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. 2009 Oct 30;139(3):499-511.
doi: 10.1016/j.cell.2009.08.039.

Tetherin inhibits HIV-1 release by directly tethering virions to cells

David Perez-Caballero  1 , Trinity Zang, Alaleh Ebrahimi, Matthew W McNatt, Devon A Gregory, Marc C Johnson, Paul D Bieniasz
Affiliations

Tetherin inhibits HIV-1 release by directly tethering virions to cells

David Perez-Caballero et al. Cell. .

Abstract

Tetherin is an interferon-induced protein whose expression blocks the release of HIV-1 and other enveloped viral particles. The underlying mechanism by which tetherin functions and whether it directly or indirectly causes virion retention are unknown. Here, we elucidate the mechanism by which tetherin exerts its antiviral activity. We demonstrate, through mutational analyses and domain replacement experiments, that tetherin configuration rather than primary sequence is critical for antiviral activity. These findings allowed the design of a completely artificial protein, lacking sequence homology with native tetherin, that nevertheless mimicked its antiviral activity. We further show that tetherin is incorporated into HIV-1 particles as a parallel homodimer using either of its two membrane anchors. These results indicate that tetherin functions autonomously and directly and that infiltration of virion envelopes by one or both of tetherin's membrane anchors is necessary, and likely sufficient, to tether enveloped virus particles that bud through the plasma membrane.

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Figures

Figure 1
Figure 1. Schematic representation of tetherin and analysis of post-translational modifications
(A) Schematic representation of the tetherin dimer, indicating the transmembrane (TM) and coiled coil (CC) domains as well as glycophosphatidylinositol (GPI, green), glycosylation (blue) and disulfide bond (red) modifications. A tetherin derivative that incorporated an extracellular HA tag was used throughout, unless otherwise indicated. (B) Western blot analysis (anti-HA) of 293T cells transiently transfected with 200ng of plasmids expressing WT tetherin and variants bearing mutations at putative glycosylation sites. Additionally, lysates of 293T cells stably expressing WT-tetherin were run on the same gel. A longer exposure of this lane is also shown. (C) Western blot analysis (anti-HA) of 293T cells stably transduced with LHCX-based retroviral vectors expressing WT tetherin and tetherin variants bearing mutations at putative glycosylation sites. (D) Western blot analysis (anti-HA) of tetherin following deglycosylation. Lysates of 293T ells stably expressing HA tagged tetherin were incubated in the presence or absence of peptide-N-glycosidase-F (PNGase), as described in the supplemental information, prior to analysis. (E) Western blot analysis (anti-HA) of parental (CHO) and PIGL-defective (ΔPIGL CHO) cells transfected with plasmids (200ng) expressing WT or delGPI tetherin in the absence (-) or presence (+) of 100ng of a plasmid expressing rat PIGL. A GFP expression plasmid (100ng) was cotransfected and blots were probed with anti-GFP (lower panel) verify equal transfection efficiency and gel loading. (F) Immunofluorescence analysis (anti-HA, red) of parental (CHO) and PIGL-defective (ΔPIGL CHO) cells transfected with plasmids expressing WT or delGPI tetherin in the absence (mock) or presence (+PIGL) of a plasmid expressing rat PIGL. Cells were transfected as in (E) and nuclei were stained with DAPI (blue) Scale bar =10μm. (G) Immunofluorescence analysis (anti-HA, green) of parental (CHO) and PIGL-defective (ΔPIGL CHO) cells transfected with plasmids expressing WT tetherin and an ER-localized DsRed2 fluorescent protein. Cells were transfected as in (E) and nuclei were stained with DAPI (blue). Scale bar =10μm. The inset shows an expanded portion of the image indicated by the white square in the lower right image. For (F) and (G), at least 50 cells were inspected and all exhibited similar patterns of tetherin localization. (H) Western blot analysis (anti-HA) of 293T cells transiently transfected with 200ng of plasmids expressing WT tetherin or tetherin variants bearing mutations at the indicated cysteine residues. Samples were untreated or treated with β-mercaptoethanol (β-ME) prior to analysis. Numbers to the left of panels (B), (C), (D), (E) and (H) represent the positions and sizes (in kDa) of molecular weight markers.
Figure 2
Figure 2. Tetherin features required for antiviral activity
(A) Schematic representation of the tetherin mutants panel, indicating the positions of glycophosphatidylinositol (GPI, green), glycosylation (blue) and disulfide bond (red) modifications. The tetherin proteins bore point mutations at cysteine residues involved in disulfide bonding (C53, C63 and/or C91) or N-linked glycosylation sites (N65 and/or N92). Alternatively, the tetherin coiled-coil was deleted (delCC) or replaced with a coiled-coil from dystrophia myotonica protein kinase (DMPK CC). The membrane anchors were removed (delGPI and delTM) or replaced by the the C-terminal GPI modification signal from urokinase Plasminogen Activator Receptor (delGPI(uPAR)). (B), (C) Effect of WT and mutant tetherin proteins on HIV-1 release. 293T cells were transfected with HIV-1(WT) or HIV-1(delVpu) proviral plasmids and increasing amount of plasmids expressing WT or mutant tetherin proteins (0, 25, 75 and 200ng for each). Infectious virion yield, measured using TZM-Bl indicator cells is given in relative light units (RLU). Error bars indicate the range of duplicate determinations and are representative of 2 to 5 experiments for each tetherin mutant. (C) Quantitative western blot analysis (LICOR, anti-p24) of 293T cells and corresponding virions after transfection with HIV-1(WT) or HIV-1(delVpu) proviral plasmids and 50ng of the WT and mutant tetherin plasmids. Numbers below each lane indicate the amounts of p24 in virion pellets, in arbitrary units and are representative of three experiments.
Figure 3
Figure 3. An artificial tetherin-like protein has potent antiviral activity
(A) Design of an artificial tetherin (art-tetherin) protein using domains of similar configuration from the transferrin receptor (TfR), dystrophia myotonica protein kinase (DMPK) and urokinase Plasminogen Activator Receptor (uPAR). An HA epitope tag was inserted between the DMPK and uPAR domains. (B) Western blot analysis (anti-HA) of 293T cells transfected with plasmids expressing WT tetherin or art-tetherin and subjected to SDS PAGE in the absence or presence of β-mercaptoethanol. A presumed degradation product from art-tetherin is indicated by an asterisk. (C) Immunofluorescence analysis (anti-HA, red) of HT1080 cells transfected with plasmids expressing WT tetherin or art-tetherin. Nuclei were stained with DAPI (blue) and the images are representative of at least 50 cells that were inspected for each expressed protein. Scale bar = 10μm (D) Western blot analysis (anti-HA and anti-tubulin, upper panels and anti-p24, lower panels) of cell lysates and corresponding released virions following transfection of 293T cells with HIV-1(WT) or HIV-1(delVpu) proviral plasmids and increasing amounts (0ng 25ng, 50ng, 100ng) of plasmids expressing WT tetherin or art-tetherin. (E) Infectious virion release, measured using TZM-Bl indicator cells, following transfection of 293T cells with HIV-1(WT) or HIV-1(delVpu) proviral plasmids and increasing amounts (0ng 25ng, 50ng, 100ng) of plasmids expressing WT tetherin, art-tetherin or art-tetherin mutants. Error bars indicate the range of duplicate determinations and are representative of 2 to 5 experiments for each protein. (F) Western blot analysis (anti-HA upper panels and anti-p24, center and lower panels) of cell lysates and corresponding released virions following transfection of 293T cells with an HIV-1(delVpu) proviral plasmid and increasing amounts (0ng 25ng, 50ng, 100ng) of plasmids expressing or art-tetherin and mutant derivatives. Numbers to the left of panels (B) and (F) represent the positions and sizes (in kDa) of molecular weight markers. (G) Western blot analysis of 293T cells (anti-p24 and anti-HA) and virions (anti-p24) following transfection with HIV-1(WT) or HIV-1(delVpu) and plasmids expressing WT tetherin or art-tetherin. Virions that were constitutively released, or released following incubation of the cells with subtilisin (bottom panels), were pelleted through sucrose prior to analysis. (H) Scanning electron micrograph of HT1080 cells transfected with plasmids expressing HIV-1 Gag in the presence or absence of either WT or art-tetherin. Scale bar indicates 1μm. At least ten particle expressing cells were evaluated in each of two independent experiments and representative images are shown.
Figure 4
Figure 4. Incorporation of tetherin variants into HIV-1 particles
(A) Transmission electron microscopic analysis of HT1080 cells stably expressing tetherin-HA and infected with HIV-1(delVpu). Infected cells were fixed and stained with anti-HA and anti mouse-IgG-gold particles prior to sectioning. Scale bars = 200nm. (B) Analysis of mutant tetherin incorporation into HIV-1 particles. 293T cells and corresponding released virions were harvested following transfection with HIV-1(delVpu) and increasing amounts of plasmids (0, 3.125, 6.25, 12.5, 25 and 50ng) expressing HA-tagged WT and mutant tetherin proteins. Virions were pelleted through sucrose prior to analysis. Cells and virions were subjected to Western blot analysis (anti-p24 and anti-HA, as indicated). (C) Western blot analysis (anti-p24 and anti-HA) of virions derived from HIV-1(delVpu) infected 293T cells stably expressing delTM or delGPI tetherin proteins. Virions were purified on Optiprep gradients and sixteen fractions were collected for western blot analysis of p24 and tetherin content. (D) Analysis of WT tetherin incorporation into virions. Unmodified 293T cells (left panels), or 293T cells stably expressing full-length tetherin-HA (center panels), were transfected with a plasmid expressing codon-optimized HIV-1 Gag. VLPs were pelleted through sucrose and purified on linear (10-30%) optiprep gradient. Ten fractions were collected, precipitated and subjected to a western blot analysis using anti-p24 and anti-HA antibodies. As a control, mock transfected 293T cells stably expressing full-length tetherin-HA, were subjected to the same procedure (right panels).
Figure 5
Figure 5. Configuration of tetherin in virions and exclusion by Vpu
(A) Analysis of virions derived from HIV-1(delVpu) infected 293T cells stably expressing N-terminally HA-tagged delGPI, and C-terminally HA-tagged delTM tetherin proteins. Virions were pelleted through sucrose, treated with (+) or without (-) subtilisin, pelleted again though sucrose and subjected to SDS PAGE in the presence or absence of β-mercaptoethanol prior to Western blot analysis with anti-p24 and anti-HA antibodies. (B) Analysis of virions recovered from the surface of 293T cells stably expressing N-terminally HA-tagged WT tetherin. Cells were infected with HIV-1(delVpu) and virions that were constitutively released, or released following incubation of the cells with subtilisin, were pelleted through sucrose prior to analysis. Virions and corresponding cell lysates were subjected to western blot analysis with anti-p24 and anti-HA antibodies. (C) Analysis of the effect of Vpu on virions derived from HIV-1 infected 293T cells stably expressing HA-tagged delGPI, delGI/T45I/delGPI, or delTM tetherin proteins. Cells were infected with HIV-1 (WT) or HIV-1(delVpu) and virions were pelleted through sucrose prior to analysis. Virions and corresponding cell lysates were subjected to western blot analysis with anti-p24 and anti-HA antibodies. Numbers to the left of panels (A), (B), and (C) represent the positions and sizes (in kDa) of molecular weight markers. (D) Immunofluorescence analysis (anti-HA) of virions harvested from HIV-1/MA-YFP(WT) or HIV-1/MA-YFP(delVpu) infected 293T cells stably expressing delTM and delGPI tetherin proteins. Virions were pelleted, applied to poly-D-lysine coated coverslips prior to analysis. Scale bars = 2μm. A proportion of representative fields observed in each of two experiments is shown. (E) Quantitative analysis of tetherin incorporation into virions. The proportion of HIV-1/MA-YFP(WT) or HIV-1/MA-YFP(delVpu) virions that were positive for delTM and delGPI tetherin proteins was quantified. The mean and standard deviation of the percentage of YFP-positive virions that were positive for tetherin is shown. Three fields containing a total of at least 600 YFP-positive virions for each condition were evaluated, in each of two independent experiments.
Figure 6
Figure 6. Scanning electron microscopic analysis of tetherin interaction with budding virions and models for tetherin incorporation
(A) Scanning electron microscopic analysis of 293T cells transiently expressing HA-tagged WT, delTM or delGPI tetherin proteins and HIV-1 Gag bearing mutations in the PTAP L-domain sequence. Cells were fixed and stained with anti-HA primary antibody and anti-mouse IgG-gold conjugate. Surface topography (upper panels) reveals virion particles, while backscatter electron detection (lower panels) reveals gold particles (white) marking the position of tetherin molecules. Scale bar = 500nm (B) Models for incorporation and virion tethering by the tetherin protein. Several stages of HIV-1 assembly are depicted for model 1, and only tethered virions are shown for models 2-4. In model 1, the TM domains of a tetherin dimer are incorporated into the virion envelope, and the GPI anchors remain embedded in the host-cell membrane. In model 2, the reverse situation occurs. In model 3, only one of a pair of disulfide linked tetherin molecules has both membrane anchors incorporated into the virion envelope. In model 4, one disulfide linked tetherin dimer incorporated into the virion envelope interacts with another dimer in the host-cell membrane via coiled-coil based interactions.
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Comment in

  • Tetherin is as tetherin does.
    Hammonds J, Spearman P. Hammonds J, et al. Cell. 2009 Oct 30;139(3):456-7. doi: 10.1016/j.cell.2009.10.011. Cell. 2009. PMID: 19879831

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