Limits...
The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

Jin Q, Chen H, Wang X, Zhao L, Xu Q, Wang H, Li G, Yang X, Ma H, Wu H, Ji X - PLoS ONE (2015)

Bottom Line: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization.We also explored the possible mechanisms underlying the observed antiviral effects.This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu Province, China; Department of Neurology, The People's Hospital of Jiangsu Province, 300 Guangzhou Road, Nanjing, Jiangsu Province, China; Department of Neurology, Nanjing First Hospital, 68 Changle Road, Nanjing, Jiangsu Province, China.

ABSTRACT

Background: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.

Methods: We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.

Results: Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.

Conclusions: Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

No MeSH data available.


Related in: MedlinePlus

Recombinant CCR5-T4L acts as a HIV-1 co-receptor and down-regulates WT-CCR5 expression on the 3T3T4 cell surface.A) HIV-1 co-receptor function was estimated using cell-cell fusion assays. 3T3.T4 cells were transfected with 15 μg pSC-CCR5-T4L, then infected with vaccinia virus encoding LacZ gene under T7 promotor and used as target cells. The target cells were mixed with HeLa effector cells, expressing BaL Env protein and bacteriophage T7 RNA polymerase. After incubation for 2.5 h at 37°C, the amount of β-galactosidase activity was measured. WT-CCR5 was used as a positive control. B) CCR5 expression on the cell surface was confirmed using FACS. The level of CCR5 expression on above target 3T3.T4 cells was measured by flow cytometry using either phycoerythrin (PE)-conjugated 2D7 or phycoerythrin (PE)-conjugated 3A9 monoclonal antibodies. C and D) CCR5-T4L was more sensitive to CCR5 agonists maraviroc and TAK-779 inhibition. Different concentrations of maraviroc or TAK-779 were used to pretreat target 3T3.T4 cells transfected with pSC-CCR5-T4L or pSC-CCR5 for 1 h at 37°C. After washing with PBS, cell-cell fusion was performed. E and F) CCR5-T4L inhibited WT-CCR5 by reducing its expression on the cell surface. Different amounts of CCR5-T4L or WT-CCR5 cDNA (0 μg、 1 μg、 2.5 μg、5 μg、 7.5 μg and 10 μg) were co-transfected into 3T3.T4 cells. The effect of R5 HIV-1 Env-mediated cell-cell fusion was examined (E) and the level of CCR5 expression on the cell surface was measured by flow cytometry using a PE-conjugated monoclonal antibody (2D7) (F). G) The inhibition by CCR5-T4L decreased as the amounts of CCR5-T4L and WT-CCR5 increased. CCR5 expression was measured using flow cytometry and a PE-conjugated monoclonal antibody (2D7). The averaged mean fluorescence values (MFVs) for CCR5 from three experiments are plotted as a bar diagram, where WT-CCR5 expression after transfection with WT-CCR5 plus empty vector is set at 100. H and I) The interaction between CCR5-T4L and WT-CCR5 was tested using co-immunoprecipitation. 3T3.T4 cells co-transfected with CCR5-T4L and WT-CCR5 were lysed with RIPA buffer. Lysates were prepared and immunoprecipitated with the CCR5 C-terminal antibody, fractionated by SDS-PAGE, and immunoblotted. Blots were probed with the N-terminal CCR5 antibody (H), stripped, and reprobed with the anti-6×His antibody (I). Following the primary antibody reaction, blots were washed and probed with the anti-mouse antibody conjugated to HRP. Blots were exposed to X-ray film after reaction with the HRP substrate.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496087&req=5

pone.0131894.g001: Recombinant CCR5-T4L acts as a HIV-1 co-receptor and down-regulates WT-CCR5 expression on the 3T3T4 cell surface.A) HIV-1 co-receptor function was estimated using cell-cell fusion assays. 3T3.T4 cells were transfected with 15 μg pSC-CCR5-T4L, then infected with vaccinia virus encoding LacZ gene under T7 promotor and used as target cells. The target cells were mixed with HeLa effector cells, expressing BaL Env protein and bacteriophage T7 RNA polymerase. After incubation for 2.5 h at 37°C, the amount of β-galactosidase activity was measured. WT-CCR5 was used as a positive control. B) CCR5 expression on the cell surface was confirmed using FACS. The level of CCR5 expression on above target 3T3.T4 cells was measured by flow cytometry using either phycoerythrin (PE)-conjugated 2D7 or phycoerythrin (PE)-conjugated 3A9 monoclonal antibodies. C and D) CCR5-T4L was more sensitive to CCR5 agonists maraviroc and TAK-779 inhibition. Different concentrations of maraviroc or TAK-779 were used to pretreat target 3T3.T4 cells transfected with pSC-CCR5-T4L or pSC-CCR5 for 1 h at 37°C. After washing with PBS, cell-cell fusion was performed. E and F) CCR5-T4L inhibited WT-CCR5 by reducing its expression on the cell surface. Different amounts of CCR5-T4L or WT-CCR5 cDNA (0 μg、 1 μg、 2.5 μg、5 μg、 7.5 μg and 10 μg) were co-transfected into 3T3.T4 cells. The effect of R5 HIV-1 Env-mediated cell-cell fusion was examined (E) and the level of CCR5 expression on the cell surface was measured by flow cytometry using a PE-conjugated monoclonal antibody (2D7) (F). G) The inhibition by CCR5-T4L decreased as the amounts of CCR5-T4L and WT-CCR5 increased. CCR5 expression was measured using flow cytometry and a PE-conjugated monoclonal antibody (2D7). The averaged mean fluorescence values (MFVs) for CCR5 from three experiments are plotted as a bar diagram, where WT-CCR5 expression after transfection with WT-CCR5 plus empty vector is set at 100. H and I) The interaction between CCR5-T4L and WT-CCR5 was tested using co-immunoprecipitation. 3T3.T4 cells co-transfected with CCR5-T4L and WT-CCR5 were lysed with RIPA buffer. Lysates were prepared and immunoprecipitated with the CCR5 C-terminal antibody, fractionated by SDS-PAGE, and immunoblotted. Blots were probed with the N-terminal CCR5 antibody (H), stripped, and reprobed with the anti-6×His antibody (I). Following the primary antibody reaction, blots were washed and probed with the anti-mouse antibody conjugated to HRP. Blots were exposed to X-ray film after reaction with the HRP substrate.

Mentions: To assay the co-receptor function of CCR5-T4L, we transfected CCR5-T4L into 3T3.T4 cells and measured fusion efficiency using Env-mediated cell-cell fusion assays. Wild type CCR5 was used as a positive control. Expression of CCR5-T4L in 3T3.T4 cells resulted in efficient fusion of 3T3.T4 cells with HeLa-R5-BaL Env cells (Fig 1A), which can be detected on the cell surface using 2D7 and 3A9 monoclonal antibodies (Fig 1B). As shown in Fig 1B, both CCR5-T4L and WT-CCR5 were expressed on the 3T3.T4 cell surface. The results showed that CCR5-T4L has the same HIV co-receptor function as WT-CCR5. Furthermore, CCR5-T4L was more sensitive to the CCR5 agonists maraviroc and TAK-779 inhibition compared to WT-CCR5. As shown in Fig 1C and 1D, in CCR5-T4L expressing cells, IC50 values for maraviroc and TAK-779 were 0.6±0.23 nM and 1.67±0.49 nM, respectively. In contrast, wild type CCR5-expressing cells had IC50 values of 5.3±1.02 nM and 11.5±2.3 nM, respectively.


The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

Jin Q, Chen H, Wang X, Zhao L, Xu Q, Wang H, Li G, Yang X, Ma H, Wu H, Ji X - PLoS ONE (2015)

Recombinant CCR5-T4L acts as a HIV-1 co-receptor and down-regulates WT-CCR5 expression on the 3T3T4 cell surface.A) HIV-1 co-receptor function was estimated using cell-cell fusion assays. 3T3.T4 cells were transfected with 15 μg pSC-CCR5-T4L, then infected with vaccinia virus encoding LacZ gene under T7 promotor and used as target cells. The target cells were mixed with HeLa effector cells, expressing BaL Env protein and bacteriophage T7 RNA polymerase. After incubation for 2.5 h at 37°C, the amount of β-galactosidase activity was measured. WT-CCR5 was used as a positive control. B) CCR5 expression on the cell surface was confirmed using FACS. The level of CCR5 expression on above target 3T3.T4 cells was measured by flow cytometry using either phycoerythrin (PE)-conjugated 2D7 or phycoerythrin (PE)-conjugated 3A9 monoclonal antibodies. C and D) CCR5-T4L was more sensitive to CCR5 agonists maraviroc and TAK-779 inhibition. Different concentrations of maraviroc or TAK-779 were used to pretreat target 3T3.T4 cells transfected with pSC-CCR5-T4L or pSC-CCR5 for 1 h at 37°C. After washing with PBS, cell-cell fusion was performed. E and F) CCR5-T4L inhibited WT-CCR5 by reducing its expression on the cell surface. Different amounts of CCR5-T4L or WT-CCR5 cDNA (0 μg、 1 μg、 2.5 μg、5 μg、 7.5 μg and 10 μg) were co-transfected into 3T3.T4 cells. The effect of R5 HIV-1 Env-mediated cell-cell fusion was examined (E) and the level of CCR5 expression on the cell surface was measured by flow cytometry using a PE-conjugated monoclonal antibody (2D7) (F). G) The inhibition by CCR5-T4L decreased as the amounts of CCR5-T4L and WT-CCR5 increased. CCR5 expression was measured using flow cytometry and a PE-conjugated monoclonal antibody (2D7). The averaged mean fluorescence values (MFVs) for CCR5 from three experiments are plotted as a bar diagram, where WT-CCR5 expression after transfection with WT-CCR5 plus empty vector is set at 100. H and I) The interaction between CCR5-T4L and WT-CCR5 was tested using co-immunoprecipitation. 3T3.T4 cells co-transfected with CCR5-T4L and WT-CCR5 were lysed with RIPA buffer. Lysates were prepared and immunoprecipitated with the CCR5 C-terminal antibody, fractionated by SDS-PAGE, and immunoblotted. Blots were probed with the N-terminal CCR5 antibody (H), stripped, and reprobed with the anti-6×His antibody (I). Following the primary antibody reaction, blots were washed and probed with the anti-mouse antibody conjugated to HRP. Blots were exposed to X-ray film after reaction with the HRP substrate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496087&req=5

pone.0131894.g001: Recombinant CCR5-T4L acts as a HIV-1 co-receptor and down-regulates WT-CCR5 expression on the 3T3T4 cell surface.A) HIV-1 co-receptor function was estimated using cell-cell fusion assays. 3T3.T4 cells were transfected with 15 μg pSC-CCR5-T4L, then infected with vaccinia virus encoding LacZ gene under T7 promotor and used as target cells. The target cells were mixed with HeLa effector cells, expressing BaL Env protein and bacteriophage T7 RNA polymerase. After incubation for 2.5 h at 37°C, the amount of β-galactosidase activity was measured. WT-CCR5 was used as a positive control. B) CCR5 expression on the cell surface was confirmed using FACS. The level of CCR5 expression on above target 3T3.T4 cells was measured by flow cytometry using either phycoerythrin (PE)-conjugated 2D7 or phycoerythrin (PE)-conjugated 3A9 monoclonal antibodies. C and D) CCR5-T4L was more sensitive to CCR5 agonists maraviroc and TAK-779 inhibition. Different concentrations of maraviroc or TAK-779 were used to pretreat target 3T3.T4 cells transfected with pSC-CCR5-T4L or pSC-CCR5 for 1 h at 37°C. After washing with PBS, cell-cell fusion was performed. E and F) CCR5-T4L inhibited WT-CCR5 by reducing its expression on the cell surface. Different amounts of CCR5-T4L or WT-CCR5 cDNA (0 μg、 1 μg、 2.5 μg、5 μg、 7.5 μg and 10 μg) were co-transfected into 3T3.T4 cells. The effect of R5 HIV-1 Env-mediated cell-cell fusion was examined (E) and the level of CCR5 expression on the cell surface was measured by flow cytometry using a PE-conjugated monoclonal antibody (2D7) (F). G) The inhibition by CCR5-T4L decreased as the amounts of CCR5-T4L and WT-CCR5 increased. CCR5 expression was measured using flow cytometry and a PE-conjugated monoclonal antibody (2D7). The averaged mean fluorescence values (MFVs) for CCR5 from three experiments are plotted as a bar diagram, where WT-CCR5 expression after transfection with WT-CCR5 plus empty vector is set at 100. H and I) The interaction between CCR5-T4L and WT-CCR5 was tested using co-immunoprecipitation. 3T3.T4 cells co-transfected with CCR5-T4L and WT-CCR5 were lysed with RIPA buffer. Lysates were prepared and immunoprecipitated with the CCR5 C-terminal antibody, fractionated by SDS-PAGE, and immunoblotted. Blots were probed with the N-terminal CCR5 antibody (H), stripped, and reprobed with the anti-6×His antibody (I). Following the primary antibody reaction, blots were washed and probed with the anti-mouse antibody conjugated to HRP. Blots were exposed to X-ray film after reaction with the HRP substrate.
Mentions: To assay the co-receptor function of CCR5-T4L, we transfected CCR5-T4L into 3T3.T4 cells and measured fusion efficiency using Env-mediated cell-cell fusion assays. Wild type CCR5 was used as a positive control. Expression of CCR5-T4L in 3T3.T4 cells resulted in efficient fusion of 3T3.T4 cells with HeLa-R5-BaL Env cells (Fig 1A), which can be detected on the cell surface using 2D7 and 3A9 monoclonal antibodies (Fig 1B). As shown in Fig 1B, both CCR5-T4L and WT-CCR5 were expressed on the 3T3.T4 cell surface. The results showed that CCR5-T4L has the same HIV co-receptor function as WT-CCR5. Furthermore, CCR5-T4L was more sensitive to the CCR5 agonists maraviroc and TAK-779 inhibition compared to WT-CCR5. As shown in Fig 1C and 1D, in CCR5-T4L expressing cells, IC50 values for maraviroc and TAK-779 were 0.6±0.23 nM and 1.67±0.49 nM, respectively. In contrast, wild type CCR5-expressing cells had IC50 values of 5.3±1.02 nM and 11.5±2.3 nM, respectively.

Bottom Line: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization.We also explored the possible mechanisms underlying the observed antiviral effects.This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu Province, China; Department of Neurology, The People's Hospital of Jiangsu Province, 300 Guangzhou Road, Nanjing, Jiangsu Province, China; Department of Neurology, Nanjing First Hospital, 68 Changle Road, Nanjing, Jiangsu Province, China.

ABSTRACT

Background: Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.

Methods: We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.

Results: Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.

Conclusions: Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

No MeSH data available.


Related in: MedlinePlus