tropic NL4-3 (A) or CCR5-tropic RHPA.c/2635 (B), and the proportion of transgene-expressing cells was monitored over time by flow cytometry. (C) Survival curve of the transduced CEMss-CCR5 cells. Data from a representative experiment of at least two independent experiments are shown.
![Figure 5. Selective survival of human CD4+ T cells expressing GPI-10E8 during CXCR4-tropic or CCR5-tropic HIV-1 infection. CEMss-CCR5 cells were transduced with GPI-10E8/GFP and mixed with untransduced cells at a ratio of approximately 20% GFP-positive cells. The mixed population was challenged with 1,000 TCID50 of CXCR4-tropic NL4-3 (A) or CCR5-tropic RHPA.c/2635 (B), and the proportion of transgene-expressing cells was monitored over time by flow cytometry. (C) Survival curve of the transduced CEMss-CCR5 cells. Data from a representative experiment of at least two independent experiments are shown.](/cms/asset/b93be934-fda9-417a-bd32-7a1b22ff06ff/temi_a_2011616_f0005_oc.jpg)
Figure 6. Design and characterization of GPI-anchored bifunctional inhibitors targeting gp41. (A) Design strategy. The sequence of a fusion inhibitor peptide P41 was genetically connected to the N- or C-termini of 10E8 scFv through a flexible (GGGGS)n linker, generating eight GPI-anchored bifunctional constructs named CMI01 ∼ CMI08. (B) The expression of CMI constructs was detected in the cell lysates and culture supernatants of transduced TZM-bl cells by western blotting with an anti-His tag antibody. (C) Surface expression of CMI constructs in transduced TZM-bl cells with or without PI-PLC treatment was determined by FACS analysis using an anti-His tag antibody. (D) Effects of CMI constructs on the surface expression of CD4, CCR5, and CXCR4 in transduced TZM-bl cells were detected by FACS analysis using a PE-conjugated anti-human CD4, CCR5, or CXCR4 antibody and judged by fluorescence intensity.
![Figure 6. Design and characterization of GPI-anchored bifunctional inhibitors targeting gp41. (A) Design strategy. The sequence of a fusion inhibitor peptide P41 was genetically connected to the N- or C-termini of 10E8 scFv through a flexible (GGGGS)n linker, generating eight GPI-anchored bifunctional constructs named CMI01 ∼ CMI08. (B) The expression of CMI constructs was detected in the cell lysates and culture supernatants of transduced TZM-bl cells by western blotting with an anti-His tag antibody. (C) Surface expression of CMI constructs in transduced TZM-bl cells with or without PI-PLC treatment was determined by FACS analysis using an anti-His tag antibody. (D) Effects of CMI constructs on the surface expression of CD4, CCR5, and CXCR4 in transduced TZM-bl cells were detected by FACS analysis using a PE-conjugated anti-human CD4, CCR5, or CXCR4 antibody and judged by fluorescence intensity.](/cms/asset/d41c757e-f2e0-49d6-964c-8fae16debb0c/temi_a_2011616_f0006_oc.jpg)
Figure 7. Antiviral activity of GPI-anchored bifunctional inhibitors in transduced cells. The inhibitory activities of CMI constructs against two HIV-1 pseudoviruses (A), two replicative HIV-2 isolates (B), and two SIV pseudoviruses (C), as well as the inhibitory activities of the CMI02 and CMI06 mutants with the disruptive P41 mutations on the HIV-1SF162 and SIVmac239 pseudoviruses (D) and their Env-mediated cell-cell fusion (E), and the cell-cell HIV-1 transmission of three replicative HIV-1 isolates (F) were respectively determined in transduced TZM-bl or 293FTtarget cells. (G) The inhibitory activities of CMI02 and CMI06 constructs against the cell-cell fusion mediated by a panel of HIV-1 Envs that were partially resistant to GPI-10E8. (H) The inhibitory activities of GPI-10E8 and CMI constructs on VSV-G infection. (I-K) The anti-HIV activities of CMI02 and CMI06 in human primary CD4+ T cells. Transduced human primary CD4+ T cells were infected with 1,000 TCID50 of NL4-3, and intracellular p24 and GFP expression were monitored over time by flow cytometry. Infection curves of the transduced CD4+ T cells (I) represent the means ± SD from three independent experiments with triplicate samples, and statistical comparison was only performed between GPI-FluIgG03-ransduced cells and CMI02 or CMI06-transduced cells at day 6 after NL4-3 infection (****, P < 0.0001) as the most GPI-FluIgG03-transduced cells were dead at day 9. The survival curves of human CD4+ T cells transduced with CMI02 (J) and CMI06 (K) were statistically analyzed. The results of three independent experiments are shown with different colours of lines.
![Figure 7. Antiviral activity of GPI-anchored bifunctional inhibitors in transduced cells. The inhibitory activities of CMI constructs against two HIV-1 pseudoviruses (A), two replicative HIV-2 isolates (B), and two SIV pseudoviruses (C), as well as the inhibitory activities of the CMI02 and CMI06 mutants with the disruptive P41 mutations on the HIV-1SF162 and SIVmac239 pseudoviruses (D) and their Env-mediated cell-cell fusion (E), and the cell-cell HIV-1 transmission of three replicative HIV-1 isolates (F) were respectively determined in transduced TZM-bl or 293FTtarget cells. (G) The inhibitory activities of CMI02 and CMI06 constructs against the cell-cell fusion mediated by a panel of HIV-1 Envs that were partially resistant to GPI-10E8. (H) The inhibitory activities of GPI-10E8 and CMI constructs on VSV-G infection. (I-K) The anti-HIV activities of CMI02 and CMI06 in human primary CD4+ T cells. Transduced human primary CD4+ T cells were infected with 1,000 TCID50 of NL4-3, and intracellular p24 and GFP expression were monitored over time by flow cytometry. Infection curves of the transduced CD4+ T cells (I) represent the means ± SD from three independent experiments with triplicate samples, and statistical comparison was only performed between GPI-FluIgG03-ransduced cells and CMI02 or CMI06-transduced cells at day 6 after NL4-3 infection (****, P < 0.0001) as the most GPI-FluIgG03-transduced cells were dead at day 9. The survival curves of human CD4+ T cells transduced with CMI02 (J) and CMI06 (K) were statistically analyzed. The results of three independent experiments are shown with different colours of lines.](/cms/asset/5fc6b7e3-e978-4e73-a9ce-6da75105e310/temi_a_2011616_f0007_oc.jpg)
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