Neuronal BST2: A Pruritic Mediator alongside Protease-Activated Receptor 2 in the IL-27-Driven Itch Pathway.

Chronic itch is a common and complex symptom often associated with skin diseases such as atopic dermatitis (AD). Although IL-27 is linked to AD, its role and clinical significance in itch remain undefined. We sought to investigate IL-27 function in itch using tissue-specific transgenic mice, various itch models, behavior scoring, RNA sequencing, and cytokine/kinase array. Our findings show that IL-27 receptors were overexpressed in human AD skin. Intradermal IL-27 injection failed to directly induce itch in mice but upregulated skin protease-activated receptor 2 (PAR2) transcripts, a key factor in itch and AD. IL-27 activated human keratinocytes, increasing PAR2 transcription and activity. Coinjection of SLIGRL (PAR2 agonist) and IL-27 in mice heightened PAR2-mediated itch. In addition, IL-27 boosted BST2 transcription in sensory neurons and keratinocytes. BST2 was upregulated in AD skin, and its injection in mice induced itch-like response. BST2 colocalized with sensory nerve branches in AD skin from both human and murine models. Sensory neurons released BST2, and mice with sensory neuron-specific BST2 knockout displayed reduced itch responses. Overall, this study provides evidence that skin IL-27/PAR2 and neuronal IL-27/BST2 axes are implicated in cutaneous inflammation and pruritus. The discovery of neuronal BST2 in pruritus shed light on BST2 in the itch cascade.

Human BST2 inhibits rabies virus release independently of cysteine-linked dimerization and asparagine-linked glycosylation.

The innate immune response is a first-line defense mechanism triggered by rabies virus (RABV). Interferon (IFN) signaling and ISG products have been shown to confer resistance to RABV at various stages of the virus's life cycle. Human tetherin, also known as bone marrow stromal cell antigen 2 (hBST2), is a multifunctional transmembrane glycoprotein induced by IFN that has been shown to effectively counteract many viruses through diverse mechanisms. Here, we demonstrate that hBST2 inhibits RABV budding by tethering new virions to the cell surface. It was observed that release of virus-like particles (VLPs) formed by RABV G (RABV-G VLPs), but not RABV M (RABV-G VLPs), were suppressed by hBST2, indicating that RABV-G has a specific effect on the hBST2-mediated restriction of RABV. The ability of hBST2 to prevent the release of RABV-G VLPs and impede RABV growth kinetics is retained even when hBST2 has mutations at dimerization and/or glycosylation sites, making hBST2 an antagonist to RABV, with multiple mechanisms possibly contributing to the hBST2-mediated suppression of RABV. Our findings expand the knowledge of host antiviral mechanisms that control RABV infection.

Independent loss events of a functional tetherin gene in galliform birds.

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation.

Unbalanced protein homeostasis (proteostasis) networks are frequently linked to tumorigenesis, making cancer cells more susceptible to treatments that target proteostasis regulators. Proteasome inhibition is the first licensed proteostasis-targeting therapeutic strategy, and has been proven effective in hematological malignancy patients. However, drug resistance almost inevitably develops, pressing for a better understanding of the mechanisms that preserve proteostasis in tumor cells. Here we report that CD317, a tumor-targeting antigen with a unique topology, was upregulated in hematological malignancies and preserved proteostasis and cell viability in response to proteasome inhibitors (PIs). Knocking down CD317 lowered Ca2+ levels in the endoplasmic reticulum (ER), promoting PIs-induced proteostasis failure and cell death. Mechanistically, CD317 interacted with calnexin (CNX), an ER chaperone protein that limits calcium refilling via the Ca2+ pump SERCA, thereby subjecting CNX to RACK1-mediated autophagic degradation. As a result, CD317 decreased the level of CNX protein, coordinating Ca2+ uptake and thus favoring protein folding and quality control in the ER lumen. Our findings reveal a previously unrecognized role of CD317 in proteostasis control and imply that CD317 could be a promising target for resolving PIs resistance in the clinic.

The Feline Immunodeficiency Virus Envelope Signal Peptide Is a Tetherin Antagonizing Protein.

Signal peptides are N-terminal peptides, generally less than 30 amino acids in length, that direct translocation of proteins into the endoplasmic reticulum and secretory pathway. The envelope glycoprotein (Env) of the nonprimate lentivirus feline immunodeficiency virus (FIV) contains the longest signal peptide of all eukaryotic, prokaryotic, and viral proteins (175 amino acids), yet the reason is unknown. Tetherin is a dual membrane-anchored host protein that inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved three antagonists: the small accessory proteins Vpu and Nef, and in the case of HIV-2, Env. Here, we identify the FIV Env signal peptide (Fsp) as the FIV tetherin antagonist. A short deletion in the central portion of Fsp had no effect on viral replication in the absence of tetherin, but severely impaired virion budding in its presence. Fsp is necessary and sufficient, acting as an autonomous accessory protein with the rest of Env dispensable. In contrast to primate lentivirus tetherin antagonists, its mechanism is to stringently block the incorporation of this restriction factor into viral particles rather than by degrading it or downregulating it from the plasma membrane. IMPORTANCE The study of species- and virus-specific differences in restriction factors and their antagonists has been central to deciphering the nature of these key host defenses. FIV is an AIDS-causing lentivirus that has achieved pandemic spread in the domestic cat. We now identify its tetherin antagonist as the signal sequence of the Envelope glycoprotein, thus identifying the fourth lentiviral anti-tetherin protein and the first new lentiviral accessory protein in decades. Fsp is necessary and sufficient and functions by stringently blocking particle incorporation of tetherin, which differs from the degradation or surface downregulation mechanisms used by primate lentiviruses. Fsp also is a novel example of signal peptide dual function, being both a restriction factor antagonist and a mediator of protein translocation into the endoplasmic reticulum.

BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain.

Bone Marrow Stromal Cell Antigen 2 (BST2) is a type II transmembrane protein expressed on various cell types that tethers the release of viruses. Natural killer (NK) cells express low levels of BST2 under normal conditions but exhibit increased expression of BST2 upon activation. In this study, we show for the first time that murine BST2 can control the cytotoxicity of NK cells. The cytoplasmic tail of murine BST2 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). The absence of BST2 on NK cells can enhance their cytotoxicity against tumor cells compared to wild type NK cells. NK cells isolated from NZW mice, which express ITIM-deficient BST2, also showed higher cytotoxicity than wild type NK cells. In addition, we found that galectin-8 and galectin-9 were ligands of BST2, since blocking galectin-8 or -9 with monoclonal antibodies enhanced the cytotoxicity of NK cells. These results suggested that BST2 might be a novel NK cell inhibitory receptor as it was involved in regulating NK cell cytotoxicity through its interaction with galectins.

Unique Evolution of Antiviral Tetherin in Bats.

Bats are recognized as important reservoirs of viruses deadly to other mammals, including humans. These infections are typically nonpathogenic in bats, raising questions about host response differences that might exist between bats and other mammals. Tetherin is a restriction factor which inhibits the release of a diverse range of viruses from host cells, including retroviruses, coronaviruses, filoviruses, and paramyxoviruses, some of which are deadly to humans and transmitted by bats. Here, we characterize the tetherin genes from 27 bat species, revealing that they have evolved under strong selective pressure, and that fruit bats and vesper bats express unique structural variants of the tetherin protein. Tetherin was widely and variably expressed across fruit bat tissue types and upregulated in spleen tissue when stimulated with Toll-like receptor agonists. The expression of two computationally predicted splice isoforms of fruit bat tetherin was verified. We identified an additional third unique splice isoform which includes a C-terminal region that is not homologous to known mammalian tetherin variants but was functionally capable of restricting the release of filoviral virus-like particles. We also report that vesper bats possess and express at least five tetherin genes, including structural variants, more than any other mammal reported to date. These findings support the hypothesis of differential antiviral gene evolution in bats relative to other mammals. IMPORTANCE Bats are an important host of various viruses which are deadly to humans and other mammals but do not cause outward signs of illness in bats. Furthering our understanding of the unique features of the immune system of bats will shed light on how they tolerate viral infections, potentially informing novel antiviral strategies in humans and other animals. This study examines the antiviral protein tetherin, which prevents viral particles from escaping their host cell. Analysis of tetherin from 27 bat species reveals that it is under strong evolutionary pressure, and we show that multiple bat species have evolved to possess more tetherin genes than other mammals, some of which encode structurally unique tetherins capable of activity against different viral particles. These data suggest that bat tetherin plays a potentially broad and important role in the management of viral infections in bats.

Dual Role of HIV-1 Envelope Signal Peptide in Immune Evasion.

HIV-1 Env signal peptide (SP) is an important contributor to Env functions. Env is generated from Vpu/Env encoded bicistronic mRNA such that the 5' end of Env-N-terminus, that encodes for Env-SP overlaps with 3' end of Vpu. Env SP displays high sequence diversity, which translates into high variability in Vpu sequence. This study aimed to understand the effect of sequence polymorphism in the Vpu-Env overlapping region (VEOR) on the functions of two vital viral proteins: Vpu and Env. We used infectious molecular clone pNL4.3-CMU06 and swapped its SP (or VEOR) with that from other HIV-1 isolates. Swapping VEOR did not affect virus production in the absence of tetherin however, presence of tetherin significantly altered the release of virus progeny. VEOR also altered Vpu's ability to downregulate CD4 and tetherin. We next tested the effect of these swaps on Env functions. Analyzing the binding of monoclonal antibodies to membrane embedded Env revealed changes in the antigenic landscape of swapped Envs. These swaps affected the oligosaccharide composition of Env-N-glycans as shown by changes in DC-SIGN-mediated virus transmission. Our study suggests that genetic diversity in VEOR plays an important role in the differential pathogenesis and also assist in immune evasion by altering Env epitope exposure.

Tetherin downmodulation by SIVmac Nef lost with the H196Q escape variant is restored by an upstream variant.

The H196 residue in SIVmac239 Nef is conserved across the majority of HIV and SIV isolates, lies immediately adjacent to the AP-2 (adaptor protein 2) binding di-leucine domain (ExxxLM195), and is critical for several described AP-2 dependent Nef functions, including the downregulation of tetherin (BST-2/CD317), CD4, and others. Surprisingly, many stocks of the closely related SIVmac251 swarm virus harbor a nef allele encoding a Q196. In SIVmac239, this variant is associated with loss of multiple AP-2 dependent functions. Publicly available sequences for SIVmac251 stocks were mined for variants linked to Q196 that might compensate for functional defects associated with this residue. Variants were engineered into the SIVmac239 backbone and in Nef expression plasmids and flow cytometry was used to examine surface tetherin expression in primary CD4 T cells and surface CD4 expression in SupT1 cells engineered to express rhesus CD4. We found that SIVmac251 stocks that encode a Q196 residue in Nef uniformly also encode an upstream R191 residue. We show that R191 restores the ability of Nef to downregulate tetherin in the presence of Q196 and has a similar but less pronounced impact on CD4 expression. However, a published report showed Q196 commonly evolves to H196 in vivo, suggesting a fitness cost. R191 may represent compensatory evolution to restore the ability to downregulate tetherin lost in viruses harboring Q196.

Antiviral Activity and Adaptive Evolution of Avian Tetherins.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

The N-glycosylation of Equine Tetherin Affects Antiviral Activity by Regulating Its Subcellular Localization.

Tetherin is an interferon-inducible type II transmembrane glycoprotein which inhibits the release of viruses, including retroviruses, through a "physical tethering" model. However, the role that the glycosylation of tetherin plays in its antiviral activity remains controversial. In this study, we found that mutation of N-glycosylation sites resulted in an attenuation of the antiviral activity of equine tetherin (eqTHN), as well as a reduction in the expression of eqTHN at the plasma membrane (PM). In addition, eqTHN N-glycosylation mutants colocalize obviously with ER, CD63, LAMP1 and endosomes, while WT eqTHN do not. Furthermore, we also found that N-glycosylation impacts the transport of eqTHN in the cell not by affecting the endocytosis, but rather by influencing the anterograde trafficking of the protein. These results suggest that the N-glycosylation of eqTHN is important for the antiviral activity of the protein through regulating its normal subcellular localization. This finding will enhance our understanding of the function of this important restriction factor.

Activation of the ILT7 receptor and plasmacytoid dendritic cell responses are governed by structurally-distinct BST2 determinants.

Type I interferons (IFN-I) are key innate immune effectors predominantly produced by activated plasmacytoid dendritic cells (pDCs). By modulating immune responses at their foundation, IFNs can widely reshape immunity to control infectious diseases and malignancies. Nevertheless, their biological activities can also be detrimental to surrounding healthy cells, as prolonged IFN-I signaling is associated with excessive inflammation and immune dysfunction. The interaction of the human pDC receptor immunoglobulin-like transcript 7 (ILT7) with its IFN-I-regulated ligand, bone marrow stromal cell antigen 2 (BST2) plays a key role in controlling the IFN-I amounts produced by pDCs in response to Toll-like receptor (TLR) activation. However, the structural determinants and molecular features of BST2 that govern ILT7 engagement and activation are largely undefined. Using two functional assays to measure BST2-stimulated ILT7 activation as well as biophysical studies, here we identified two structurally-distinct regions of the BST2 ectodomain that play divergent roles during ILT7 activation. We found that although the coiled-coil region contains a newly defined ILT7-binding surface, the N-terminal region appears to suppress ILT7 activation. We further show that a stable BST2 homodimer binds to ILT7, but post-binding events associated with the unique BST2 coiled-coil plasticity are required to trigger receptor signaling. Hence, BST2 with an unstable or a rigid coiled-coil fails to activate ILT7, whereas substitutions in its N-terminal region enhance activation. Importantly, the biological relevance of these newly defined domains of BST2 is underscored by the identification of substitutions having opposing potentials to activate ILT7 in pathological malignant conditions.

Canine Influenza Virus is Mildly Restricted by Canine Tetherin Protein.

Tetherin (BST2/CD317/HM1.24) has emerged as a key host-cell ·defence molecule that acts by inhibiting the release and spread of diverse enveloped virions from infected cells. We analysed the biological features of canine tetherin and found it to be an unstable hydrophilic type I transmembrane protein with one transmembrane domain, no signal peptide, and multiple glycosylation and phosphorylation sites. Furthermore, the tissue expression profile of canine tetherin revealed that it was particularly abundant in immune organs. The canine tetherin gene contains an interferon response element sequence that can be regulated and expressed by canine IFN-α. A CCK-8 assay showed that canine tetherin was effective in helping mitigate cellular damage caused by canine influenza virus (CIV) infection. Additionally, we found that the overexpression of canine tetherin inhibited replication of the CIV and that interference with the canine tetherin gene enhanced CIV replication in cells. The impact of canine tetherin on CIV replication was mild. However, these results elucidate the role of the innate immune factor, canine tetherin, during CIV infection for the first time.

Vaccination with a DNA vaccine encoding CD317-targeting HBs antigen elicits enhanced immunity in mice.

BACKGROUND: Conventional hepatitis B virus (HBV) vaccines fail to induce protective antibody titers in 5-10% of immune-competent vaccines. Therefore, safe and effective HBV vaccines are still clinically needed. METHODS: In this study, we developed a plasmid DNA vaccine encoding CD317 single-chain fragment variable (α317scFv) linked with the hepatitis B surface antigen (HBsAg) and detected the humoral and cellular immune responses elicited by this vaccine in BALB/c mice. RESULTS: Vaccination with this fusion DNA vaccine in BALB/c mice induced more robust antiviral T cell and antibody immunity against HBsAg. Compared with mice vaccinated with control vaccine encoding HBsAg, the level of serum-circulating anti-HBsAg antibody (HBsAb) was nearly double in fusion DNA-vaccinated mice. More interesting, splenic lymphocytes isolated from fusion DNA-vaccinated mice showed more potent proliferation and IFN-γ production after being re-stimulated with recombinant HBsAg in vitro. And not only that, the cytotoxicity of fusion DNA vaccine-sensitized splenocytes was ∼3-fold higher than that of controls. CONCLUSION: Taken together, our results reveal that the fusion DNA vaccine can induce more effective immunological protection against HBV, and is a promising candidate for preventing HBV infection.

Polymorphisms in Rhesus Macaque Tetherin Are Associated with Differences in Acute Viremia in Simian Immunodeficiency Virus Δnef-Infected Animals.

Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on simian immunodeficiency virus (SIV) infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIVmac239 and 47 animals infected with SIVmac239Δnef Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Acute viremia did not differ significantly among the wild-type SIV-infected animals; however, differences in acute viral loads were associated with polymorphisms in tetherin among the animals infected with SIVΔnef In particular, polymorphisms at positions 43 and 111 (P43 and H111) were associated with lower acute-phase viral loads for SIVΔnef infection. These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef.IMPORTANCE As a consequence of ongoing evolutionary conflict with viral pathogens, tetherin has accumulated numerous species-specific differences that represent important barriers to the transmission of viruses between species. This study reveals extensive polymorphism in rhesus macaque tetherin and identifies specific alleles that are associated with lower viral loads during the first few weeks after infection with nef-deleted SIV. These observations suggest that the variable selective pressure of viral pathogens, in addition to driving the diversification of tetherin among species, also operates within certain species to maintain sequence variation in tetherin.

Strain-Specific Antagonism of the Human H1N1 Influenza A Virus against Equine Tetherin.

Tetherin/BST-2/CD317 is an interferon-induced host restriction factor that can block the budding of enveloped viruses by tethering them to the cell surface. Many viruses use certain proteins to counteract restriction by tetherin from their natural hosts, but not from other species. The influenza A virus (FLUAV) has a wide range of subtypes with different host tropisms. Human tetherin (huTHN) has been reported to restrict only specific FLUAV strains and the viral hemagglutinin (HA) and neuraminidase (NA) genes determine the sensitivity to huTHN. Whether tetherins from other hosts can block human FLUAV is still unknown. Here, we evaluate the impact of equine tetherin (eqTHN) and huTHN on the replication of A/Sichuan/1/2009 (H1N1) and A/equine/Xinjiang/1/2007 (H3N8) strains. Our results show that eqTHN had higher restriction activity towards both viruses, and its shorter cytoplasmic tail contributed to that activity. We further demonstrated that HA and NA of A/Hamburg/4/2009 (H1N1) could counteract eqTHN. Notably, our results indicate that four amino acids, 13T and 49L of HA and 32T and 80V of NA, were involved in blocking the restriction activity of eqTHN. These findings reveal interspecies restriction by eqTHN towards FLUAV, and the role of the HA and NA proteins in overcoming this restriction.

Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity.

HIV-1 causes chronic inflammation and AIDS in humans, whereas related simian immunodeficiency viruses (SIVs) replicate efficiently in their natural hosts without causing disease. It is currently unknown to what extent virus-specific properties are responsible for these different clinical outcomes. Here, we incorporate two putative HIV-1 virulence determinants, i.e., a Vpu protein that antagonizes tetherin and blocks NF-κB activation and a Nef protein that fails to suppress T cell activation via downmodulation of CD3, into a non-pathogenic SIVagm strain and test their impact on viral replication and pathogenicity in African green monkeys. Despite sustained high-level viremia over more than 4 years, moderately increased immune activation and transcriptional signatures of inflammation, the HIV-1-like SIVagm does not cause immunodeficiency or any other disease. These data indicate that species-specific host factors rather than intrinsic viral virulence factors determine the pathogenicity of primate lentiviruses.

Cloning, identification, and functional analysis of bone marrow stromal cell antigen-2 from sika deer (Cervus nippon).

BST-2(tetherin/CD317/HM1.24) has been identified as a cellular antiviral factor that inhibits the release of a wide range of enveloped viruses from infected cells. Orthologs of BST-2 have been identified in several species including humans, monkeys, cows, sheep, pigs, and mice. In this study, we cloned the gene and characterized the protein of the BST-2 homolog from sika deer (Cervus nippon). cnBST-2 shares 37.8% and 74.2% identity with the BST-2 homologs from Homo sapiens and Ovis aries, respectively. The extracellular domain of cnBST-2 has two putative N-linked glycosylation sites and three potential dimerization sites. cnBST-2 was shown to be expressed on the cell surface, like human BST-2. Exogenous expression of cnBST-2 resulted in potent inhibition of HIV-1 particle release in 293T cells; however, this activity resisted antagonism by HIV-1 Vpu. Moreover, cnBST-2 was not able to activate nuclear factor-κB, in contrast to human BST-2. This study is the first report of the isolation and characterization of BST-2 from C. nippon.

High-Mannose But Not Complex-Type Glycosylation of Tetherin Is Required for Restriction of HIV-1 Release.

Tetherin is an interferon-inducible antiviral protein that inhibits the release of a broad spectrum of enveloped viruses by retaining virions at the surface of infected cells. While the role of specific tetherin domains in antiviral activity is clearly established, the role of glycosylation in tetherin function is not clear. In this study, we carried out a detailed investigation of this question by using tetherin variants in which one or both sites of N-linked glycosylation were mutated (N65A, N92A, and N65,92A), and chemical inhibitors that prevent glycosylation at specific stages of oligosaccharide were added or modified. The single N-linked glycosylation mutants, N65A and N92A, efficiently inhibited the release of Vpu-defective human immunodeficiency virus type 1 (HIV-1). In contrast, the non-glycosylated double mutant, N65,92A, lost its ability to block HIV-1 release. The inability of the N65,92A mutant to inhibit HIV-1 release is associated with a lack of cell-surface expression. A role for glycosylation in cell-surface tetherin expression is supported by tunicamycin treatment, which inhibits the first step of N-linked glycosylation and impairs both cell-surface expression and antiviral activity. Inhibition of complex-type glycosylation with kifunensine, an inhibitor of the oligosaccharide processing enzyme mannosidase 1, had no effect on either the cell-surface expression or antiviral activity of tetherin. These results demonstrate that high-mannose modification of a single asparagine residue is necessary and sufficient, while complex-type glycosylation is dispensable, for cell-surface tetherin expression and antiviral activity.