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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

Antiviral activities of soluble recombinant CCR5-T4L against R5-tropic and X4-tropic virus.A and D) The inhibitory activities using cell-cell fusion assays. Effector HeLa cells co-expressing the R5 viral envelope BaL (A) or X4 (D) envelope LAV were pre-incubated for 30 min at 37°C with increasing concentrations of soluble recombinant CCR5-T4L plus 2 nM of soluble CD4. After washing with PBS, cell-cell fusion was performed. B and E) The inhibitory activities using single cycle viral infection assays in THP1 cells. Cells were pre-cultured overnight and infected with BaL or IIIB at 100 TCID50 in the presence or absence of soluble recombinant CCR5-T4L. Luciferase activity was analyzed using a luciferase kit 8 days post-infection. C and F) The inhibitory activities using replication competent viral assays with BaL strain virus- (C) and IIIB strain virus- (F) in THP1 cells. CCR5-tropic HIV-1 ADA was used as a positive control. THP1 cells (1×106 cells per well) were pre-cultured overnight and infected with BaL or IIIB at 100 TCID50 in the presence or absence of soluble recombinant CCR5-T4L overnight. Supernatants were collected 7 days post-infection and tested for p24 antigen using ELISA. Data are the average from three independent experiments. Recombinant ligand CCL5 and PBS were used as controls.
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pone.0131894.g003: Antiviral activities of soluble recombinant CCR5-T4L against R5-tropic and X4-tropic virus.A and D) The inhibitory activities using cell-cell fusion assays. Effector HeLa cells co-expressing the R5 viral envelope BaL (A) or X4 (D) envelope LAV were pre-incubated for 30 min at 37°C with increasing concentrations of soluble recombinant CCR5-T4L plus 2 nM of soluble CD4. After washing with PBS, cell-cell fusion was performed. B and E) The inhibitory activities using single cycle viral infection assays in THP1 cells. Cells were pre-cultured overnight and infected with BaL or IIIB at 100 TCID50 in the presence or absence of soluble recombinant CCR5-T4L. Luciferase activity was analyzed using a luciferase kit 8 days post-infection. C and F) The inhibitory activities using replication competent viral assays with BaL strain virus- (C) and IIIB strain virus- (F) in THP1 cells. CCR5-tropic HIV-1 ADA was used as a positive control. THP1 cells (1×106 cells per well) were pre-cultured overnight and infected with BaL or IIIB at 100 TCID50 in the presence or absence of soluble recombinant CCR5-T4L overnight. Supernatants were collected 7 days post-infection and tested for p24 antigen using ELISA. Data are the average from three independent experiments. Recombinant ligand CCL5 and PBS were used as controls.

Mentions: As shown in Fig 3A, treatment of HeLa cells expressing R5 HIV Env BaL with low concentration (10−12 M level) of soluble recombinant CCR5-T4L protein plus 2 nM of soluble CD4 induced significant cell-cell fusion promotion. The highest fusion was achieved at 5×10−10 M. Further increases in recombinant protein concentration resulted in dose-dependent inhibition of cell fusion (Fig 3A). In the presence of CCL5 alone, only inhibition was observed. In contrast, treatment of HeLa cells expressing X4 HIV Env IIIB with same amount of protein had no effect on cell-cell fusion (Fig 3D). These data indicate that recombinant CCR5-T4L promotes R5 HIV Env-mediated cell-cell fusion, but not X4 HIV Env-mediated cell-cell fusion, at low concentrations and inhibits it at higher concentrations.


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)

Antiviral activities of soluble recombinant CCR5-T4L against R5-tropic and X4-tropic virus.A and D) The inhibitory activities using cell-cell fusion assays. Effector HeLa cells co-expressing the R5 viral envelope BaL (A) or X4 (D) envelope LAV were pre-incubated for 30 min at 37°C with increasing concentrations of soluble recombinant CCR5-T4L plus 2 nM of soluble CD4. After washing with PBS, cell-cell fusion was performed. B and E) The inhibitory activities using single cycle viral infection assays in THP1 cells. Cells were pre-cultured overnight and infected with BaL or IIIB at 100 TCID50 in the presence or absence of soluble recombinant CCR5-T4L. Luciferase activity was analyzed using a luciferase kit 8 days post-infection. C and F) The inhibitory activities using replication competent viral assays with BaL strain virus- (C) and IIIB strain virus- (F) in THP1 cells. CCR5-tropic HIV-1 ADA was used as a positive control. THP1 cells (1×106 cells per well) were pre-cultured overnight and infected with BaL or IIIB at 100 TCID50 in the presence or absence of soluble recombinant CCR5-T4L overnight. Supernatants were collected 7 days post-infection and tested for p24 antigen using ELISA. Data are the average from three independent experiments. Recombinant ligand CCL5 and PBS were used as controls.
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Related In: Results  -  Collection

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pone.0131894.g003: Antiviral activities of soluble recombinant CCR5-T4L against R5-tropic and X4-tropic virus.A and D) The inhibitory activities using cell-cell fusion assays. Effector HeLa cells co-expressing the R5 viral envelope BaL (A) or X4 (D) envelope LAV were pre-incubated for 30 min at 37°C with increasing concentrations of soluble recombinant CCR5-T4L plus 2 nM of soluble CD4. After washing with PBS, cell-cell fusion was performed. B and E) The inhibitory activities using single cycle viral infection assays in THP1 cells. Cells were pre-cultured overnight and infected with BaL or IIIB at 100 TCID50 in the presence or absence of soluble recombinant CCR5-T4L. Luciferase activity was analyzed using a luciferase kit 8 days post-infection. C and F) The inhibitory activities using replication competent viral assays with BaL strain virus- (C) and IIIB strain virus- (F) in THP1 cells. CCR5-tropic HIV-1 ADA was used as a positive control. THP1 cells (1×106 cells per well) were pre-cultured overnight and infected with BaL or IIIB at 100 TCID50 in the presence or absence of soluble recombinant CCR5-T4L overnight. Supernatants were collected 7 days post-infection and tested for p24 antigen using ELISA. Data are the average from three independent experiments. Recombinant ligand CCL5 and PBS were used as controls.
Mentions: As shown in Fig 3A, treatment of HeLa cells expressing R5 HIV Env BaL with low concentration (10−12 M level) of soluble recombinant CCR5-T4L protein plus 2 nM of soluble CD4 induced significant cell-cell fusion promotion. The highest fusion was achieved at 5×10−10 M. Further increases in recombinant protein concentration resulted in dose-dependent inhibition of cell fusion (Fig 3A). In the presence of CCL5 alone, only inhibition was observed. In contrast, treatment of HeLa cells expressing X4 HIV Env IIIB with same amount of protein had no effect on cell-cell fusion (Fig 3D). These data indicate that recombinant CCR5-T4L promotes R5 HIV Env-mediated cell-cell fusion, but not X4 HIV Env-mediated cell-cell fusion, at low concentrations and inhibits it at higher concentrations.

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