Equine Tetherin Blocks Retrovirus Release and Its Activity Is Antagonized by Equine Infectious Anemia Virus Envelope Protein

Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism.     

Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.     

Tetherin Restricts Herpes Simplex Virus 1 and Is Antagonized by Glycoprotein M

Tetherin is a broadly active antiviral effector that works by tethering nascent enveloped virions to a host cell membrane, thus preventing their release. In this study, we demonstrate that herpes simplex virus 1 (HSV-1) is targeted by tetherin. We identify the viral envelope glycoprotein M (gM) as having moderate anti-tetherin activity. We show that gM but not gB or gD efficiently removes tetherin from the plasma membrane and can functionally substitute for the human immunodeficiency virus type 1 (HIV-1) Vpu protein, the prototypic viral tetherin antagonist, in rescuing HIV-1 release from tetherin-expressing cells. Our data emphasize that tetherin is a broadly active antiviral effector and contribute to the emerging hypothesis that viruses must suppress or evade an array of host cell countermeasures in order to establish a productive infection.     

The RING-CH Ligase K5 Antagonizes Restriction of KSHV and HIV-1 Particle Release by Mediating Ubiquitin-Dependent Endosomal Degradation of Tetherin

Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi''s sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.     

束缚蛋白抗HIV机制

束缚蛋白(tetherin)是一种具有特殊功能的蛋白质,它可抑制包膜病毒从感染细胞中释放。研究发现,人的束缚蛋白可将新生的HIV-1病毒颗粒固定在细胞表面,同时,它还可以减小HIV-1子代病毒的传染性。本文将主要从分子结构、抗HIV-1病毒的作用机制和在HIV传播中的作用三方面来阐述束缚蛋白抗病毒作用的最新研究进展。     

Feline tetherin is characterized by a short N-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein

Tetherin (BST2) is the host cell factor that blocks the particle release of some enveloped viruses. Two putative feline tetherin proteins differing at the level of the N-terminal coding region have recently been described and tested for their antiviral activity. By cloning and comparing the two reported feline tetherins (called here cBST2(504) and cBST2*) and generating specific derivative mutants, this study provides evidence that feline tetherin has a shorter intracytoplasmic domain than those of other known homologues. The minimal tetherin promoter was identified and assayed for its ability to drive tetherin expression in an alpha interferon-inducible manner. We also demonstrated that cBST2(504) is able to dimerize, is localized at the cellular membrane, and impairs human immunodeficiency virus type 1 (HIV-1) particle release, regardless of the presence of the Vpu antagonist accessory protein. While cBST2(504) failed to restrict wild-type feline immunodeficiency virus (FIV) egress, FIV mutants, bearing a frameshift at the level of the envelope-encoding region, were potently blocked. The transient expression of the FIV envelope glycoprotein was able to rescue mutant particle release from feline tetherin-positive cells but did not antagonize human BST2 activity. Moreover, cBST2(504) was capable of specifically immunoprecipitating the FIV envelope glycoprotein. Finally, cBST2(504) also exerted its function on HIV-2 ROD10 and on the simian immunodeficiency virus SIVmac239. Taken together, these results show that feline tetherin does indeed have a short N-terminal region and that the FIV envelope glycoprotein is the predominant factor counteracting tetherin restriction.     

Herpes Simplex Virus 1 Counteracts Tetherin Restriction via Its Virion Host Shutoff Activity

The interferon-inducible membrane protein tetherin (Bst-2, or CD317) is an antiviral factor that inhibits enveloped virus release by cross-linking newly formed virus particles to the producing cell. The majority of viruses that are sensitive to tetherin restriction appear to be those that acquire their envelopes at the plasma membrane, although many viruses, including herpesviruses, envelope at intracellular membranes, and the effect of tetherin on such viruses has been less well studied. We investigated the tetherin sensitivity and possible countermeasures of herpes simplex virus 1 (HSV-1). We found that overexpression of tetherin inhibits HSV-1 release and that HSV-1 efficiently depletes tetherin from infected cells. We further show that the virion host shutoff protein (Vhs) is important for depletion of tetherin mRNA and protein and that removal of tetherin compensates for defects in replication and release of a Vhs-null virus. Vhs is known to be important for HSV-1 to evade the innate immune response in vivo. Taken together, our data suggest that tetherin has antiviral activity toward HSV-1 and that the removal of tetherin by Vhs is important for the efficient replication and dissemination of HSV-1.     

Cloning and characterization of the antiviral activity of feline Tetherin/BST-2

Human Tetherin/BST-2 has recently been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. In this study, we cloned a cDNA fragment encoding a feline homolog of Tetherin/BST-2 and characterized the protein product. The degree of amino acid sequence identity between human Tetherin/BST-2 and the feline homolog was 44.4%. Similar to human Tetherin/BST-2, the expression of feline Tetherin/BST-2 mRNA was inducible by type I interferon (IFN). Exogenous expression of feline Tetherin/BST-2 efficiently inhibited the release of feline endogenous retrovirus RD-114. The extracellular domain of feline Tetherin/BST-2 has two putative N-linked glycosylation sites, N79 and N119. Complete loss of N-linked glycosylation by introduction of mutations into both sites resulted in almost complete abolition of its antiviral activity. In addition, feline Tetherin/BST-2 was insensitive to antagonism by HIV-1 Vpu, although the antiviral activity of human Tetherin/BST-2 was antagonized by HIV-1 Vpu. Our data suggest that feline Tetherin/BST-2 functions as a part of IFN-induced innate immunity against virus infection and that the induction of feline Tetherin/BST-2 in vivo may be effective as a novel antiviral strategy for viral infection.     

Species-Specific Activity of HIV-1 Vpu and Positive Selection of Tetherin Transmembrane Domain Variants

Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins.     

Tetherin has negligible activity in restricting hepatitis C virus in hepatocytes

We investigated the ability of tetherin, a recently identified antiviral factor, in restricting hepatitis C virus (HCV) in the Japanese fulminant hepatitis-1 (JFH-1) infectious cell culture system. Human hepatocytes (Huh7, Huh7.5.1) expressedlow levels of endogenous tetherin, which could be induced by IFN-α. However, tetherin contributes little to IFN-α-mediated anti-HCV JFH-1 activity. Although tetherin could inhibit Vpu-deleted HIV-1 release, it had negligible activity in restricting HCV JFH-1 release from hepatocytes, which was evidenced by unaffected levels of intracellular/extracellular HCV RNA and infectious virus. The failure of tetherin's anti-HCV activity could not be related to the counteraction of HCV, as HCV infection of hepatocytes affected neither tetherin expression nor anti-HIV function of tetherin. These observations imply that tetherin has negligible activity in the restriction of HCV JFH-1 in human hepatocytes.     

Inhibition of Lassa and Marburg Virus Production by Tetherin

Recently, tetherin has been identified as an effective cellular factor that prevents the release of human immunodeficiency virus type 1. Here, we show that the production of virus-like particles induced by viral matrix proteins of Lassa virus or Marburg virus was markedly inhibited by tetherin and that N-linked glycosylation of tetherin was dispensable for this antiviral activity. Our data also suggest that viral matrix proteins or one or more components that originate from host cells are targets of tetherin but that viral surface glycoproteins are not. These results suggest that tetherin inhibits the release of a wide variety of enveloped viruses from host cells by a common mechanism.There are a number of innate host defense systems against virus infection, including interferon (IFN) and toll-like receptor signaling pathways. Cellular factors that inhibit viral replication through interactions with viral components at various steps have also been identified.Recently, tetherin (also known as BST2, CD317, or HM1.24) was identified as a cellular factor that inhibits the release of human immunodeficiency virus type 1 (HIV-1) from infected cells (6). Tetherin is a membrane-associated protein with an N-terminal transmembrane domain, a central extracellular domain with two potential N-linked glycosylation sites, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (Fig. ​(Fig.1A)1A) (3, 4), which appears to prevent HIV-1 release by retaining fully formed progeny virions on the surfaces of infected cells (6, 11). Tetherin is constitutively present on the surfaces of HeLa and CEM cells, while its cell surface expression is induced by alpha IFN (IFN-α) in HEK293, 293T, HOS, HT1080, and COS-7 cells. Tetherin expression has also been reported to be stimulated by IFN in various tissues, including those of the liver, lung, placenta, heart, pancreas, kidney, skeletal muscle, and brain (1, 3), suggesting that it may function as part of IFN-induced innate immunity against enveloped viruses in vivo.Open in a separate windowFIG. 1.Inhibitory effects of tetherin and its mutants against Lassa VLP release. (A) Tetherin (WT) contains an N-terminal intracellular domain (ID), a transmembrane domain (TM), a central extracellular domain (ED), and a C-terminal GPI anchor (GPI). Arrowheads indicate the predicted sites of cleavage prior to the addition of the GPI anchor. Tetherin possesses two potential N-linked glycosylation sites at positions 65 and 92 in the ED. N65A and N92A are mutants with the loss of a glycosylation site by an Asn-to-Ala substitution at positions 65 and 92, respectively. N65A/N92A is a nonglycosylated mutant with the loss of both glycosylation sites. (B and D) The Lassa virus Z and GP-C expression plasmids were cotransfected with the expression plasmid for WT or mutant tetherin or an empty vector (Control) into COS-7 cells (B) or 293T cells (D). Extracellular VLPs induced by Lassa virus Z/GP-C were pelleted from the culture fluids. Cell- or VLP-associated Z and GP-C (GP-2) were detected by Western blotting using rabbit anti-Z antiserum and mouse anti-GP-2 monoclonal antibody. WB using anti-FLAG antibody was also performed to examine the expression of WT and mutant tetherin in cells. WB for actin was done as the internal control. (C) The intensities of the bands for VLP-associated Z or GP-2 in panel B were quantified using a LAS3000 imaging system (Fujifilm). The level of Z or GP-2 in VLPs released from cells cotransfected with control vector was set to 100%. The data are shown as averages and standard deviations for three independent experiments. (E) COS-7 cells were cotransfected with the Lassa virus Z expression plasmid and the expression plasmid for tetherin (WT) or the empty vector (Control). VLPs induced by Z alone were examined by WB as described above. (F) 293T cells were cotransfected with pCLV-Z and the empty vector (left) or the expression plasmid for tetherin (right). At 48 h posttransfection, cells were observed by electron microscopy, which was performed as described previously (9). Mock, mock infected; Teth, tetherin. Bars, 500 nm.The antiviral activity of tetherin is antagonized by HIV-1 Vpu due to the downregulation of cell surface expression of tetherin by Vpu (6, 11). Previously, the IFN-α-induced cell surface retention of virus-like particles (VLPs) induced by Ebola virus matrix protein VP40 was shown to be overcome by Vpu expression (5). Thus, the release of enveloped viruses other than HIV-1 may also be inhibited by tetherin.Lassa and Marburg viruses are emerging viruses belonging to the families Arenaviridae and Filoviridae, respectively, that cause hemorrhagic fever with high mortality rates. No approved vaccines or antiviral drugs are available to prevent or treat these viral diseases. Similar to HIV-1, both are enveloped viruses that exit the host cells by membrane extrusion, known as budding, from the plasma membrane. Therefore, having an antiviral effect against Lassa and Marburg viruses would make tetherin a potent tool for novel antiviral strategies against a wide variety of enveloped viruses.We examined the antiviral activities of tetherin against Lassa and Marburg viruses and analyzed the characteristics required for its antiviral activity in order to gain insight into its antiviral mechanism of action.     

Immunoelectron Microscopic Evidence for Tetherin/BST2 as the Physical Bridge between HIV-1 Virions and the Plasma Membrane

Tetherin/BST2 was identified in 2008 as the cellular factor responsible for restricting HIV-1 replication at a very late stage in the lifecycle. Tetherin acts to retain virion particles on the plasma membrane after budding has been completed. Infected cells that express large amounts of tetherin display large strings of HIV virions that remain attached to the plasma membrane. Vpu is an HIV-1 accessory protein that specifically counteracts the restriction to virus release contributed by tetherin. Tetherin is an unusual Type II transmembrane protein that contains a GPI anchor at its C-terminus and is found in lipid rafts. The leading model for the mechanism of action of tetherin is that it functions as a direct physical tether bridging virions and the plasma membrane. However, evidence that tetherin functions as a physical tether has thus far been indirect. Here we demonstrate by biochemical and immunoelectron microscopic methods that endogenous tetherin is present on the viral particle and forms a bridge between virion particles and the plasma membrane. Endogenous tetherin was found on HIV particles that were released by partial proteolytic digestion. Immunoelectron microscopy performed on HIV-infected T cells demonstrated that tetherin forms an apparent physical link between virions and connects patches of virions to the plasma membrane. Linear filamentous strands that were highly enriched in tetherin bridged the space between some virions. We conclude that tetherin is the physical tether linking HIV-1 virions and the plasma membrane. The presence of filaments with which multiple molecules of tetherin interact in connecting virion particles is strongly suggested by the morphologic evidence.     

In COS Cells Vpu Can Both Stabilize Tetherin Expression and Counteract Its Antiviral Activity

The interferon-inducible cellular protein tetherin (CD317/BST-2) inhibits the release of a broad range of enveloped viruses. The HIV-1 accessory protein Vpu enhances virus particle release by counteracting this host restriction factor. While the antagonism of human tetherin by Vpu has been associated with both proteasomal and lysosomal degradation, the link between Vpu-mediated tetherin degradation and the ability of Vpu to counteract the antiviral activity of tetherin remains poorly understood. Here, we show that human tetherin is expressed at low levels in African green monkey kidney (COS) cells. However, Vpu markedly increases tetherin expression in this cell line, apparently by sequestering it in an internal compartment that bears lysosomal markers. This stabilization of tetherin by Vpu requires the transmembrane sequence of human tetherin. Although Vpu stabilizes human tetherin in COS cells, it still counteracts the ability of tetherin to suppress virus release. The enhancement of virus release by Vpu in COS cells is associated with a modest reduction in cell-surface tetherin expression, even though the overall expression of tetherin is higher in the presence of Vpu. This study demonstrates that COS cells provide a model system in which Vpu-mediated enhancement of HIV-1 release is uncoupled from Vpu-mediated tetherin degradation.     

Compensatory changes in the cytoplasmic tail of gp41 confer resistance to tetherin/BST-2 in a pathogenic nef-deleted SIV

Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. Whereas HIV-1 Vpu and HIV-2 Env counteract human tetherin, most SIVs use Nef to antagonize the tetherin proteins of their nonhuman primate hosts. Here, we show that compensatory changes in the cytoplasmic domain of SIV gp41, acquired by a nef-deleted virus that regained a pathogenic phenotype in infected rhesus macaques, restore resistance to tetherin. These changes facilitate virus release in the presence of rhesus tetherin, but not human tetherin, and enhance virus replication in interferon-treated primary lymphocytes. The substitutions in gp41 result in a selective physical association with rhesus tetherin, and the internalization and sequestration of rhesus tetherin by a mechanism that depends on a conserved endocytosis motif in gp41. These results are consistent with HIV-2 Env antagonism of human tetherin and suggest that the ability to oppose tetherin is important for lentiviral pathogenesis.     

Influenza virus partially counteracts restriction imposed by tetherin/BST-2

Influenza virus infections lead to a burst of type I interferon (IFN) in the human respiratory tract, which most probably accounts for a rapid control of the virus. Although in mice, IFN-induced Mx1 factor mediates a major part of this response, the situation is less clear in humans. Interestingly, a recently identified IFN-induced cellular protein, tetherin (also known as CD317, BST-2, or HM1.24), exerts potent antiviral activity against a broad range of retroviruses, as well as several other enveloped viruses, by impeding the release of newly generated viral particles from the cell surface. Here we show that influenza virus belongs to the targets of this potent antiviral factor. Ectopic expression of tetherin strongly inhibited fully replicative influenza virus. In addition, depleting endogenous tetherin increased viral production of influenza virions, both in cells constitutively expressing tetherin and upon its induction by IFN. We further demonstrate, by biochemical and morphological means, that tetherin exerts its antiviral action by tethering newly budded viral particles, a mechanism similar to the one that operates against HIV-1. In addition, we determined that the magnitude of tetherin antiviral activity is comparable with or higher than the one of several previously identified anti-influenza cellular factors, such as MxA, ADAR1, ISG15, and viperin. Finally, we demonstrate that influenza virus reduces the impact of tetherin-mediated restriction on its replication by several mechanisms. First, the influenza virus NS1 protein impedes IFN-mediated tetherin induction. Second, influenza infection leads to a decrease of tetherin steady state levels, and the neuraminidase surface protein partly counteracts its activity. Overall, our study helps to delineate the intricate molecular battle taking place between influenza virus and its host cells.     

Two retroviruses packaged in one cell line can combined inhibit the replication of HIV-1 in TZM-bl cells

The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In this study, two retroviral vector plasmids were constructed. One inhibited the vpu gene expression; the other one over-expressed the tetherin. Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses. The retroviral vector plasmids’ functions of tetherin over-expression or vpu-RNAi were detected at the cell level. Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3T0V, and then mixed retroviruses were harvested. The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bl cells. The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.