Separable Determinants of Subcellular Localization and Interaction Account for the Inability of Group O HIV-1 Vpu To Counteract Tetherin

Abstract

Tetherin (BST-2/CD317) is thought to restrict retroviral particle release by cross-linking nascent viral and cellular membranes. Unlike the Vpu proteins encoded by human immunodeficiency virus type 1 (HIV-1) group M strains (M-Vpu), those from the nonpandemic HIV-1 group O (O-Vpu) are not able to counteract tetherin activity. Here, we characterized the basis of this defect in O-Vpu. O-Vpu differs from M-Vpu in that it fails to interact with tetherin and downregulate it from the cell surface. Unlike M-Vpu, O-Vpu localizes to the endoplasmic reticulum (ER) rather than the trans-Golgi network (TGN). Interestingly M-Vpu bearing an ER retention signal at the C terminus localizes similarly to O-Vpu. While it still interacts with tetherin, it fails to promote virus release, suggesting that O-Vpu deficiency correlates with its cellular distribution in the endoplasmic reticulum as well as its failure to bind tetherin. O-Vpu-M-Vpu chimeras were designed to identify the minimal changes required to restore tetherin antagonism. While several chimeric proteins bearing residues of the M-Vpu transmembrane domain into the O-Vpu transmembrane domain recovered tetherin binding in coimmunoprecipitation studies, efficient antagonism required an additional glutamic acid-to-lysine change in the membrane-proximal hinge region of the O-Vpu cytoplasmic tail that was sufficient to abolish ER retention and permit TGN localization.

INTRODUCTION

Tetherin is a recently identified antiviral factor that interferes with the late stages of the human immunodeficiency virus type 1 (HIV-1) replication cycle by preventing the release of nascent viral particles from infected cells (38, 59). It is an interferon-induced type II membrane glycoprotein (3, 25, 40) composed of a short cytoplasmic tail (CT), a transmembrane domain (TMD), and an extracellular domain mainly consisting of a coiled-coil region (19, 47, 56, 64), followed by a putative glycophosphatidylinositol (GPI) lipid anchor at the C terminus embedded in cholesterol-rich microdomains at the cellular membrane (25). Tetherin localizes to the plasma membrane (PM) and multiple membrane compartments, including the trans-Golgi network (TGN), and recycles via the clathrin-adaptor complex (AP-1 and AP-2) endocytosis machinery (25, 43). The protein's function essentially relies on its unusual structural topology, underscored by the ability to create a completely artificial tetherin-like protein from unrelated protein subunits with similar restriction activity (42). Tetherin is thought to inhibit virus release by being incorporated into nascent virions and directly cross-linking the viral and the cellular membranes (11, 16, 42), leading to the accumulation of fully mature virions at the plasma membrane, followed by internalization and subsequent lysosomal degradation (37, 38). Interestingly, since tetherin does not directly interact with any virally encoded structural protein, it can block a broad spectrum of mammalian enveloped viruses, including retroviruses, Ebola virus, Marburg virus, Lassa fever virus, and Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) (22, 23, 30, 41, 44). Concomitantly, many of these viruses have evolved countermeasures that target tetherin function (5). The prototype of these antagonists is the HIV-1 accessory protein Vpu (38, 59).

Vpu is a 16-kDa phosphoprotein consisting of an N-terminal transmembrane domain and a cytoplasmic tail that forms two alpha helices linked by a conserved DGSNES motif that can be phosphorylated by casein kinase II (55). The interaction of this motif with β-TrCP1 and -2 is required to mediate endoplasmic reticulum (ER)-associated degradation of CD4 (2, 31, 48, 63). The Vpu TM domain is thought to oligomerize to form pentameric cation channels (10, 28, 50) and localizes predominantly to internal membrane structures, including the TGN and endosomes (9, 61). Vpu is not incorporated into HIV-1 particles and therefore must perform its functions within the infected cells. Vpu induces tetherin to be downregulated from the cell surface (59) and degraded (6, 13, 20, 29, 34), although whether this degradation is an absolute requirement to overcome tetherin restriction is still unclear (35). The large fraction of Vpu colocalization with tetherin in the TGN suggests that newly synthesized or recycled tetherin from the cell surface might be sequestered in the TGN (8), similarly to what has been observed with HIV-2 Env (17, 26). Consistent with this, TGN localization of Vpu correlates with antitetherin function (9). Tetherin and Vpu interact with each other via their transmembrane domains (8, 20, 24, 62). Some of the determinants required for the interaction have recently been mapped and form single faces of both proteins' respective TM domains. In the tetherin TM domain, mutations on residues I34, L37, and L41 affect its sensitivity for Vpu (24). In the viral protein, residues A14, W22, and, to a lesser extent, A18 that form one face of the Vpu TMD are required for tetherin interaction and antagonism (62).

The HIV-1 Vpu protein is able to counteract only human, chimpanzee, and gorilla tetherins. This species specificity resides in the aforementioned TM domain interactions, and several of the determinants in the tetherin TM domain have been subjected to positive selection during primate evolution (14, 33). In simian immunodeficiency virus (SIV) cpz, the direct precursor of HIV-1, the Nef protein rather than Vpu antagonizes chimpanzee tetherin, and the ability of Vpu to target human tetherin is an adaptation that presumably occurred during the zoonosis of this virus to humans (21, 33, 45, 65, 66). To add further to this complexity, only Vpu proteins from HIV-1 group M, the viruses predominantly responsible for the HIV-1 pandemic, can both antagonize tetherin efficiently and degrade CD4. In HIV-1 group O, Vpu proteins are unable to target tetherin but still retain the ability to degrade CD4, whereas this situation is reversed in HIV-1 group N, with Vpu retaining some level of tetherin antagonism but losing activity against CD4 (45). HIV-1 group O infections are limited mainly to Cameroon. While HIV-1 group O-infected individuals can progress to AIDS, the spread of this virus appears to be inefficient compared to group M (32). It has recently been speculated that Vpu adaptation to human tetherin may have therefore been important for the pandemic spread of HIV/AIDS (46).

In this study, we characterized the cell biological basis for the defects in the Vpu proteins from HIV-1 group O (O-Vpu) that impair its ability to antagonize tetherin. We found that O-Vpu is defective both for tetherin binding and also in its subcellular localization to the TGN. These attributes are separable to the TM domain and the membrane-proximal hinge region of the first alpha helix of the cytoplasmic tail, respectively. Simultaneous replacement of these domains in O-Vpu with those from Vpu proteins encoded by HIV-1 group M strains (M-Vpu) are required to reconstitute tetherin antagonism.

MATERIALS AND METHODS

Cells and plasmids.

All cells were maintained at 37°C with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, United Kingdom) supplemented with 10% fetal calf serum (FCS) and gentamicin. HEK293T (293T) cells and HeLa cells were obtained from the American Type Culture Collection (ATCC), and the HIV-1 reporter cell line HeLa-TZMbl was kindly provided by John Kappes through the NIH AIDS Reagents Repository Program (ARRP). HeLa/CD4 cells were provided by A. Akrigg through the National Institute of Biological Standards and Controls (NIBSC) Centre for AIDS Reagents (Potters Bar, United Kingdom). The HIV-1 molecular clone plasmid pNL4.3 was obtained from the NIH ARRP, and the Vpu-defective counterpart has been described previously (37). pCR3.1, encoding a codon-optimized HIV-1 NL4.3 Vpu tagged at the C terminus with an HA epitope (pCR3.1-Vpu-HA), mentioned in this study as M-Vpu Wt, was derived from pVphu (kindly provided by K. Strebel through the NIH ARRP) (39). Codon-optimized Vpu group O (O-Vpu) genes derived from HIV-1 group O strains 9435 and HJ001 as well as a consensus sequence assembled from 32 full-length O-Vpu sequences available in the HIV Sequence Database (http://www.hiv.lanl.gov) were synthesized by multiple overlapping PCRs and cloned into the expression vector pCR3.1. O-Vpu mutants and chimeras were generated by QuikChange site-directed mutagenesis and overlapping PCR, respectively, by standard methods using Phusion II polymerase (New England BioLabs). All the sequences were confirmed.

Virus release assays.

Subconfluent HEK293T cells were transfected with 500 ng of proviral plasmid in combination with 50 ng of pCR3.1 human tetherin and variable concentrations of pCR3.1-Vpu-HA or mutants thereof using polyethylenimine (1 mg/ml; Polysciences). The medium was replaced 16 h after transfection, and viral supernatants and cell lysates were harvested at 48 h posttransfection and analyzed for infectivity using HeLa-TZMbl indicator cells and particle production by Western blotting and as described previously (62).

Flow cytometry.

Subconfluent HeLa cells in 6-well dishes were transfected with 400 ng pCR3.1 encoding enhanced green fluorescent protein (pCR3.1-EGFP) and 400 ng pCR3.1-Vpu-HA. At 48 h posttransfection, the cells were harvested and stained for surface tetherin using a specific anti-BST2 monoclonal IgG2a antibody (Abnova) and a goat anti-mouse IgG2a-allphycocyanin Alexa 633 conjugated as a secondary antibody (Molecular Probes, Invitrogen, United Kingdom). Tetherin expression in green fluorescent protein (GFP)-positive cells was then analyzed using a FACSCalibur flow cytometer (Becton Dickinson) and FlowJo software.

Immunofluorescence microscopy.

HeLa cells plated on glass coverslips were transfected with 100 ng of Vpu-HA expression vector. At 24 h posttransfection, the cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and immunostained using mouse or rabbit anti-HA antibodies (Covance), a sheep anti-human TGN46 (Serotec), and a mouse anti-protein disulfide isomerase (anti-PDI; Molecular Probes, Invitrogen), followed by the appropriate donkey secondary antibodies coupled to Alexa 488 and 594 fluorophores (Molecular Probes, Invitrogen, United Kingdom). The coverslips were then mounted on slides using ProLong antifade reagent containing 4′,6-diamidino-2-phenylindole (Molecular Probes, Invitrogen, United Kingdom) and examined on a Leica DM-IRE2 confocal microscope.

Immunoprecipitation.

Coimmunoprecipitation of tetherin with Vpu-HA from transfected HEK293T cells was performed as described previously (62). Cell lysates and immunoprecipitates were Western blotted for Vpu using mouse anti-HA and tetherin using rabbit anti-BST2 (kindly provided by K. Strebel through the NIH ARRP).

RESULTS

O-Vpu 9435 fails to interact with tetherin or mediate its cell surface downregulation and localizes to ER-associated compartments.

HIV-1 group O Vpu has been shown to lack the ability to counteract human tetherin but can still induce CD4 degradation (45). To confirm this, a Vpu gene from HIV-1 group O strain 9435 was synthesized with a C-terminal HA tag and tested for its ability to block tetherin antiviral function. 293T cells were cotransfected with a fixed dose of tetherin plasmid, increasing amounts of Vpu expression vectors, and a proviral plasmid of HIV-1 NL4.3 deleted for the Vpu gene [HIV-1(Vpu⁻)]. At 48 h posttransfection, cells were lysed and supernatants were analyzed by Western blotting for p24 and Vpu detection or titrated on HeLa-TZMbl indicator cells (Fig. 1A). As expected, a prototype M-Vpu (NL4.3) efficiently rescues virus production in the presence of tetherin. In contrast, O-Vpu of strain 9435 (O-Vpu 9435) was unable to rescue particle release from tetherin restriction, even at higher plasmid inputs, and was equivalent to an M-Vpu A14L-W22A TM mutant that we have shown previously to be defective for tetherin antagonism (62). All proteins were expressed at comparable levels, and O-Vpu 9435 expression did not affect intracellular Gag protein synthesis (Fig. 1A). Whereas M-Vpu runs on a Western blot as a single species, O-Vpu 9435 appears as a doublet. M-Vpu overcomes human tetherin by mediating its removal from the cell surface and blocking its incorporation into nascent virions. To address whether O-Vpu fails to antagonize tetherin because it cannot induce its downregulation, tetherin-positive HeLa cells were cotransfected with an empty vector control or a Vpu expression vector in combination with a GFP marker plasmid. At 48 h posttransfection, cells were harvested and stained for surface tetherin levels (Fig. 1B). As expected in GFP-positive cells, expression of M-Vpu Wt but not M-Vpu A14L-W22A leads to a decrease of tetherin levels at the cell surface. Expression of O-Vpu 9435 had little effect on surface tetherin levels. Finally, Vpu mediates tetherin downregulation through interaction via their transmembrane domains. Mutations impairing this interaction render the viral protein incapable of allowing coimmunoprecipitation of tetherin from cell lysates (62). We then assessed the ability of O-Vpu 9435 to interact with tetherin in this assay. 293T cells were cotransfected with tetherin and Vpu-HA expression vectors. Two days after transfection, cells were lysed and Vpu proteins were immunoprecipitated via the HA epitope and analyzed by Western blotting (Fig. 1C). As expected, tetherin coimmunoprecipitates with M-Vpu Wt and not with the transmembrane Vpu mutant (M-Vpu A14L-W22A). Similarly, tetherin also failed to coimmunoprecipitate with O-Vpu 9435, suggesting the lack of a direct interaction between the tetherin TMD and O-Vpu TMD. Finally, despite these defects in tetherin antagonism, as expected, this O-Vpu construct retained the ability to induce CD4 downregulation (Fig. 1D).

Fig. 1.

Defects in tetherin antagonism, binding, and downregulation by O-Vpu 9435. (A) 293T cells were transiently transfected with 500 ng of HIV-1(Vpu⁻) provirus, 50 ng of tetherin plasmid, and increasing doses of the indicated Vpu expression vector. At 48 h posttransfection, the resulting viral supernatants were assayed for infectivity on HeLa-TZMbl indicator cells by measuring beta-galactosidase activity at 48 h postinfection. Cell lysates and pelleted virions from cells transfected with 50 ng of Vpu plasmid were subjected to SDS-PAGE and blotted for Vpu-HA, p24-CA, and Hsp90 protein, which served as a loading control, and analyzed by a LiCor quantitative imager. Relative virus release was calculated as a percentage of the virion p24 band intensity of the no-tetherin control. RLU, relative light units. (B) HeLa cells were cotransfected with 400 ng of GFP marker plasmid and the indicated Vpu expression vector. At 48 h posttransfection, endogenous surface tetherin was immunostained and expression levels were quantified by flow cytometry. Histograms represent the tetherin levels on GFP-positive gated cells in empty vector control cells (black peak) or in Vpu-expressing cells (overlaid gray peak). Median fluorescence intensities of the overlaid histogram are indicated in the top right corner. (C) 293T cells were transiently transfected with 500 ng of human tetherin-encoding plasmid and 500 ng of the indicated Vpu expression vector. After 2 days of incubation at 37°C, Vpu was immunoprecipitated (IP) via the HA tag from cell lysates and subjected to SDS-PAGE. Total cell lysates and immunoprecipitates were then Western blotted for tetherin and Vpu-HA. Molecular mass markers are indicated, and blots are a representative example of three independent experiments. (D) As in panel B, but the effects of O-Vpu on surface CD4 were examined in transfected HeLa/CD4 cells.

Vpu localization in the TGN has been shown to be important to suppress tetherin restriction activity, consistent with Vpu inducing sequestration of tetherin in the TGN (8, 9). Here we addressed whether the O-Vpu 9435 cellular localization accounts for its inability to block tetherin function. HeLa cells were transfected with a Vpu-HA-encoding vector. Twenty-four hours later cells were fixed and immunostained for Vpu-HA and either the ER marker, protein disulfide isomerase (PDI), or the trans-Golgi network marker, TGN46 (Fig. 2). As expected, M-Vpu localized in perinuclear compartments that mainly overlay with TGN46-positive compartments but not with PDI. However, although a minor fraction of O-Vpu protein 9435 localizes in the TGN, the majority forms a reticular staining pattern that partially overlays with PDI-positive compartments, suggesting that, unlike M-Vpu, O-Vpu 9435 localizes to ER-associated compartments. Thus, the failure of O-Vpu 9435 to antagonize tetherin correlates both with its inability to interact directly with tetherin and with its lack of accumulation in the TGN.

Fig. 2.

O-Vpu 9435 localizes to the ER. HeLa cells were transfected by either 100 ng of M-Vpu Wt-HA or O-Vpu Wt-HA plasmid. Twenty-four hours later, the cells were fixed and stained for Vpu detection with anti-HA antibody (green), a TGN marker (TGN46; red), or an ER marker antibody (PDI; red) and examined by confocal microscopy.

Retention of M-Vpu in the endoplasmic reticulum prevents tetherin antagonism but does not block their interaction.

We next investigated the functional consequences of restricting Vpu expression to the ER. Vpu-induced virus release in T cells is sensitive to brefeldin A (51), suggesting that Vpu function requires post-ER trafficking. However, the broad inhibition of the secretory pathway will also affect tetherin trafficking, potentially confounding this conclusion. To circumvent this, we made a M-Vpu protein bearing an ER retention signal derived from the bovine foamy virus envelope protein (-KKDQ) at the C terminus (12). As expected, M-Vpu KKDQ shows a reticular staining overlapping with PDI-positive compartments, indicative of an ER localization (Fig. 3A), with only a minor fraction of M-Vpu KKDQ proteins localizing with the TGN marker. M-Vpu KKDQ displays weak activity against tetherin restriction, despite increasing doses of plasmid (Fig. 3B), indicating that post-ER trafficking of Vpu is essential for tetherin antagonism. Similarly, transfection of tetherin-positive HeLa cells reveals that M-Vpu KKDQ fails to downregulate tetherin from the plasma membrane (Fig. 3C). However, preventing M-Vpu from leaving the ER does not impair its ability to interact with tetherin in coimmunoprecipitates from transient transfections (Fig. 3D). This was not due to tetherin-Vpu interactions occurring during the immunoprecipitation, as subsequent mixing of cell lysates from 293T cells transfected with either Vpu or tetherin individually failed to yield tetherin coimmunoprecipitating with Vpu-HA (see supplemental Fig. 1 [http://www.kcl.ac.uk/schools/medicine/research/diiid/depts/infectious/groups/neil/]). These data therefore suggest that Vpu and tetherin may interact early in the secretory pathway, but only after exit from the ER is Vpu able to exert its inhibitory effect on tetherin activity. Furthermore, these results suggest that O-Vpu 9435 localization in the endoplasmic reticulum may contribute to its deficiency in tetherin antagonism but does not account for its lack of tetherin interaction.

Fig. 3.

Retention of M-Vpu in the ER inhibits tetherin antagonism but does not block interaction. (A) HeLa cells were transfected by 100 ng of M-Vpu KKDQ-HA plasmid. Twenty-four hours later, the cells were fixed and stained for Vpu detection with anti-HA antibody (green), a TGN marker (TGN46; red), or an ER marker antibody (PDI; red) and the appropriate secondary antibodies and examined by confocal microscopy. (B) 293T cells were transiently transfected with 500 ng of HIV-1(Vpu⁻) provirus, 50 ng of tetherin-encoding plasmid, and increasing amounts of the indicated Vpu expression vector. Cells and supernatant-containing viral particles were harvested at 48 h posttransfection. The resulting infectious virions in culture supernatants were titrated on HeLa-TZMbl indicator cells by measuring beta-galactosidase activity at 48 h postinfection. Vpu protein expression in cell lysates was analyzed by Western blotting using an anti-HA antibody. (C) The indicated Vpu constructs were cotransfected in HeLa cells with a GFP-encoding vector, and surface tetherin levels were quantified by flow cytometry at 48 h posttransfection. (D) 293T cells were transiently transfected with 500 ng of human tetherin-encoding plasmid and 500 ng of the indicated Vpu expression vector. After 2 days of incubation at 37°C, Vpu was immunoprecipitated via the HA tag from cell lysates and subjected to SDS-PAGE. Total cell lysates and immunoprecipitates were then Western blotted for tetherin and Vpu-HA detection. Molecular mass markers are indicated, and blots are a representative example of three independent experiments.

Defects in tetherin interaction and TGN localization map to the TM domain and first alpha helix of O-Vpu, respectively.

We then went on to assess the minimal changes needed in O-Vpu 9435 to allow it to antagonize human tetherin. O-Vpu 9435 and M-Vpu proteins derive from distinct SIVcpz zoonoses and as such are very diverse at the amino acid level. However, they do have basic features known to be important for CD4 degradation (7) (Fig. 4A): first, the conserved W residue in the TM domain (58, 62) and, second, the dual serine motif phosphorylated by CKII that binds to β-TrCP (7). After the TM domain in Vpu is a putative hinge region followed by an amphipathic alpha helix (H1) which has been proposed to lie along the face of the membrane with the nonpolar residues partially submerged and the charged residues interacting with the polar phospholipid heads (4). In contrast, in H1 of O-Vpu the TM-proximal part of the helix is extended by a run of alternating basic and acidic residues that breaks its amphipathicity. Chimeric proteins, combining M-Vpu TMD and O-Vpu CT or vice versa, were designed (Fig. 4A). 293T cells were cotransfected with a fixed amount of tetherin, Vpu, and the HIV-1(Vpu⁻) proviral plasmid. Forty-eight hours later cells were analyzed by Western blotting and titrated on HeLa-TZMbl indicator cells (Fig. 4B). Substitution of the two key residues from M-Vpu TMD, A14 and A18, essential for tetherin interaction (62), into O-Vpu TMD did not result in a functional O-Vpu mutant (O-Vpu B), demonstrating that these residues are not sufficient to confer tetherin antagonism. Although they localized to the TGN (data not shown), chimeras composed of O-Vpu TMD and M-Vpu CT were unable to enhance particle release in tetherin-expressing cells. The same mutant designed by adding the equivalent determinants of M-Vpu TMD into O-Vpu TMD (Vpu OBTM-MCT) did not gain function to suppress tetherin restriction. However, two chimeras that were both composed of an intact M-Vpu TMD gained function against tetherin either in the context of the O-Vpu cytoplasmic tail (Vpu MTM-OCT) or with the M-Vpu first alpha helix (Vpu MTM-MH1-OH2). To characterize these proteins further, Vpu MTM-OCT and Vpu MTM-MH1-OH2 mutants were rescreened against a fixed dose of tetherin but with various expression levels of Vpu. Despite both chimeras being effective at coimmunoprecipitating tetherin (Fig. 4C), only Vpu MTM-MH1-OH2 gained function against tetherin restriction at lower plasmid inputs (Fig. 4D) and at higher expression had activity almost to the levels of M-Vpu Wt. In contrast Vpu MTM-OCT achieved only a low efficiency of tetherin antagonism, even at the highest plasmid inputs. Consistent with the importance of Vpu association with the TGN for tetherin antagonism (9), Vpu MTM-MH1-OH2 but not Vpu MTM-OCT localized predominantly to the TGN rather than the ER (Fig. 4E). Together these data suggest that amino acid differences in the first alpha helix between O-Vpu 9435 and M-Vpu retain the former in ER-like compartments. Thus, the simultaneous replacement of the tetherin interaction domain (TM) in the context of the first alpha helix of M-Vpu sequences that permit TGN localization is required for full antagonism of tetherin.

Fig. 4.

Defects in O-Vpu can be rescued by replacement of the TM domain in the context of the M-Vpu first alpha helix. (A) Schematic representation of the Vpu topology and alignments comparing Vpu sequences from HIV-1 group M and group O or the indicated mutants. (B) 293T cells were transfected with the HIV-1(Vpu⁻) provirus, a fixed dose of tetherin plasmid, and the indicated Vpu construct. After 2 days, viral supernatants were assayed on HeLa-TZMbl indicator cells and cells lysates were analyzed by Western blotting for Vpu-HA and Hsp90. Numbers below indicate the Vpu expression levels normalized with Hsp90 expression levels compared to the M-Vpu Wt protein expression. (C) The indicated Vpu constructs were coexpressed in 293T cells with 500 ng of human tetherin-encoding plasmid. At 48 h posttransfection, cells were lysed and Vpu proteins were isolated via immunoprecipitation and subjected to SDS-PAGE. Both tetherin and Vpu-HA expressions were visualized on a Western blot using the appropriate antibodies. (D) Same as in panel B but with various doses of the indicated Vpu mutants added in trans. (E) The indicated Vpu-HA chimeras (green) were transiently expressed in HeLa cells and coimmunostained either with a TGN marker (TGN46; red) or with an ER marker (PDI; red).

A single glutamic acid-to-lysine change in the putative hinge region of O-Vpu proteins confers localization to the TGN.

We next investigated the determinants in the H1 domain of O-Vpu that account for its localization to the ER. First, we assessed whether ER retention of our O-Vpu 9435 was representative of the known available sequences. To this end, we synthesized an HA-tagged consensus O-Vpu (O-Vpu cons) from all the full-length sequences available in the HIV Sequence Database (http://www.hiv.lanl.gov) (n = 32) and, additionally, the Vpu from strain HJ001 (45). As expected, both proteins were defective for counteracting tetherin from transiently transfected 293T cells (Fig. 5A) and failed to interact in coimmunoprecipitations (Fig. 5B), consistent with our previous observations. When we examined the subcellular localization, however, we observed that while Vpu-O cons was again retained in the ER, HJ001 Vpu displayed more prominent localization to the TGN (Fig. 5C). Alignment of the H1 domains of these proteins revealed that a major difference in HJ001 was a membrane-proximal K residue at position 32 instead of an E in Vpu-O cons and 9435 (Fig. 6A). This lysine was present in only a minority of O-Vpu sequences, suggesting that the acidic residue is representative of HIV-1 group O (Fig. 6A). Interestingly, position 32 in O-Vpu is equivalent to K31 in M-Vpu, which is embedded within the putative membrane-proximal hinge YRKILR. Mutation of R30-K31 to alanines in HIV-1 NL4.3 Vpu was previously shown to lead to an endosomal localization and a concomitant decrease in antitetherin activity (9). To determine whether this residue also plays a role in O-Vpu localization, we replaced the hinge region with the equivalent part of M-Vpu in the context of the M-Vpu TM domain (MTM-RKILR-OCT). In addition, we also made the E32K point mutation in O-Vpu 9435 and MTM-OCT. When transfected into 293T cells with tetherin, MTM-RKILR-OCT and MTM-OCT E32K both regained function equivalent to that of the MTM-MH1-OH2 chimera, indicating that this single amino acid change was sufficient to enhance tetherin antagonism (Fig. 6B). As expected, the E32K change on its own did not confer tetherin antagonism to O-Vpu due to its TM domain's inability to interact with tetherin (data not shown). Examination of the localization of these Vpu proteins revealed that unlike the parental O-Vpu, MTM-RKILR-OCT, MTM-OCT E32K, and O-Vpu E32K all displayed localization to TGN46-positive compartments (Fig. 6C). In contrast, the reciprocal mutation, K31E, in the context of M-Vpu was not sufficient to restrict the protein's localization to ER-associated compartments (data not shown). Thus, a single acidic residue present in the hinge region of the majority of O-Vpu sequences precludes the protein from leaving ER-associated compartments and is responsible for their poor activity against human tetherin even when TM-mediated interaction is restored.

Fig. 5.

Antitetherin activities and subcellular localizations of a consensus O-Vpu and O-Vpu HJ001. (A) Counteraction of tetherin-mediated restriction of HIV-1(Vpu⁻) from transfected 293T cells by the indicated O-Vpu-HA construct and corresponding Western blots of cell lysates and pelleted virions as described in the legend to Fig. 1. (B) Coimmunoprecipitation of O-Vpu-HA proteins with tetherin from transiently transfected 293T cells as described in the text. (C) Subcellular localization of the indicated O-Vpu-HA (green) in transfected HeLa cells costained either with a TGN marker (TGN46; red) or with an ER marker (PDI; red).

Fig. 6.

An E32K point mutation confers TGN localization and tetherin antagonism to O-Vpu bearing the group M TM domain. (A) Alignment of the consensus O-Vpu, O-Vpu 9435, and O-Vpu HJ001 sequences (above) and expanded logoplot of the amino acid sequences of the first alpha helix of publicly available O-Vpu sequences (n = 32) (below). Position E32 is indicated (red arrow). (B) 293T cells were transfected with 500 ng of HIV-1(Vpu⁻), 50 ng of tetherin, and various doses of the indicated Vpu-HA construct and processed as described in the legend to Fig. 4. (C) Subcellular localization of the indicated O-Vpu-HA (green) in transfected HeLa cells costained with a TGN marker (TGN46; red).

DISCUSSION

The ability to counteract tetherin is a conserved attribute of primate lentiviruses, although the viral protein that performs this function varies (46). While tetherin antagonism is associated with the Vpu proteins of the SIVgsn/mon/mus lineage, their descendant, SIVcpz Vpu, lacks this activity, probably due to redundancy with a Nef protein acquired via recombination from a SIVrcm-related virus (45). Adaptation of HIV-1 Vpu to human tetherin is therefore associated with the genetic changes that accompanied the zoonosis of SIVcpz to humans, which has happened at least four times in the last century, giving rise to groups M, N, O, and P (18). The Vpu proteins of most group M viruses tested can counteract tetherin (45). In contrast, group O Vpu proteins are defective for this attribute, and in the few sequences from group N, Vpu counteraction of tetherin is variable (45). In this study, we have addressed the molecular and cell biological basis for the difference between M-Vpu and O-Vpu proteins. We have shown that O-Vpu is defective for tetherin antagonism for two reasons. First and most important, its TM domain lacks the capacity to interact with tetherin in coimmunoprecipitations. However, the ability to bind tetherin is not sufficient to confer antagonism to O-Vpu. O-Vpu appears to be retained in the ER and fails to localize to the TGN. This maps to the first alpha helix of the cytoplasmic tail, specifically, a glutamic acid residue at position 32, found in the majority of O-Vpu sequences. Replacement of this region is also required for efficient tetherin antagonism. Second, we further show that ER retention of M-Vpu fails to antagonize tetherin, despite maintaining the ability to interact with tetherin.

We showed previously that mutation of conserved residues A14, A18, and W22, which form one face of the M-Vpu TM domain, impairs tetherin interaction and antagonism (62). The A14 and A18 positions are conserved in M- and N-Vpu proteins but not in group O or SIVcpz Vpu proteins, suggesting that changes to this face of the TM domain helix may have been driven by adaptation of HIV-1 Vpu to human tetherin. The O-Vpu TM domain failed to interact with human tetherin in coimmunoprecipitations; however, replacement of these residues is not sufficient to confer tetherin interaction to O-Vpu. Moreover, in attempts to delineate the minimal requirements to render the O-Vpu TM domain capable of mediating tetherin antagonism in the context of MTM-MH1, no chimeric TM domain gained function (data not shown). These results suggest that the functional binding interface of Vpu with tetherin is likely to be contextually dependent on the entire conformation of the TM domain.

The retention of O-Vpu in ER-associated compartments confers a defect to antagonism even when interaction with tetherin is mediated through a chimeric TM domain. This can be partially overcome by increased Vpu expression, which we interpret as being due to minor amounts of O-Vpu being observable in the TGN at high expression levels. Several years ago Schubert and Strebel demonstrated that brefeldin A inhibited Vpu-mediated HIV-1 release from infected T cells (51), and the same laboratory has recently confirmed these data, in the light of the discovery of tetherin (1). However, because brefeldin A blocks the bulk flow of secretory proteins from the ER, including tetherin, we attempted to alleviate any potential confounding factors by appending a strong ER retention signal to M-Vpu. ER-retained M-Vpu was clearly defective for tetherin antagonism, but unlike O-Vpu, it was still able to interact with tetherin in coimmunoprecipitates, in contrast to a recent report (54). This suggests that while tetherin and Vpu can interact in the ER, antagonism of tetherin function requires trafficking of Vpu-tetherin complexes into TGN compartments. Recent data from the Strebel group have further shown that under overexpression conditions, Vpu can induce ER-associated degradation of newly synthesized tetherin, but this does not happen in virus-infected cells (1). Thus, if Vpu and tetherin do interact prior to ER exit, the appending of the -KKDQ motif leads to disruption of this interaction when the Vpu is retrieved from the cis-Golgi network. However, to definitively show whether this is the case will require further fluorescence resonance energy transfer-based microscopy studies of Vpu-tetherin interactions in living cells.

The inability of O-Vpu to exit the ER maps to the membrane-proximal region of the first alpha helix of the cytoplasmic tail. The amphipathic nature of helix 1 is thought to allow it to lie partially buried along the face of the membrane with the basic residues in contact with the phospholipid heads (4). Between the TM domain and the first alpha helix is a putative hinge region, the basic residues of which in subtype B Vpu proteins have been implicated in endosome-to-TGN localization when mutated to hydrophobic residues (9). We found that replacement of the hinge region in O-Vpu 9435 with the corresponding RKILR of M-Vpu conferred both TGN localization and efficient tetherin antagonism when combined with the M-Vpu TM domain. This phenotype mapped to an acidic residue (E32) in the position equivalent to M-Vpu K31 that is conserved in the majority of O-Vpu proteins. Since the reverse mutation in M-Vpu did not lead to its ER retention, these data suggest to us that it is unlikely that this is a specific TGN-targeting motif itself. Rather, we suggest that the distribution of basic and acidic residues in the membrane-proximal region of the O-Vpu may influence the overall conformation of the cytoplasmic tail in relation to the membrane and that the retention of O-Vpu in ER-associated compartments may be related to such a structural change. It is interesting to note that O-Vpu 9435 and chimeric molecules bearing its first alpha-helical region run as a doublet on SDS-polyacrylamide gels, perhaps suggesting potential differences in conformation or phosphorylation. Since O-Vpu still downmodulates CD4, a process that requires interaction with the Vpu cytoplasmic tail and its phosphorylation, such putative conformational differences do not affect this ER-associated process. However, they do preclude tetherin antagonism without high overexpression. Interestingly, while the majority of O-Vpu proteins have an E at position 32, a minority of sequences has K at this position and hence display increased TGN localization (exemplified by O-Vpu HJ001). The lack of a TMD-mediated interaction still precludes tetherin antagonism in this case.

The Vpu protein of SIVcpz is able to downregulate CD4 but cannot target tetherin, presumably because this function became redundant when the ancestral virus acquired a tetherin-antagonizing Nef protein from the SIVrcm lineage (45). Unlike the result with the TM domain of O-Vpu, replacement of the TM domain of consensus SIVcpzUS Vpu with that of HIV-1 M-Vpu is sufficient to confer tetherin antagonism (27). Groups M, N, O, and P represent four distinct zoonoses of SIVcpz strains to humans. Group O is also highly related to SIVgor, suggesting that both viruses have derived from the same SIVcpz strain relatively recently (less than 200 years) (57, 60). Whether group O was acquired directly from gorillas or whether these are separate zoonoses of the same virus from chimpanzees is not clear. However, they were transmitted to humans, and in each case the zoonotic virus would have initially been unable to target human tetherin due to the loss of the Nef-targeting determinant in the human protein's cytoplasmic tail (21, 45, 66). In the case of groups M and N, it is likely that the TM adaptation of the SIVcpz Vpu proteins was sufficient to adapt to tetherin antagonism, although N-Vpu proteins appear to have lost the ability to degrade CD4, the reason for which is unclear at present (45). CD4 targeting is conserved in all other known HIV-1/SIV Vpu proteins, indicating that this is also an essential function and is maintained, despite Nef performing a similar role, further suggesting spatial and temporal differences for CD4 targeting. It is interesting to speculate that the competing pressure to maintain CD4 degradation in the more distantly related SIVcpz that gave rise to HIV-1 group O precluded its adaptation to human tetherin because of its ER retention. In SIVcpz infections in chimpanzees, the requirement of the protein to leave the ER efficiently may have been under less pressure to be maintained because SIVcpz Nef antagonized tetherin in this species and was lost. Alternatively, if group O was primarily derived from SIVgor, these differences in Vpu may be a reflection of the SIVcpz Vpu process of adaptation to new hosts in relatively quick succession. To understand this further, more detailed molecular and cellular characterization of SIV Vpu proteins is required. Furthermore, Vpu, like Nef, may have other targets, in addition to CD4 and tetherin. Recent studies have implicated a role for Vpu in downregulating CD1d and NTB-A to avoid killing of infected cells by NK T cells and NK cells, respectively (36, 53). Therefore, the adaptation of any SIV Vpu in a new species is liable to be subject to further pressure to maintain functions against these and other yet-to-be-identified immunomodulatory targets.

It has recently become clear that primate lentiviruses are under evolutionary pressure to maintain an activity that counteracts tetherin. That O-Vpu and at least some N-Vpu proteins have no such activity has led to speculation that this Vpu function may account for the lack of efficient spread of groups N and O in humans compared to group M (46). Furthermore, evidence from HIV-2 in human and Nef-defective SIVmac-infected macaques suggests that when tetherin antagonism is compromised, viruses that restore the activity in their envelope glycoproteins can emerge (15, 26, 52). It should be borne in mind that group N and O viruses still retain the capacity to cause AIDS in infected individuals, and at present, it is not known whether the failure of O-Vpu to adapt to human tetherin has forced the acquisition of tetherin antagonism on the O-group Env. This is all the more plausible given that there is at least one documented case of an HIV-1 envelope glycoprotein with Vpu-like activity (49).