Accessory Protein Vpu Research Articles

Minor contribution of HLA class I-associated selective pressure to the variability of HIV-1 accessory protein Vpu

Host HLA class I (HLA-I) allele-associated immune responses are major forces driving the evolution of HIV-1 proteins such as Gag and Nef. The viral protein U (Vpu) is an HIV-1 accessory protein responsible for CD4 degradation and enhancement of virion release by antagonizing tetherin/CD317. Although Vpu represents one of the most variable proteins in the HIV-1 proteome, it is still not clear to what extent HLA-I influence its evolution. To examine this issue, we enrolled 240 HLA-I-typed, treatment naïve, chronically HIV-infected subjects in Japan, and analyzed plasma HIV RNA nucleotide sequences of the vpu region. Using a phylogenetically-informed method incorporating corrections for HIV codon covariation and linkage disequilibrium among HLA alleles, we investigated HLA-associated amino acid mutations in the Vpu protein as well as in the translational products encoded by alternative reading frames. Despite substantial amino acid variability in Vpu, we identified only 4 HLA-associations in all possible translational products encoded in this region, suggesting that HLA-associated immune responses had minor effects on Vpu variability in this cohort. Rather, despite its size (81 amino acids), Vpu showed 103 codon–codon covariation associations, suggesting that Vpu conformation and function are preserved through many possible combinations of primary and secondary polymorphisms. Taken together, our study suggests that Vpu has been comparably less influenced by HLA-I-associated immune-driven evolution at the population level compared to other highly variable HIV-1 accessory proteins.

Ubiquitination of BST-2 Protein by HIV-1 Vpu Protein Does Not Require Lysine, Serine, or Threonine Residues within the BST-2 Cytoplasmic Domain

The cellular protein BST-2/CD317/Tetherin has been shown to inhibit the release of HIV-1 and other enveloped viruses from infected cells. The HIV-1 accessory protein Vpu binds to both BST-2 and βTrCP, a substrate-recognition subunit for the SCF (Skip1-Cullin1-F-box protein) E3 ubiquitin ligase complex. This interaction leads to both the degradation of BST-2 and the enhancement of viral egress. Recently BST-2 was shown to be ubiquitinated in this process. Here we have confirmed the Vpu- and βTrCP-dependent multi/polyubiquitination of BST-2. Ubiquitinated BST-2 accumulated in cells treated with a lysosomal inhibitor but not a proteasomal inhibitor. Additionally, we observed that a BST-2 mutant deleted for its cytosolically exposed lysine residues is also ubiquitinated. Subsequent experiments suggested that Vpu promotes BST-2 ubiquitination upon amino acid residues bearing hydroxyl- but not thiol-bearing side chains. However, a BST-2 mutant bearing substitutions for its cytoplasmically exposed Ser, Thr, and Lys residues was still down-regulated, ubiquitinated, and degraded in a Vpu-dependent manner. Our results suggest that Vpu may target either the BST-2 cytoplasmic Tyr residues or the NH(2) terminus itself for ubiquitination.

A Cytoplasmic Tail Determinant in HIV-1 Vpu Mediates Targeting of Tetherin for Endosomal Degradation and Counteracts Interferon-Induced Restriction

The HIV-1 accessory protein Vpu counteracts tetherin (BST-2/CD317) by preventing its incorporation into virions, reducing its surface expression, and ultimately promoting its degradation. Here we characterize a putative trafficking motif, EXXXLV, in the second alpha helix of the subtype-B Vpu cytoplasmic tail as being required for efficient tetherin antagonism. Mutation of this motif prevents ESCRT-dependent degradation of tetherin/Vpu complexes, tetherin cell surface downregulation, but not its physical interaction with Vpu. Importantly, this motif is required for efficient cell-free virion release from CD4+ T cells, particularly after their exposure to type-1 interferon, indicating that the ability to reduce surface tetherin levels and promote its degradation is important to counteract restriction under conditions that the virus likely encounters in vivo. Vpu EXXXLV mutants accumulate with tetherin at the cell surface and in endosomal compartments, but retain the ability to bind both β-TrCP2 and HRS, indicating that this motif is required for a post-binding trafficking event that commits tetherin for ESCRT-dependent degradation and prevents its transit to the plasma membrane and viral budding zones. We further found that while Vpu function is dependent on clathrin, and the entire second alpha helix of the Vpu tail can be functionally complemented by a clathrin adaptor binding peptide derived from HIV-1 Nef, none of the canonical clathrin adaptors nor retromer are required for this process. Finally we show that residual activity of Vpu EXXXLV mutants requires an intact endocytic motif in tetherin, suggesting that physical association of Vpu with tetherin during its recycling may be sufficient to compromise tetherin activity to some degree.

The UBAP1 Subunit of ESCRT-I Interacts with Ubiquitin via a SOUBA Domain

SummaryThe endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5.

SAMHD1-Dependent and -Independent Functions of HIV-2/SIV Vpx Protein

Both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) encode a unique set of accessory proteins that enhance viral replication in the host. Two similar accessory proteins, Vpx and Vpr, are encoded by HIV-2. In contrast, HIV-1 encodes Vpr but not Vpx. Recent studies have indicated that Vpx counteracts a particular host restriction factor, thereby facilitating reverse transcription in myeloid cells such as monocyte-derived macrophages and monocyte-derived dendritic cells. This mechanism of counteraction is similar to that of the accessory proteins Vif and Vpu which antagonize other host factors. In 2011, the protein SAMHD1 was identified as the restriction factor counteracted by Vpx. Studies have since revealed that SAMHD1 degrades deoxynucleoside triphosphates (dNTPs), which are components of viral genomic cDNA, in order to deprive viruses of dNTPs. Although interactions between SAMHD1 and Vpx continue to be a major research focus, Vpx has also been shown to have an apparent ability to enhance nuclear import of the viral genome in T lymphocytes. This review summarizes the current knowledge regarding SAMHD1-dependent and -independent functions of Vpx, and discusses possible reasons why HIV-2 encodes both Vpx and Vpr, unlike HIV-1.

Ebolavirus Replication and Tetherin/BST-2

Ebolavirus (EBOV) is an enveloped, non-segmented, negative-stranded RNA virus, which consists of five species: Zaire ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus. EBOV causes a lethal hemorrhagic fever in both humans and non-human primates. The EBOV RNA genome encodes seven viral proteins: NP, VP35, VP40, GP, VP30, VP24, and L. VP40 is a matrix protein and is essential for virus assembly and release from host cells. Expression of VP40 in mammalian cells is sufficient to generate extracellular virus-like particles, which resemble authentic virions. Tetherin/BST-2, which was identified as an effective cellular factor that prevents human immunodeficiency virus-1 release in the absence of viral accessory protein Vpu, has been reported to inhibit ZEBOV VP40-induced VLP release. Tetherin/BST-2 appears to inhibit virus release by physically tethering viral particles to the cell surface via its N-terminal transmembrane domain and C-terminal glycosylphosphatidylinositol anchor. Replication of ZEBOV is not inhibited by tetherin/BST-2 expression, although tetherin/BST-2 was expected to inhibit EBOV release as well as VLP release. Recently, it was reported that viral glycoprotein of EBOV, GP, antagonizes the antiviral effect of tetherin/BST-2. However, the mechanism by which GP antagonizes the antiviral activity of tetherin/BST-2 and whether GP of the other EBOV species function as antagonists of tetherin/BST-2 remain unclear.

The HIV-1 Vpu Viroporin Inhibitor BIT225 Does Not Affect Vpu-Mediated Tetherin Antagonism

Among its many roles, the HIV-1 accessory protein Vpu performs a viroporin function and also antagonizes the host cell restriction factor tetherin through its transmembrane domain. BIT225 is a small molecule inhibitor that specifically targets the Vpu viroporin function, which, in macrophages, resulted in late stage inhibition of virus release and decreased infectivity of released virus, a phenotype similar to tetherin-mediated restriction. Here, we investigated whether BIT225 might mediate its antiviral function, at least in part, via inhibition of Vpu-mediated tetherin antagonism. Using T-cell lines inducible for tetherin expression, we found that BIT225 does not exert its antiviral function by inhibiting Vpu-mediated tetherin downmodulation from the cell surface, the main site of action of tetherin activity. In addition, results from a bioluminescence resonance energy transfer (BRET) assay showed that the Vpu-tetherin interaction was not affected by BIT225. Our data provide support for the concept that tetherin antagonism and viroporin function are separable on the Vpu transmembrane and that viroporin function might be cell-type dependent. Further, this work contributes to the characterization of BIT225 as an inhibitor that specifically targets the viroporin function of Vpu.

Sequences in Gibbon Ape Leukemia Virus Envelope That Confer Sensitivity to HIV-1 Accessory Protein Vpu

HIV-1 efficiently forms pseudotyped particles with many gammaretrovirus glycoproteins, such as Friend murine leukemia virus (F-MLV) Env, but not with the related gibbon ape leukemia virus (GaLV) Env or with a chimeric F-MLV Env with a GaLV cytoplasmic tail domain (CTD). This incompatibility is modulated by the HIV-1 accessory protein Vpu. Because the GaLV Env CTD does not resemble tetherin or CD4, the well-studied targets of Vpu, we sought to characterize the modular sequence in the GaLV Env CTD required for this restriction in the presence of Vpu. Using a systematic mutagenesis scan, we determined that the motif that makes GaLV Env sensitive to Vpu is INxxIxxVKxxVxRxK. This region in the CTD of GaLV Env is predicted to form a helix. Mutations in the CTD that would break this helix abolish sensitivity to Vpu. Although many of these positions can be replaced with amino acids with similar biophysical properties without disrupting the Vpu sensitivity, the final lysine residue is required. This Vpu sensitivity sequence appears to be modular, as the unrelated Rous sarcoma virus (RSV) Env can be made Vpu sensitive by replacing its CTD with the GaLV Env CTD. In addition, F-MLV Env can be made Vpu sensitive by mutating two amino acids in its cytoplasmic tail to make it resemble more closely the Vpu sensitivity motif. Surprisingly, the core components of this Vpu sensitivity sequence are also present in the host surface protein CD4, which is also targeted by Vpu through its CTD.

Role of the Endocytic Pathway in the Counteraction of BST-2 by Human Lentiviral Pathogens

The interferon-inducible transmembrane protein BST-2 (CD317, tetherin) restricts the release of several enveloped viruses from infected cells. BST-2 is broadly active against retroviruses, including HIV-1 and HIV-2. To counteract this host defense, HIV-1 uses the accessory protein Vpu, whereas HIV-2 uses its envelope glycoprotein (Env). In both cases, viral antagonism is associated with decreased expression of BST-2 at the cell surface. Here, we provide evidence supporting a role for the clathrin-mediated endocytic pathway in the downregulation of BST-2 from the cell surface and the counteraction of restricted virion release. A catalytically inactive, dominant negative version of the vesicle "pinch-ase" dynamin 2 (dyn2K44A) inhibited the downregulation of BST-2 by Vpu, and it inhibited the release of wild-type (Vpu-expressing) HIV-1 virions. Similarly, dyn2K44A inhibited the downregulation of BST-2 by HIV-2 Env, and it inhibited the release of vpu-negative HIV-1 virions when HIV-2 Env was provided in trans. dyn2K44A inhibited Env more robustly than Vpu, suggesting that dynamin 2, while a cofactor for both Env and Vpu, might support just one of several pathways though which Vpu counteracts BST-2. In support of a role for clathrin in these effects, the C-terminal domain of the clathrin assembly protein AP180 also inhibited the downregulation of BST-2 by either Vpu or HIV-2 Env. Consistent with modulation of the postendocytic itinerary of BST-2, Vpu enhanced the accumulation of cell surface-derived BST-2 in transferrin-containing endosomes. Vpu also inhibited the transport of BST-2 from a brefeldin A-insensitive compartment to the cell surface, consistent with a block to endosomal recycling. We propose that HIV-1 Vpu, and probably HIV-2 Env, traps BST-2 in an endosomal compartment following endocytosis, reducing its level at the cell surface to counteract restricted viral release.

High-throughput assay to identify inhibitors of Vpu-mediated down-regulation of cell surface BST-2

Bone marrow stromal cell antigen 2 (BST-2, also known as Tetherin) inhibits HIV-1 release and thereby severely impairs viral replication. HIV-1 accessory protein Vpu induces the down-regulation of cell surface BST-2, and counteracts the antiviral function of BST-2. Blocking Vpu-mediated down-regulation of cell surface BST-2 is viewed as a new opportunity for developing anti-HIV drugs. In this study, we have developed a high-throughput cell-based ELISA to identify small molecules that antagonize HIV-1 Vpu function and consequently inhibit HIV-1 replication through rescuing the antiviral activity of host BST-2. This cell-ELISA shows an excellent correlation with results obtained by flow cytometry (FACS). Under optimal conditions, a Z′ factor of 0.605 was achieved in a 96-well format. Together, these results demonstrate that this assay can be used to quantify the cell surface level of BST-2 and be adapted to a high-throughput screening for novel anti-HIV compounds.

Polarity Changes in the Transmembrane Domain Core of HIV-1 Vpu Inhibits Its Anti-Tetherin Activity

Tetherin (BST-2/CD317) is an interferon-inducible antiviral protein that restricts the release of enveloped viruses from infected cells. The HIV-1 accessory protein Vpu can efficiently antagonize this restriction. In this study, we analyzed mutations of the transmembrane (TM) domain of Vpu, including deletions and substitutions, to delineate amino acids important for HIV-1 viral particle release and in interactions with tetherin. The mutants had similar subcellular localization patterns with that of wild-type Vpu and were functional with respect to CD4 downregulation. We showed that the hydrophobic binding surface for tetherin lies in the core of the Vpu TM domain. Three consecutive hydrophobic isoleucine residues in the middle region of the Vpu TM domain, I15, I16 and I17, were important for stabilizing the tetherin binding interface and determining its sensitivity to tetherin. Changing the polarity of the amino acids at these positions resulted in severe impairment of Vpu-induced tetherin targeting and antagonism. Taken together, these data reveal a model of specific hydrophobic interactions between Vpu and tetherin, which can be potentially targeted in the development of novel anti-HIV-1 drugs.

β-TrCP is dispensable for Vpu's ability to overcome the CD317/Tetherin-imposed restriction to HIV-1 release

BackgroundThe cellular transmembrane protein CD317/BST-2/HM1.24/Tetherin restricts HIV-1 infection by physically tethering mature virions to the surface of infected cells. HIV-1 counteracts this restriction by expressing the accessory protein Vpu, yet the mechanism of this antagonism is incompletely understood. β-TrCP is the substrate recognition domain of an E3 ubiquitin ligase complex that interacts with the di-serine motif S52/S56 in the cytoplasmic tail of Vpu to target the CD4 receptor for proteasomal degradation. Recently, it has been suggested that β-TrCP is also critically involved in Vpu's ability to overcome the CD317-mediated virion release block.ResultsTo test this model, we analyzed the consequences of several experimental strategies to interfere with the Vpu-β-TrCP protein-protein interaction. Under these conditions, we studied effects of Vpu on expression and localization of CD317 and CD4, as well as on its ability to promote HIV-1 release. Our results demonstrate a strict requirement for Vpu's di-serine motif for degradation of CD4 and also CD317, reduction of cell surface exposure of CD317, and HIV-1 release enhancement. We further show a critical role of β-TrCP2, but not of the structurally related β-TrCP1 isoform, for Vpu-mediated degradation of both receptors. Most importantly, Vpu remained active in downregulating CD317 from the cell surface and in overcoming the HIV-1 release restriction in β-TrCP-depleted cells.ConclusionsThese results demonstrate that β-TrCP is not strictly required for Vpu's ability to counteract the CD317-imposed virion release block and support the relevance of cell surface down-modulation of the restriction factor as a central mechanism of Vpu antagonism. Moreover, we propose the existence of a critical, yet to be identified cellular factor that interacts with Vpu via its di-serine motif to alter the trafficking of the restriction factor.

Molecular mechanisms of viral infection and propagation: An overview of the second Advanced Summer School in Africa

Molecular mechanisms of viral infection and propagation: An overview of the second Advanced Summer School in Africa

Direct internalization of cell-surface BST-2/tetherin by the HIV-1 accessory protein Vpu

The host transmembrane protein BST-2/tetherin is a powerful antiviral factor that blocks the production of enveloped viruses. The HIV-1 accessory protein Vpu inhibits the antiviral activity of BST-2; however, the degradation pathway by which Vpu downregulates BST-2 from the cell surface and the actual subcellular location where Vpu targets BST-2 for downregulation remain controversial. Whereas one study showed that Vpu acts on constitutively endocytosed BST-2, we recently reported that Vpu can internalize BST-2 from the cell surface. Because the evidence for this conclusion was derived from indirect results, we present direct evidence in this study using an antibody internalization assay with an endocytosis-defective mutant of BST-2. The internalization of the BST-2 protein into cells coexpressing wild-type Vpu was observed when the cells were preincubated with antibodies against BST-2 at 37˚C, but not at 4˚C, for 10 min. These results strongly support our previous finding that continuously expressed de novo BST-2 at the cell surface is internalized by functional Vpu protein.

CD317/Tetherin Is Enriched in the HIV-1 Envelope and Downregulated from the Plasma Membrane upon Virus Infection

CD317/Bst-2/tetherin is a host factor that restricts the release of human immunodeficiency virus type 1 (HIV-1) by trapping virions at the plasma membrane of certain producer cells. It is antagonized by the HIV-1 accessory protein Vpu. Previous light microscopy studies localized CD317 to the plasma membrane and the endosomal compartment and showed Vpu induced downregulation. In the present study, we performed quantitative immunoelectron microscopy of CD317 in cells producing wild-type or Vpu-defective HIV-1 and in control cells. Double-labeling experiments revealed that CD317 localizes to the plasma membrane, to early and recycling endosomes, and to the trans-Golgi network. CD317 largely relocated to endosomes upon HIV-1 infection, and this effect was partly counteracted by Vpu. Unexpectedly, CD317 was enriched in the membrane of viral buds and cell-associated and cell-free viruses compared to the respective plasma membrane, and this enrichment was independent of Vpu. These results suggest that the tethering activity of CD317 critically depends on its density at the cell surface and appears to be less affected by its density in the virion membrane.

Pseudotyping Incompatibility between HIV-1 and Gibbon Ape Leukemia Virus Env Is Modulated by Vpu

The Env protein from gibbon ape leukemia virus (GaLV) has been shown to be incompatible with human immunodeficiency virus type 1 (HIV-1) in the production of infectious pseudotyped particles. This incompatibility has been mapped to the C-terminal cytoplasmic tail of GaLV Env. Surprisingly, we found that the HIV-1 accessory protein Vpu modulates this incompatibility. The infectivity of HIV-1 pseudotyped with murine leukemia virus (MLV) Env was not affected by Vpu. However, the infectivity of HIV-1 pseudotyped with an MLV Env with the cytoplasmic tail from GaLV Env (MLV/GaLV Env) was restricted 50- to 100-fold by Vpu. A Vpu mutant containing a scrambled membrane-spanning domain, Vpu(RD), was still able to restrict MLV/GaLV Env, but mutation of the serine residues at positions 52 and 56 completely alleviated the restriction. Loss of infectivity appeared to be caused by reduced MLV/GaLV Env incorporation into viral particles. The mechanism of this downmodulation appears to be distinct from Vpu-mediated CD4 downmodulation because Vpu-expressing cells that failed to produce infectious HIV-1 particles nonetheless continued to display robust surface MLV/GaLV Env expression. In addition, if MLV and HIV-1 were simultaneously introduced into the same cells, only the HIV-1 particle infectivity was restricted by Vpu. Collectively, these data suggest that Vpu modulates the cellular distribution of MLV/GaLV Env, preventing its recruitment to HIV-1 budding sites.

HIV-1 Accessory Protein Vpu Internalizes Cell-surface BST-2/Tetherin through Transmembrane Interactions Leading to Lysosomes

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, beta-transducin repeat-containing protein (betaTrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a betaTrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes.

Effect of Calcium-Modulating Cyclophilin Ligand on Human Immunodeficiency Virus Type 1 Particle Release and Cell Surface Expression of Tetherin

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpu enhances virus particle release by counteracting a host factor that retains virions at the surfaces of infected cells. It was recently demonstrated that cellular protein BST-2/CD317/Tetherin restricts HIV-1 release in a Vpu-dependent manner. Calcium-modulating cyclophilin ligand (CAML) was also proposed to be involved in this process. We investigated whether CAML is involved in cell surface expression of Tetherin. Here, we show that CAML overexpression in permissive Cos-7 cells or CAML depletion in restrictive HeLa cells has no effect on HIV-1 release or on Tetherin surface expression, indicating that CAML is not required for Tetherin-mediated restriction of HIV-1 release.

Vpu Antagonizes BST-2–Mediated Restriction of HIV-1 Release via β-TrCP and Endo-Lysosomal Trafficking

The interferon-induced transmembrane protein BST-2/CD317 (tetherin) restricts the release of diverse enveloped viruses from infected cells. The HIV-1 accessory protein Vpu antagonizes this restriction by an unknown mechanism that likely involves the down-regulation of BST-2 from the cell surface. Here, we show that the optimal removal of BST-2 from the plasma membrane by Vpu requires the cellular protein β-TrCP, a substrate adaptor for a multi-subunit SCF E3 ubiquitin ligase complex and a known Vpu-interacting protein. β-TrCP is also required for the optimal enhancement of virion-release by Vpu. Mutations in the DSGxxS β-TrCP binding-motif of Vpu impair both the down-regulation of BST-2 and the enhancement of virion-release. Such mutations also confer dominant-negative activity, consistent with a model in which Vpu links BST-2 to β-TrCP. Optimal down-regulation of BST-2 from the cell surface by Vpu also requires the endocytic clathrin adaptor AP-2, although the rate of endocytosis is not increased; these data suggest that Vpu induces post-endocytic membrane trafficking events whose net effect is the removal of BST-2 from the cell surface. In addition to its marked effect on cell-surface levels, Vpu modestly decreases the total cellular levels of BST-2. The decreases in cell-surface and intracellular BST-2 are inhibited by bafilomycin A1, an inhibitor of endosomal acidification; these data suggest that Vpu induces late endosomal targeting and partial degradation of BST-2 in lysosomes. The Vpu-mediated decrease in surface expression is associated with reduced co-localization of BST-2 and the virion protein Gag along the plasma membrane. Together, the data support a model in which Vpu co-opts the β-TrCP/SCF E3 ubiquitin ligase complex to induce endosomal trafficking events that remove BST-2 from its site of action as a virion-tethering factor.

Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein

Mammalian cells employ numerous innate cellular mechanisms to inhibit viral replication and spread. Tetherin, also known as Bst-2 or CD317, is a recently identified, IFN-induced, cellular response factor that blocks release of HIV-1 and other retroviruses from infected cells. The means by which tetherin retains retroviruses on the cell surface, as well as the mechanism used by the HIV-1 accessory protein Vpu to antagonize tetherin function and promote HIV-1 release, are unknown. Here, we document that tetherin functions as a broadly acting antiviral factor by demonstrating that both human and murine tetherin potently inhibit the release of the filovirus, Ebola, from the surface of cells. Expression of the Ebola glycoprotein (GP) antagonized the antiviral effect of human and murine tetherin and facilitated budding of Ebola particles, as did the HIV-1 Vpu protein. Conversely, Ebola GP could substitute for Vpu to promote HIV-1 virion release from tetherin-expressing cells, demonstrating a common cellular target for these divergent viral proteins. Ebola GP efficiently coimmunoprecipitated with tetherin, suggesting that the viral glycoprotein directly interferes with this host antiviral factor. These results demonstrate that tetherin is a cellular antiviral factor that restricts budding of structurally diverse enveloped viruses. Additionally, Ebola has evolved a highly effective strategy to combat this antiviral response elicited in the host during infection.