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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 disrupts anterograde transport of newly synthesized CD317 to the cell surface and traps trafficking CD317 molecules in the trans-Golgi network. The nuclei of TZM-bl cells, grown on coverslips, were comicroinjected with a CD317-HAint expression plasmid together with vectors encoding either GFP (A and C) or Vpu.GFP (B and D). Approximately 200 cells were microinjected per plasmid combination. Subsequently, cells were cultivated for 1, 2, 6, or 16 h and then fixed, permeabilized, and stained with an anti-HA MAb followed by an Alexa 568-conjugated secondary Ab (red staining) to detect newly synthesized CD317-HAint. (E and F) 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 either GFP (E) or Vpu.GFP (F). Histogram bars depict the relative percentage of cells for each time point from at least 150 cells that were analyzed out of three independent microinjection experiments. (G to J) Localization studies for trafficking CD317 molecules. (G and H) Internalized antibody-CD317 complexes. TZM-bl cells grown on coverslips were transfected with expression plasmids encoding either GFP or Vpu.GFP. Twenty-four hours posttransfection, cells were stained with unconjugated anti-HM1.24/CD317 MAb at 4°C before incubation at 37°C for 180 min. Anti-HM1.24 MAb–CD317 complexes were visualized in fixed and permeabilized cells using an Alexa 660-conjugated secondary antibody (blue staining). In addition, cells were stained with an anti-TGN46 Ab, which was detected with an Alexa 568-conjugated secondary antibody (red staining). Single-channel and merged confocal microphotographs for the localization of GFP or Vpu.GFP (both green), anti-HM1.24–CD317 complexes, and TGN46 are shown. (I and J) Newly synthesized CD317. TZM-bl cells were microinjected with plasmids encoding either GFP or Vpu.GFP as described above. Subsequently, cells were cultivated for 1 or 16 h and then fixed, permeabilized, and stained with an anti-HA MAb followed by an Alexa 660-conjugated secondary Ab (blue staining) and the TGN marker TGN46 (red staining). Microphotographs shown are representative for three independent experiments. In panels A, B, D, H, I, and J, cell circumferences are indicated. Scale bars, 10 µm. “Zoomed Merge” in panels G and H refers to a higher magnification of the box depicted in the image above in which the indicated color channels are superimposed.
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f4: Vpu disrupts anterograde transport of newly synthesized CD317 to the cell surface and traps trafficking CD317 molecules in the trans-Golgi network. The nuclei of TZM-bl cells, grown on coverslips, were comicroinjected with a CD317-HAint expression plasmid together with vectors encoding either GFP (A and C) or Vpu.GFP (B and D). Approximately 200 cells were microinjected per plasmid combination. Subsequently, cells were cultivated for 1, 2, 6, or 16 h and then fixed, permeabilized, and stained with an anti-HA MAb followed by an Alexa 568-conjugated secondary Ab (red staining) to detect newly synthesized CD317-HAint. (E and F) 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 either GFP (E) or Vpu.GFP (F). Histogram bars depict the relative percentage of cells for each time point from at least 150 cells that were analyzed out of three independent microinjection experiments. (G to J) Localization studies for trafficking CD317 molecules. (G and H) Internalized antibody-CD317 complexes. TZM-bl cells grown on coverslips were transfected with expression plasmids encoding either GFP or Vpu.GFP. Twenty-four hours posttransfection, cells were stained with unconjugated anti-HM1.24/CD317 MAb at 4°C before incubation at 37°C for 180 min. Anti-HM1.24 MAb–CD317 complexes were visualized in fixed and permeabilized cells using an Alexa 660-conjugated secondary antibody (blue staining). In addition, cells were stained with an anti-TGN46 Ab, which was detected with an Alexa 568-conjugated secondary antibody (red staining). Single-channel and merged confocal microphotographs for the localization of GFP or Vpu.GFP (both green), anti-HM1.24–CD317 complexes, and TGN46 are shown. (I and J) Newly synthesized CD317. TZM-bl cells were microinjected with plasmids encoding either GFP or Vpu.GFP as described above. Subsequently, cells were cultivated for 1 or 16 h and then fixed, permeabilized, and stained with an anti-HA MAb followed by an Alexa 660-conjugated secondary Ab (blue staining) and the TGN marker TGN46 (red staining). Microphotographs shown are representative for three independent experiments. In panels A, B, D, H, I, and J, cell circumferences are indicated. Scale bars, 10 µm. “Zoomed Merge” in panels G and H refers to a higher magnification of the box depicted in the image above in which the indicated color channels are superimposed.

Mentions: Protein expression was readily detectable 1 h postinjection by confocal microscopy. At this early time point, CD317-HAint molecules showed a primarily perinuclear, ER-like distribution in GFP-coexpressing control cells, while CD317-HAint localization at the plasma membrane was still rarely observed (Fig. 4A; quantification is shown in Fig. 4E). Already at 2 h, newly synthesized CD317-HAint molecules were detectable at the surface of ~50% of cells, indicative of a rapid transport to the plasma membrane. By 16 h, a strong surface localization was apparent in over 90% of cells in addition to cytoplasmic pools of the restriction factor (Fig. 4C and 4E).


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 disrupts anterograde transport of newly synthesized CD317 to the cell surface and traps trafficking CD317 molecules in the trans-Golgi network. The nuclei of TZM-bl cells, grown on coverslips, were comicroinjected with a CD317-HAint expression plasmid together with vectors encoding either GFP (A and C) or Vpu.GFP (B and D). Approximately 200 cells were microinjected per plasmid combination. Subsequently, cells were cultivated for 1, 2, 6, or 16 h and then fixed, permeabilized, and stained with an anti-HA MAb followed by an Alexa 568-conjugated secondary Ab (red staining) to detect newly synthesized CD317-HAint. (E and F) 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 either GFP (E) or Vpu.GFP (F). Histogram bars depict the relative percentage of cells for each time point from at least 150 cells that were analyzed out of three independent microinjection experiments. (G to J) Localization studies for trafficking CD317 molecules. (G and H) Internalized antibody-CD317 complexes. TZM-bl cells grown on coverslips were transfected with expression plasmids encoding either GFP or Vpu.GFP. Twenty-four hours posttransfection, cells were stained with unconjugated anti-HM1.24/CD317 MAb at 4°C before incubation at 37°C for 180 min. Anti-HM1.24 MAb–CD317 complexes were visualized in fixed and permeabilized cells using an Alexa 660-conjugated secondary antibody (blue staining). In addition, cells were stained with an anti-TGN46 Ab, which was detected with an Alexa 568-conjugated secondary antibody (red staining). Single-channel and merged confocal microphotographs for the localization of GFP or Vpu.GFP (both green), anti-HM1.24–CD317 complexes, and TGN46 are shown. (I and J) Newly synthesized CD317. TZM-bl cells were microinjected with plasmids encoding either GFP or Vpu.GFP as described above. Subsequently, cells were cultivated for 1 or 16 h and then fixed, permeabilized, and stained with an anti-HA MAb followed by an Alexa 660-conjugated secondary Ab (blue staining) and the TGN marker TGN46 (red staining). Microphotographs shown are representative for three independent experiments. In panels A, B, D, H, I, and J, cell circumferences are indicated. Scale bars, 10 µm. “Zoomed Merge” in panels G and H refers to a higher magnification of the box depicted in the image above in which the indicated color channels are superimposed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f4: Vpu disrupts anterograde transport of newly synthesized CD317 to the cell surface and traps trafficking CD317 molecules in the trans-Golgi network. The nuclei of TZM-bl cells, grown on coverslips, were comicroinjected with a CD317-HAint expression plasmid together with vectors encoding either GFP (A and C) or Vpu.GFP (B and D). Approximately 200 cells were microinjected per plasmid combination. Subsequently, cells were cultivated for 1, 2, 6, or 16 h and then fixed, permeabilized, and stained with an anti-HA MAb followed by an Alexa 568-conjugated secondary Ab (red staining) to detect newly synthesized CD317-HAint. (E and F) 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 either GFP (E) or Vpu.GFP (F). Histogram bars depict the relative percentage of cells for each time point from at least 150 cells that were analyzed out of three independent microinjection experiments. (G to J) Localization studies for trafficking CD317 molecules. (G and H) Internalized antibody-CD317 complexes. TZM-bl cells grown on coverslips were transfected with expression plasmids encoding either GFP or Vpu.GFP. Twenty-four hours posttransfection, cells were stained with unconjugated anti-HM1.24/CD317 MAb at 4°C before incubation at 37°C for 180 min. Anti-HM1.24 MAb–CD317 complexes were visualized in fixed and permeabilized cells using an Alexa 660-conjugated secondary antibody (blue staining). In addition, cells were stained with an anti-TGN46 Ab, which was detected with an Alexa 568-conjugated secondary antibody (red staining). Single-channel and merged confocal microphotographs for the localization of GFP or Vpu.GFP (both green), anti-HM1.24–CD317 complexes, and TGN46 are shown. (I and J) Newly synthesized CD317. TZM-bl cells were microinjected with plasmids encoding either GFP or Vpu.GFP as described above. Subsequently, cells were cultivated for 1 or 16 h and then fixed, permeabilized, and stained with an anti-HA MAb followed by an Alexa 660-conjugated secondary Ab (blue staining) and the TGN marker TGN46 (red staining). Microphotographs shown are representative for three independent experiments. In panels A, B, D, H, I, and J, cell circumferences are indicated. Scale bars, 10 µm. “Zoomed Merge” in panels G and H refers to a higher magnification of the box depicted in the image above in which the indicated color channels are superimposed.
Mentions: Protein expression was readily detectable 1 h postinjection by confocal microscopy. At this early time point, CD317-HAint molecules showed a primarily perinuclear, ER-like distribution in GFP-coexpressing control cells, while CD317-HAint localization at the plasma membrane was still rarely observed (Fig. 4A; quantification is shown in Fig. 4E). Already at 2 h, newly synthesized CD317-HAint molecules were detectable at the surface of ~50% of cells, indicative of a rapid transport to the plasma membrane. By 16 h, a strong surface localization was apparent in over 90% of cells in addition to cytoplasmic pools of the restriction factor (Fig. 4C and 4E).

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