Expression of HIV-1 Vpu Leads to Loss of the Viral Restriction Factor CD317/Tetherin from Lipid Rafts and Its Enhanced Lysosomal Degradation

CD317/tetherin (aka BST2 or HM1.24 antigen) is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts). It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i) no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii) the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii) internalised tetherin is present in non-raft fractions, iv) expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v) internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi) lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface

The cytosolic N-terminus of CD317/tetherin is a membrane microdomain exclusion motif.

The integral membrane protein CD317/tetherin has been associated with a plethora of biological processes, including restriction of enveloped virus release, regulation of B cell growth, and organisation of membrane microdomains. CD317 possesses both a conventional transmembrane (TM) domain and a glycophosphatidylinositol (GPI) anchor. We confirm that the GPI anchor is essential for CD317 to associate with membrane microdomains, and that the TM domain of CD44 is unable to rescue proper microdomain association of a ΔGPI-CD317 construct. Additionally, we demonstrate that the cytosolic amino terminal region of CD317 can function as a 'microdomain-excluding' motif, when heterologously expressed as part of a reporter construct. Finally, we show that two recently described isoforms of CD317 do not differ in their affinity for membrane microdomains. Together, these data help further our understanding of the fundamental cell biology governing membrane microdomain association of CD317

Cartoon representation of the effect of Vpu on tetherin trafficking following internalisation from the cell surface.

<p>The left hand side of the cartoon (green) shows what happens to cell surface tetherin in the absence of Vpu. It is internalised and delivered to early endosomes. During this process it exits lipid rafts. The majority of tetherin molecules then end up being recycled to the cell surface, probably via one or more intermediate compartment (as indicated by the thick arrows) whilst some tetherin ends up in multivesicular bodies (MVBs)/late endosomes and is ultimately destined for lysosomal degradation. Thus the fate of internalised tetherin is finely balanced between recycling to the cell surface and lysosomal degradation. The right hand side of the cartoon (pink) shows what happens to cell surface tetherin in the presence of Vpu. Both are internalised and delivered to early endosomes (with tetherin once again exiting lipid rafts along the way) where they associate with one another. This association leads to a shift in the balance between recycling and lysosomal degradation for tetherin, with the majority of tetherin now destined for delivery to MVBs/late endosomes and then to lysosomes (as indicated by the thick black arrows). NB this cartoon illustrates only the effect of Vpu on the fate of internalised tetherin.</p

Tetherin is in lipid rafts at the cell surface.

<p><b>A</b>. HeLa cells were labeled with a membrane impermeant biotin reagent at 4°C prior to either immediate lysis in ice cold Triton X-100, or incubation for 10 min at 37°C before lysis in ice cold Triton X-100. Lysates were then separated on sucrose density gradients and 1 ml fractions were taken. Fractions 1-4 were pooled as raft fractions, 5-8 and 9-12 as two separate non-raft fractions. Lysates were then incubated with streptavidin-agarose beads to separate biotinylated (cell surface in the case of immediate lysis after biotinylation at 4°C, or internalised in the case of the 10 min chase at 37°C, proteins) from non-biotinylated proteins. Total, plasma membrane, and internalised fractions were then subjected to immunoblot analysis using antibodies to endogenous tetherin, Transferrin receptor or Flotillin 2 (the latter two as controls) as indicated. The total population of tetherin molecules is ~ 35% raft localized (1-4 in Total), however the tetherin that is at the cell surface (i.e. the biotinylated tetherin at 0 min uptake at 37°C) is almost exclusively raft localized (1-4 in Plasma Membrane). After 10 min uptake at 37°C a significant proportion of biotinylated tetherin is present in non-raft fractions (Figure 4A Internalised). <b>B</b>. As in A, but using HeLa cells transfected to express Vpu-GFP.</p

Immunolocalisation of internalized tetherin in the presence and absence of Vpu.

<p><b>A</b>-<b>D</b>. HeLa cells that had been transiently transfected to express Vpu-GFP and which had been incubated in the presence of lysosomal enzyme inhibitors for 24 hours were incubated at 4°C for 60 min with a monoclonal antibody (HM1.24) that recognises the extracellular domain of tetherin. Cells were then transferred to 37°C for various times and processed for immunofluorescence analysis using polyclonal antibodies to markers of early (EEA-1, <b>A, B</b>) or late (LAMP-1, <b>C, D</b>) endosomes. Some of the internalised tetherin can be seen to colocalise with both Vpu and EEA1 after 5 (<b>A</b>) or 10 (<b>B</b>) min at 37°C (see white arrows; bottom sets of panels represent magnifications of the boxed regions from the corresponding images above and are 20µ²); however, there is greater evidence of colocalisation between internalized tetherin and LAMP-1 at both time points (<b>C</b> and <b>D</b>). This is quantified by Pearson’s correlation coefficient (PCC) and percentage pixel overlay (%O) in <b>E</b>. This co-localisation is greatest in cells that also express Vpu at both 5min (PCC P=0.027, %O P=0.0015 n=3) and 10min (PCC P=0.0117, %O P=0.0034 n=3) antibody uptake. It is of note that there is limited evidence of any co-localisation between Vpu and LAMP-1 (<b>C</b> and <b>D</b>). Bar = 10µ.</p

The effect of expression of Vpu on the lipid raft localisation of endogenous tetherin.

<p><b>A</b>. HeLa cells were subjected to extraction in ice-cold Triton X-100 (1%) prior to separation by sucrose density gradient centrifugation, then analysis of fractions from the gradients by immunoblot. Fractions 1-4 are considered as lipid raft fractions, fractions 5-12 as non-raft fractions. The top panel shows an immunoblot, using an antibody to tetherin, of fractions from untransfected HeLa cells and confirms that a significant proportion of endogenous tetherin is present in lipid rafts (i.e. in fractions 1-4). The second panel shows an immunoblot, using an antibody to tetherin, of fractions from HeLa cells that had been transfected to express Vpu-GFP and shows that the majority of endogenous tetherin is lost from lipid rafts (i.e. fractions 1-4) in the presence of Vpu-GFP: this experiment was performed in the absence of lysosomal enzyme inhibitors, the blot was exposed longer to ensure detection of tetherin. Panel 3 shows the distribution of Vpu-GFP across the sucrose gradient; it has a broad distribution across the gradient, indicating that it is present in a range of membrane environments, both raft and non-raft. Expression of another viroporin protein (the M2 protein of influenza virus) does not lead to a redistribution of tetherin from lipid rafts, as shown by the bottom two panels. Panel 4 shows an immunoblot, using an antibody to tetherin, of fractions from HeLa cells that had been transfected to express M2-GFP and shows that the distribution of endogenous tetherin is similar to that in non-transfected cells (top panel). The bottom panel shows the distribution of M2-GFP across the sucrose gradient. <b>B</b>. Graphical representation of the proportions of tetherin molecules in raft vs. non-raft fractions on control HeLa cell and HeLa cells expressing Vpu-GFP (as indicated, n=5).</p

Lysosomal degradation of tetherin.

<p><b>A</b>.and <b>B</b>. Triple label fluorescence analysis (antibody detection of Vpu, tetherin and TGN46, and LysoTracker detection of lysosomes) of HeLa cells that had been incubated in the presence of lysosomal inhibitors for 24 hours prior to processing for immunofluorescence analysis. Cells expressing Vpu demonstrated significant accumulation of tetherin in lysosomes (<b>B</b>), but not in the TGN (<b>A</b>). Bar = 10µ. <b>C</b>. Quantification of co-localisation between LysoTracker-594 and tetherin, using both Pearson’s correlation coefficient and percentage pixel overlay. <b>D</b>. Immunoblot analysis, using an antibody to endogenous tetherin, of lysates from control HeLa cells, or HeLa cells expressing Vpu-GFP (Vpu) that had been incubated in the presence of lysosomal (24 hours) or proteosomal (12 hours) inhibitors (as indicated). The lower panels show immunoblots for tubulin as loading controls. <b>E</b>. Graphical representation of quantification of the data presented in <b>D</b>, P=0.0002, n = 3.</p

Tetherin interacts with Vpu in whole cells, but does not do so at the cell surface.

<div><p><b>A</b>. Immunoblot of tetherin in whole cell lysates from HeLa cells transfected to express Vpu-GFP (left panel) or GFP alone (right panel) and incubated in the presence of proteosomal inhibitors (PI) or lysosomal inhibitors (LI) as indicated. These inhibitors were included in order to block any Vpu-mediated degradation of tetherin, thereby ensuring sufficient tetherin remains for detection by immunoblot. Tetherin was detected in whole cell lysates (10% of input; GFP-TRP – lanes) and in co-immunoprecipitates with Vpu-GFP, but not in co-immunoprecipitates with GFP (GFP-TRP + lanes).</p> <p><b>B</b>. Flow diagram to illustrate procedure for isolation and identification of any biotinylated tetherin that interacts with Vpu, either at the cell surface (biotinylation at 4°C) or in an endosomal compartment following internalization from the cell surface (biotinylation at 4°C followed by a 10 min chase at 37°C).</p> <p><b>C</b>. HeLa cells were processed as outlined in B (above) and samples were processed for immunoblot analysis using an antibody to tetherin. Tetherin is detected in the whole cell lysate (10% of input) from cells labeled with biotin at 4°C (0 min) (lane 1) and from cells labeled with biotin at 4°C followed by a 10 min chase at 37°C (10 min) (lane 4). Tetherin is not detected in the GFP Trap and streptavidin pull-down from cells labeled with biotin at 4°C (0 min) (lane 2), indicating that cell surface tetherin does not interact with Vpu. Multiple cell surface proteins were biotinylated by the procedure used, as shown by their detection in the whole cell lysate using streptavidin HRP (lane 3). Tetherin is detected in the GFP Trap and streptavidin pull-down from cells labeled with biotin at 4°C then subjected to a 10 min chase at 37°C (10 min) (lane 5), indicating that tetherin does interact with Vpu upon internalisation. Tetherin can be detected in material isolated from the 10 min chase lysate by streptavidin-coated beads (lane 6).</p></div