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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

Vpu’s ability to block CD317 recycling and anterograde transport depends on its diserine motif but not on proteasomal degradation of CD317 or β-TrCP. (A and B) Recycling CD317. TZM-bl cells were transfected with expression plasmids encoding either Vpu.GFP, Vpu2/6.GFP, or GFP. Twenty-four hours posttransfection, GFP-expressing cells were analyzed for surface steady-state levels of CD317 (A) or in the CD317 recycling assay (B). Shown are arithmetic means ± standard deviations of triplicates of one representative of three independent experiments. (C, D, and E) Newly synthesized CD317. TZM-bl cells were comicroinjected with CD317-HAint and Vpu2/6.GFP expression plasmids. Cells were cultivated for 1, 2, 6, or 16 h and then fixed. Next, cells were permeabilized and stained with an Alexa 568-conjugated anti-HA MAb (red staining) to detect newly synthesized CD317-HAint molecules. Microphotographs shown are representative for three independent experiments. Scale bars, 10 µm. By staining, cells were categorized as those which, besides intracellular staining, also displayed a clear plasma membrane staining (“plasma membrane”) or those with an exclusive intracellular staining (“intracellular”) of CD317-HAint upon coexpression of Vpu2/6.GFP. For each time point, at least 150 cells out of three independent microinjection experiments were counted. (F) Inhibition of proteasomal degradation by ALLN (N-acetylleucylleucylnorleucinal) does not interfere with Vpu’s ability to block CD317 recycling. TZM-bl cells were transfected with expression plasmids encoding either Vpu.GFP or GFP. At 6 h posttransfection, either ALLN (50 µM) or DMSO solvent was added to the culture medium for 18 h. Twenty-four hours posttransfection, Vpu.GFP/GFP-expressing cells were analyzed in the CD317 recycling assay or stained for CD4 surface expression (Fig. S4A). Shown is one representative of two independent experiments. (G) β-TrCP is not strictly required for Vpu’s ability to block CD317 recycling. TZM-bl cells were transfected twice with siRNAs targeting β-TrCP1 and β-TrCP2 (β-TrCP siRNA) or control siRNA. At the second transfection, expression plasmids encoding either Vpu.GFP or GFP were added. Three days following the first transfection, TZM-bl cells were harvested and analyzed in the CD317 recycling assay or stained for CD317 (Fig. S4B) or CD4 (Fig. S4C) surface expression. Shown is one representative experiment of four independent experiments, and arithmetic means ± standard deviations of triplicates are depicted. Significance by Student’s t test: *, P < 0.05; **, P < 0.02; ***, P < 0.002.
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f5: Vpu’s ability to block CD317 recycling and anterograde transport depends on its diserine motif but not on proteasomal degradation of CD317 or β-TrCP. (A and B) Recycling CD317. TZM-bl cells were transfected with expression plasmids encoding either Vpu.GFP, Vpu2/6.GFP, or GFP. Twenty-four hours posttransfection, GFP-expressing cells were analyzed for surface steady-state levels of CD317 (A) or in the CD317 recycling assay (B). Shown are arithmetic means ± standard deviations of triplicates of one representative of three independent experiments. (C, D, and E) Newly synthesized CD317. TZM-bl cells were comicroinjected with CD317-HAint and Vpu2/6.GFP expression plasmids. Cells were cultivated for 1, 2, 6, or 16 h and then fixed. Next, cells were permeabilized and stained with an Alexa 568-conjugated anti-HA MAb (red staining) to detect newly synthesized CD317-HAint molecules. Microphotographs shown are representative for three independent experiments. Scale bars, 10 µm. By staining, cells were categorized as those which, besides intracellular staining, also displayed a clear plasma membrane staining (“plasma membrane”) or those with an exclusive intracellular staining (“intracellular”) of CD317-HAint upon coexpression of Vpu2/6.GFP. For each time point, at least 150 cells out of three independent microinjection experiments were counted. (F) Inhibition of proteasomal degradation by ALLN (N-acetylleucylleucylnorleucinal) does not interfere with Vpu’s ability to block CD317 recycling. TZM-bl cells were transfected with expression plasmids encoding either Vpu.GFP or GFP. At 6 h posttransfection, either ALLN (50 µM) or DMSO solvent was added to the culture medium for 18 h. Twenty-four hours posttransfection, Vpu.GFP/GFP-expressing cells were analyzed in the CD317 recycling assay or stained for CD4 surface expression (Fig. S4A). Shown is one representative of two independent experiments. (G) β-TrCP is not strictly required for Vpu’s ability to block CD317 recycling. TZM-bl cells were transfected twice with siRNAs targeting β-TrCP1 and β-TrCP2 (β-TrCP siRNA) or control siRNA. At the second transfection, expression plasmids encoding either Vpu.GFP or GFP were added. Three days following the first transfection, TZM-bl cells were harvested and analyzed in the CD317 recycling assay or stained for CD317 (Fig. S4B) or CD4 (Fig. S4C) surface expression. Shown is one representative experiment of four independent experiments, and arithmetic means ± standard deviations of triplicates are depicted. Significance by Student’s t test: *, P < 0.05; **, P < 0.02; ***, P < 0.002.

Mentions: Since the highly conserved serine residues 52 and 56 in the cytoplasmic tail of Vpu are critical for antagonizing the CD317-imposed restriction on HIV-1 particle release when expressed in cis or trans (26, 34, 55, 56), we tested whether this diserine motif affects Vpu’s ability to inhibit the recycling and anterograde transport of CD317. Consistent with a number of recent reports (29, 31, 34, 55, 56), we observed that the reduction of CD317 steady-state surface levels in TZM-bl cells depended on Vpu’s diserine motif, as alanine replacements of serine residues 52 and 56 (Vpu2/6) largely abrogated its downmodulating activity (Fig. 5A).


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)

Vpu’s ability to block CD317 recycling and anterograde transport depends on its diserine motif but not on proteasomal degradation of CD317 or β-TrCP. (A and B) Recycling CD317. TZM-bl cells were transfected with expression plasmids encoding either Vpu.GFP, Vpu2/6.GFP, or GFP. Twenty-four hours posttransfection, GFP-expressing cells were analyzed for surface steady-state levels of CD317 (A) or in the CD317 recycling assay (B). Shown are arithmetic means ± standard deviations of triplicates of one representative of three independent experiments. (C, D, and E) Newly synthesized CD317. TZM-bl cells were comicroinjected with CD317-HAint and Vpu2/6.GFP expression plasmids. Cells were cultivated for 1, 2, 6, or 16 h and then fixed. Next, cells were permeabilized and stained with an Alexa 568-conjugated anti-HA MAb (red staining) to detect newly synthesized CD317-HAint molecules. Microphotographs shown are representative for three independent experiments. Scale bars, 10 µm. By staining, cells were categorized as those which, besides intracellular staining, also displayed a clear plasma membrane staining (“plasma membrane”) or those with an exclusive intracellular staining (“intracellular”) of CD317-HAint upon coexpression of Vpu2/6.GFP. For each time point, at least 150 cells out of three independent microinjection experiments were counted. (F) Inhibition of proteasomal degradation by ALLN (N-acetylleucylleucylnorleucinal) does not interfere with Vpu’s ability to block CD317 recycling. TZM-bl cells were transfected with expression plasmids encoding either Vpu.GFP or GFP. At 6 h posttransfection, either ALLN (50 µM) or DMSO solvent was added to the culture medium for 18 h. Twenty-four hours posttransfection, Vpu.GFP/GFP-expressing cells were analyzed in the CD317 recycling assay or stained for CD4 surface expression (Fig. S4A). Shown is one representative of two independent experiments. (G) β-TrCP is not strictly required for Vpu’s ability to block CD317 recycling. TZM-bl cells were transfected twice with siRNAs targeting β-TrCP1 and β-TrCP2 (β-TrCP siRNA) or control siRNA. At the second transfection, expression plasmids encoding either Vpu.GFP or GFP were added. Three days following the first transfection, TZM-bl cells were harvested and analyzed in the CD317 recycling assay or stained for CD317 (Fig. S4B) or CD4 (Fig. S4C) surface expression. Shown is one representative experiment of four independent experiments, and arithmetic means ± standard deviations of triplicates are depicted. Significance by Student’s t test: *, P < 0.05; **, P < 0.02; ***, P < 0.002.
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Related In: Results  -  Collection

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f5: Vpu’s ability to block CD317 recycling and anterograde transport depends on its diserine motif but not on proteasomal degradation of CD317 or β-TrCP. (A and B) Recycling CD317. TZM-bl cells were transfected with expression plasmids encoding either Vpu.GFP, Vpu2/6.GFP, or GFP. Twenty-four hours posttransfection, GFP-expressing cells were analyzed for surface steady-state levels of CD317 (A) or in the CD317 recycling assay (B). Shown are arithmetic means ± standard deviations of triplicates of one representative of three independent experiments. (C, D, and E) Newly synthesized CD317. TZM-bl cells were comicroinjected with CD317-HAint and Vpu2/6.GFP expression plasmids. Cells were cultivated for 1, 2, 6, or 16 h and then fixed. Next, cells were permeabilized and stained with an Alexa 568-conjugated anti-HA MAb (red staining) to detect newly synthesized CD317-HAint molecules. Microphotographs shown are representative for three independent experiments. Scale bars, 10 µm. By staining, cells were categorized as those which, besides intracellular staining, also displayed a clear plasma membrane staining (“plasma membrane”) or those with an exclusive intracellular staining (“intracellular”) of CD317-HAint upon coexpression of Vpu2/6.GFP. For each time point, at least 150 cells out of three independent microinjection experiments were counted. (F) Inhibition of proteasomal degradation by ALLN (N-acetylleucylleucylnorleucinal) does not interfere with Vpu’s ability to block CD317 recycling. TZM-bl cells were transfected with expression plasmids encoding either Vpu.GFP or GFP. At 6 h posttransfection, either ALLN (50 µM) or DMSO solvent was added to the culture medium for 18 h. Twenty-four hours posttransfection, Vpu.GFP/GFP-expressing cells were analyzed in the CD317 recycling assay or stained for CD4 surface expression (Fig. S4A). Shown is one representative of two independent experiments. (G) β-TrCP is not strictly required for Vpu’s ability to block CD317 recycling. TZM-bl cells were transfected twice with siRNAs targeting β-TrCP1 and β-TrCP2 (β-TrCP siRNA) or control siRNA. At the second transfection, expression plasmids encoding either Vpu.GFP or GFP were added. Three days following the first transfection, TZM-bl cells were harvested and analyzed in the CD317 recycling assay or stained for CD317 (Fig. S4B) or CD4 (Fig. S4C) surface expression. Shown is one representative experiment of four independent experiments, and arithmetic means ± standard deviations of triplicates are depicted. Significance by Student’s t test: *, P < 0.05; **, P < 0.02; ***, P < 0.002.
Mentions: Since the highly conserved serine residues 52 and 56 in the cytoplasmic tail of Vpu are critical for antagonizing the CD317-imposed restriction on HIV-1 particle release when expressed in cis or trans (26, 34, 55, 56), we tested whether this diserine motif affects Vpu’s ability to inhibit the recycling and anterograde transport of CD317. Consistent with a number of recent reports (29, 31, 34, 55, 56), we observed that the reduction of CD317 steady-state surface levels in TZM-bl cells depended on Vpu’s diserine motif, as alanine replacements of serine residues 52 and 56 (Vpu2/6) largely abrogated its downmodulating activity (Fig. 5A).

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