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HIV-1 Vpu blocks recycling and biosynthetic transport of the intrinsic immunity factor CD317/tetherin to overcome the virion release restriction.

Schmidt S, Fritz JV, Bitzegeio J, Fackler OT, Keppler OT - MBio (2011)

Bottom Line: Expression of Vpu results in a reduction of CD317 surface levels; however, the mechanism of this Vpu activity and its contribution to the virological antagonism are incompletely understood.The subversion of both CD317 transport pathways was dependent on the highly conserved diserine S52/S56 motif of Vpu; however, it did not require recruitment of the diserine motif interactor and substrate adaptor of the SCF-E3 ubiquitin ligase complex, β-TrCP.Investigating the mechanism by which Vpu overcomes the virion release restriction imposed by CD317, we find that Vpu subverts recycling and anterograde trafficking pathways of CD317, resulting in surface levels of the restriction factor insufficient to block HIV-1 spread.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT

Unlabelled: The intrinsic immunity factor CD317 (BST-2/HM1.24/tetherin) imposes a barrier to HIV-1 release at the cell surface that can be overcome by the viral protein Vpu. Expression of Vpu results in a reduction of CD317 surface levels; however, the mechanism of this Vpu activity and its contribution to the virological antagonism are incompletely understood. Here, we characterized the influence of Vpu on major CD317 trafficking pathways using quantitative antibody-based endocytosis and recycling assays as well as a microinjection/microscopy-based kinetic de novo expression approach. We report that HIV-1 Vpu inhibited both the anterograde transport of newly synthesized CD317 and the recycling of CD317 to the cell surface, while the kinetics of CD317 endocytosis remained unaffected. Vpu trapped trafficking CD317 molecules at the trans-Golgi network, where the two molecules colocalized. The subversion of both CD317 transport pathways was dependent on the highly conserved diserine S52/S56 motif of Vpu; however, it did not require recruitment of the diserine motif interactor and substrate adaptor of the SCF-E3 ubiquitin ligase complex, β-TrCP. Treatment of cells with the malaria drug primaquine resulted in a CD317 trafficking defect that mirrored that induced by Vpu. Importantly, primaquine could functionally replace Vpu as a CD317 antagonist and rescue HIV-1 particle release.

Importance: HIV efficiently replicates in the human host and induces the life-threatening immunodeficiency AIDS. Mammalian genomes encode proteins such as CD317 that can inhibit viral replication at the cellular level. As a countermeasure, HIV has evolved genes like vpu that can antagonize these intrinsic immunity factors. Investigating the mechanism by which Vpu overcomes the virion release restriction imposed by CD317, we find that Vpu subverts recycling and anterograde trafficking pathways of CD317, resulting in surface levels of the restriction factor insufficient to block HIV-1 spread. This describes a novel mechanism of immune evasion by HIV.

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Related in: MedlinePlus

CD317 recycling is rapid and sensitive to primaquine. (A) Schematic representation of an antibody-based recycling assay. For assay validation, TZM-bl cells were transfected with a GFP-expressing plasmid. Twenty-four hours posttransfection, cells’ CD317 surface pool was first labeled with saturating concentrations of unconjugated anti-HM1.24/CD317 MAb at 4°C before incubation at 37°C for the indicated time intervals (t = 0 to 12 min) to allow recycling of previously intracellular, unlabeled CD317 molecules (blue). Subsequently, cells were stained with the identical anti-HM1.24/CD317 MAb, covalently conjugated to APC, at 4°C, and the MFI of GFP-positive cells was quantified by flow cytometry. Vesicles with recently internalized cargo are shown in magenta; recycling vesicles are shown in gray. (B) Representative FACS dot plots of CD317-APC staining of transfected TZM-bl cells in the recycling assay at steady state (left), at t = 0 min (middle), and at t = 12 min (right). The red gate depicts the high-GFP-expressing cells, and the respective MFI values are indicated within the gate. (C and D) Anti-HM1.24/CD317 MAb is not shed from antibody-CD317 complexes. (C) TZM-bl cells were first stained with unconjugated anti-HM1.24/CD317 MAb at 4°C, washed, and incubated for either 0 min or 40 min on ice. Antibody-CD317 complexes at the cell surface were subsequently detected with an APC-conjugated secondary antibody at 4°C and quantified by flow cytometry. (D) TZM-bl cells were transfected with expression plasmids encoding either GFP (filled circles) or the dominant negative dynamin K44A.GFP mutant (open circles). Twenty-four hours posttransfection, cells were analyzed in the endocytosis assay as described for Fig. S1A and C. The graph depicts the kinetics of relative CD317 levels remaining at the surface of viable, GFP-positive, or dynamin K44A.GFP-positive cells as relative percentages of the respective MFIs at t = 0 min, which were set to 100%. Shown are the arithmetic means ± standard deviations of triplicates from one experiment. Two independent assays were performed. (E) Recycling assay validation. pGFP-transfected TZM-bl cells were pretreated with either brefeldin A (0.2 mM, 2 h), cycloheximide (0.2 mM, 2 h), or primaquine (0.5 mM, 45 min) or were left untreated and subsequently analyzed in the CD317 recycling assay. The graph depicts the relative cell surface expression of CD317 detected by the APC-conjugated anti-HM1.24/CD317 MAb and is presented as the factor of enhancement of the MFIs at different time points relative to the MFI at t = 0, which was set to 1. Shown are the arithmetic means ± standard deviations of triplicates of one out of three independent experiments. (F) Microphotographs of brefeldin A-treated (0.2 mM, 2 h) and untreated control cells stained for the Golgi apparatus marker β-COP (red). Nuclei were counterstained with Hoechst stain. Scale bars, 10 µm.
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f1: CD317 recycling is rapid and sensitive to primaquine. (A) Schematic representation of an antibody-based recycling assay. For assay validation, TZM-bl cells were transfected with a GFP-expressing plasmid. Twenty-four hours posttransfection, cells’ CD317 surface pool was first labeled with saturating concentrations of unconjugated anti-HM1.24/CD317 MAb at 4°C before incubation at 37°C for the indicated time intervals (t = 0 to 12 min) to allow recycling of previously intracellular, unlabeled CD317 molecules (blue). Subsequently, cells were stained with the identical anti-HM1.24/CD317 MAb, covalently conjugated to APC, at 4°C, and the MFI of GFP-positive cells was quantified by flow cytometry. Vesicles with recently internalized cargo are shown in magenta; recycling vesicles are shown in gray. (B) Representative FACS dot plots of CD317-APC staining of transfected TZM-bl cells in the recycling assay at steady state (left), at t = 0 min (middle), and at t = 12 min (right). The red gate depicts the high-GFP-expressing cells, and the respective MFI values are indicated within the gate. (C and D) Anti-HM1.24/CD317 MAb is not shed from antibody-CD317 complexes. (C) TZM-bl cells were first stained with unconjugated anti-HM1.24/CD317 MAb at 4°C, washed, and incubated for either 0 min or 40 min on ice. Antibody-CD317 complexes at the cell surface were subsequently detected with an APC-conjugated secondary antibody at 4°C and quantified by flow cytometry. (D) TZM-bl cells were transfected with expression plasmids encoding either GFP (filled circles) or the dominant negative dynamin K44A.GFP mutant (open circles). Twenty-four hours posttransfection, cells were analyzed in the endocytosis assay as described for Fig. S1A and C. The graph depicts the kinetics of relative CD317 levels remaining at the surface of viable, GFP-positive, or dynamin K44A.GFP-positive cells as relative percentages of the respective MFIs at t = 0 min, which were set to 100%. Shown are the arithmetic means ± standard deviations of triplicates from one experiment. Two independent assays were performed. (E) Recycling assay validation. pGFP-transfected TZM-bl cells were pretreated with either brefeldin A (0.2 mM, 2 h), cycloheximide (0.2 mM, 2 h), or primaquine (0.5 mM, 45 min) or were left untreated and subsequently analyzed in the CD317 recycling assay. The graph depicts the relative cell surface expression of CD317 detected by the APC-conjugated anti-HM1.24/CD317 MAb and is presented as the factor of enhancement of the MFIs at different time points relative to the MFI at t = 0, which was set to 1. Shown are the arithmetic means ± standard deviations of triplicates of one out of three independent experiments. (F) Microphotographs of brefeldin A-treated (0.2 mM, 2 h) and untreated control cells stained for the Golgi apparatus marker β-COP (red). Nuclei were counterstained with Hoechst stain. Scale bars, 10 µm.

Mentions: To determine if Vpu affects the trafficking of endocytosed CD317 molecules back to the cell surface, we developed an antibody-based recycling assay (see schematic in Fig. 1A). This assay, in contrast to previously reported approaches (45–48), does not rely on harsh protease treatment, acid stripping, radioactive or biotin labeling, or ligand-induced endocytosis. Instead, it kinetically detects CD317 molecules surfacing at the plasma membrane in transfected or infected cells from intracellular pools, into which they had recently been endocytosed. In detail, TZM-bl cells were first incubated at 4°C with saturating concentrations of the unconjugated anti-HM1.24/CD317 MAb. Subsequently, a temperature shift to 37°C for various time periods (t = 0 to 12 min) allowed, besides endocytosis of antibody-CD317 complexes, recycling of intracellular and thus unlabeled CD317 molecules back to the cell surface (Fig. 1A, blue CD317 molecules). These newly surface-exposed molecules were then directly detected using the identical anti-HM1.24/CD317 MAb, which had been covalently coupled to the allophycocyanin (APC) fluorophore. It is important to realize that molecules remaining at the cell surface that still carry the unlabeled MAb cannot be recognized by the identical MAb coupled to APC.


HIV-1 Vpu blocks recycling and biosynthetic transport of the intrinsic immunity factor CD317/tetherin to overcome the virion release restriction.

Schmidt S, Fritz JV, Bitzegeio J, Fackler OT, Keppler OT - MBio (2011)

CD317 recycling is rapid and sensitive to primaquine. (A) Schematic representation of an antibody-based recycling assay. For assay validation, TZM-bl cells were transfected with a GFP-expressing plasmid. Twenty-four hours posttransfection, cells’ CD317 surface pool was first labeled with saturating concentrations of unconjugated anti-HM1.24/CD317 MAb at 4°C before incubation at 37°C for the indicated time intervals (t = 0 to 12 min) to allow recycling of previously intracellular, unlabeled CD317 molecules (blue). Subsequently, cells were stained with the identical anti-HM1.24/CD317 MAb, covalently conjugated to APC, at 4°C, and the MFI of GFP-positive cells was quantified by flow cytometry. Vesicles with recently internalized cargo are shown in magenta; recycling vesicles are shown in gray. (B) Representative FACS dot plots of CD317-APC staining of transfected TZM-bl cells in the recycling assay at steady state (left), at t = 0 min (middle), and at t = 12 min (right). The red gate depicts the high-GFP-expressing cells, and the respective MFI values are indicated within the gate. (C and D) Anti-HM1.24/CD317 MAb is not shed from antibody-CD317 complexes. (C) TZM-bl cells were first stained with unconjugated anti-HM1.24/CD317 MAb at 4°C, washed, and incubated for either 0 min or 40 min on ice. Antibody-CD317 complexes at the cell surface were subsequently detected with an APC-conjugated secondary antibody at 4°C and quantified by flow cytometry. (D) TZM-bl cells were transfected with expression plasmids encoding either GFP (filled circles) or the dominant negative dynamin K44A.GFP mutant (open circles). Twenty-four hours posttransfection, cells were analyzed in the endocytosis assay as described for Fig. S1A and C. The graph depicts the kinetics of relative CD317 levels remaining at the surface of viable, GFP-positive, or dynamin K44A.GFP-positive cells as relative percentages of the respective MFIs at t = 0 min, which were set to 100%. Shown are the arithmetic means ± standard deviations of triplicates from one experiment. Two independent assays were performed. (E) Recycling assay validation. pGFP-transfected TZM-bl cells were pretreated with either brefeldin A (0.2 mM, 2 h), cycloheximide (0.2 mM, 2 h), or primaquine (0.5 mM, 45 min) or were left untreated and subsequently analyzed in the CD317 recycling assay. The graph depicts the relative cell surface expression of CD317 detected by the APC-conjugated anti-HM1.24/CD317 MAb and is presented as the factor of enhancement of the MFIs at different time points relative to the MFI at t = 0, which was set to 1. Shown are the arithmetic means ± standard deviations of triplicates of one out of three independent experiments. (F) Microphotographs of brefeldin A-treated (0.2 mM, 2 h) and untreated control cells stained for the Golgi apparatus marker β-COP (red). Nuclei were counterstained with Hoechst stain. Scale bars, 10 µm.
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f1: CD317 recycling is rapid and sensitive to primaquine. (A) Schematic representation of an antibody-based recycling assay. For assay validation, TZM-bl cells were transfected with a GFP-expressing plasmid. Twenty-four hours posttransfection, cells’ CD317 surface pool was first labeled with saturating concentrations of unconjugated anti-HM1.24/CD317 MAb at 4°C before incubation at 37°C for the indicated time intervals (t = 0 to 12 min) to allow recycling of previously intracellular, unlabeled CD317 molecules (blue). Subsequently, cells were stained with the identical anti-HM1.24/CD317 MAb, covalently conjugated to APC, at 4°C, and the MFI of GFP-positive cells was quantified by flow cytometry. Vesicles with recently internalized cargo are shown in magenta; recycling vesicles are shown in gray. (B) Representative FACS dot plots of CD317-APC staining of transfected TZM-bl cells in the recycling assay at steady state (left), at t = 0 min (middle), and at t = 12 min (right). The red gate depicts the high-GFP-expressing cells, and the respective MFI values are indicated within the gate. (C and D) Anti-HM1.24/CD317 MAb is not shed from antibody-CD317 complexes. (C) TZM-bl cells were first stained with unconjugated anti-HM1.24/CD317 MAb at 4°C, washed, and incubated for either 0 min or 40 min on ice. Antibody-CD317 complexes at the cell surface were subsequently detected with an APC-conjugated secondary antibody at 4°C and quantified by flow cytometry. (D) TZM-bl cells were transfected with expression plasmids encoding either GFP (filled circles) or the dominant negative dynamin K44A.GFP mutant (open circles). Twenty-four hours posttransfection, cells were analyzed in the endocytosis assay as described for Fig. S1A and C. The graph depicts the kinetics of relative CD317 levels remaining at the surface of viable, GFP-positive, or dynamin K44A.GFP-positive cells as relative percentages of the respective MFIs at t = 0 min, which were set to 100%. Shown are the arithmetic means ± standard deviations of triplicates from one experiment. Two independent assays were performed. (E) Recycling assay validation. pGFP-transfected TZM-bl cells were pretreated with either brefeldin A (0.2 mM, 2 h), cycloheximide (0.2 mM, 2 h), or primaquine (0.5 mM, 45 min) or were left untreated and subsequently analyzed in the CD317 recycling assay. The graph depicts the relative cell surface expression of CD317 detected by the APC-conjugated anti-HM1.24/CD317 MAb and is presented as the factor of enhancement of the MFIs at different time points relative to the MFI at t = 0, which was set to 1. Shown are the arithmetic means ± standard deviations of triplicates of one out of three independent experiments. (F) Microphotographs of brefeldin A-treated (0.2 mM, 2 h) and untreated control cells stained for the Golgi apparatus marker β-COP (red). Nuclei were counterstained with Hoechst stain. Scale bars, 10 µm.
Mentions: To determine if Vpu affects the trafficking of endocytosed CD317 molecules back to the cell surface, we developed an antibody-based recycling assay (see schematic in Fig. 1A). This assay, in contrast to previously reported approaches (45–48), does not rely on harsh protease treatment, acid stripping, radioactive or biotin labeling, or ligand-induced endocytosis. Instead, it kinetically detects CD317 molecules surfacing at the plasma membrane in transfected or infected cells from intracellular pools, into which they had recently been endocytosed. In detail, TZM-bl cells were first incubated at 4°C with saturating concentrations of the unconjugated anti-HM1.24/CD317 MAb. Subsequently, a temperature shift to 37°C for various time periods (t = 0 to 12 min) allowed, besides endocytosis of antibody-CD317 complexes, recycling of intracellular and thus unlabeled CD317 molecules back to the cell surface (Fig. 1A, blue CD317 molecules). These newly surface-exposed molecules were then directly detected using the identical anti-HM1.24/CD317 MAb, which had been covalently coupled to the allophycocyanin (APC) fluorophore. It is important to realize that molecules remaining at the cell surface that still carry the unlabeled MAb cannot be recognized by the identical MAb coupled to APC.

Bottom Line: Expression of Vpu results in a reduction of CD317 surface levels; however, the mechanism of this Vpu activity and its contribution to the virological antagonism are incompletely understood.The subversion of both CD317 transport pathways was dependent on the highly conserved diserine S52/S56 motif of Vpu; however, it did not require recruitment of the diserine motif interactor and substrate adaptor of the SCF-E3 ubiquitin ligase complex, β-TrCP.Investigating the mechanism by which Vpu overcomes the virion release restriction imposed by CD317, we find that Vpu subverts recycling and anterograde trafficking pathways of CD317, resulting in surface levels of the restriction factor insufficient to block HIV-1 spread.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT

Unlabelled: The intrinsic immunity factor CD317 (BST-2/HM1.24/tetherin) imposes a barrier to HIV-1 release at the cell surface that can be overcome by the viral protein Vpu. Expression of Vpu results in a reduction of CD317 surface levels; however, the mechanism of this Vpu activity and its contribution to the virological antagonism are incompletely understood. Here, we characterized the influence of Vpu on major CD317 trafficking pathways using quantitative antibody-based endocytosis and recycling assays as well as a microinjection/microscopy-based kinetic de novo expression approach. We report that HIV-1 Vpu inhibited both the anterograde transport of newly synthesized CD317 and the recycling of CD317 to the cell surface, while the kinetics of CD317 endocytosis remained unaffected. Vpu trapped trafficking CD317 molecules at the trans-Golgi network, where the two molecules colocalized. The subversion of both CD317 transport pathways was dependent on the highly conserved diserine S52/S56 motif of Vpu; however, it did not require recruitment of the diserine motif interactor and substrate adaptor of the SCF-E3 ubiquitin ligase complex, β-TrCP. Treatment of cells with the malaria drug primaquine resulted in a CD317 trafficking defect that mirrored that induced by Vpu. Importantly, primaquine could functionally replace Vpu as a CD317 antagonist and rescue HIV-1 particle release.

Importance: HIV efficiently replicates in the human host and induces the life-threatening immunodeficiency AIDS. Mammalian genomes encode proteins such as CD317 that can inhibit viral replication at the cellular level. As a countermeasure, HIV has evolved genes like vpu that can antagonize these intrinsic immunity factors. Investigating the mechanism by which Vpu overcomes the virion release restriction imposed by CD317, we find that Vpu subverts recycling and anterograde trafficking pathways of CD317, resulting in surface levels of the restriction factor insufficient to block HIV-1 spread. This describes a novel mechanism of immune evasion by HIV.

Show MeSH
Related in: MedlinePlus