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Tetherin Is as Tetherin Does

2009; Cell Press; Volume: 139; Issue: 3 Linguagem: Inglês

10.1016/j.cell.2009.10.011

1097-4172

J. Hammonds, Paul Spearman,

Mosquito-borne diseases and control

Tetherin is a cellular restriction factor that inhibits the release of HIV and other enveloped viruses from host cells. A paper from Perez-Caballero et al., 2009Perez-Caballero D. Zang T. Ebrahimi A. McNatt M.W. Gregory A.D. Johnson M.C. Bieniasz P.D. Cell. 2009; (this issue)PubMed Google Scholar in this issue of Cell clarifies how this factor works. The authors show that the aptly named tetherin directly tethers viral particles to the plasma membrane. Tetherin is a cellular restriction factor that inhibits the release of HIV and other enveloped viruses from host cells. A paper from Perez-Caballero et al., 2009Perez-Caballero D. Zang T. Ebrahimi A. McNatt M.W. Gregory A.D. Johnson M.C. Bieniasz P.D. Cell. 2009; (this issue)PubMed Google Scholar in this issue of Cell clarifies how this factor works. The authors show that the aptly named tetherin directly tethers viral particles to the plasma membrane. Studies on the interplay between viruses and host cells have yielded numerous insights into fundamental principles in biology, and the examination of specific aspects of viral replication have brought to light unexpected features of the virus-host cell conflict. One such remarkable clue came to light last year through the discovery that the host cell protein BST-2/CD317 or “tetherin” potently restricts HIV-1 replication by preventing the escape of viral particles from infected cells. Earlier studies had shown that cells either require (restrictive cells) or do not require (permissive cells) expression of the HIV-1 accessory protein Vpu for efficient release of viral particles. Neil et al., 2008Neil S.J. Zang T. Bieniasz P.D. Nature. 2008; 451: 425-430Crossref PubMed Scopus (1297) Google Scholar then identified the “restricting” activity as tetherin, an interferon-inducible gene product that is differentially expressed in restrictive cells. Vpu downregulates tetherin from the cell surface (Van Damme et al., 2008Van Damme N. Goff D. Katsura C. Jorgenson R.L. Mitchell R. Johnson M.C. Stephens E.B. Guatelli J. Cell Host Microbe. 2008; 3: 245-252Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar), adding weight to the conclusion that it was the correct Vpu-responsive cellular factor. Since these initial reports of the importance of tetherin in restricting HIV particle release, it has become clear that tetherin inhibits the release of a variety of enveloped viruses, and that viruses have in turn evolved diverse strategies to overcome this cellular restriction to replication (Le Tortorec and Neil, 2009Le Tortorec A. Neil S.J. J. Virol. 2009; (Published online September 9, 2009)PubMed Google Scholar, Zhang et al., 2009Zhang F. Wilson S.J. Landford W.C. Virgen B. Gregory D. Johnson M.C. Munch J. Kirchhoff F. Bieniasz P.D. Hatziioannou T. Cell Host Microbe. 2009; 6: 54-67Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Tetherin expression in cells that lack baseline expression, such as 293T cells, leads to massive accumulation of virion particles at the plasma membrane in the absence of counteracting factors like Vpu. But how does tetherin retain viral particles? Does tetherin function as a physical tether as implied by the name? Or does it act with cellular cofactors in an indirect fashion to induce the physical tethering of particles? In this issue of Cell, Perez-Caballero et al., 2009Perez-Caballero D. Zang T. Ebrahimi A. McNatt M.W. Gregory A.D. Johnson M.C. Bieniasz P.D. Cell. 2009; (this issue)PubMed Google Scholar provide some answers to these key questions in HIV biology. As these investigators show, the name tetherin fits the molecule's action like a glove. Perez-Caballero and colleagues used a systematic approach to dissect the functional domains of this unusual protein. Tetherin is a type II membrane protein with unusual features (Figure 1), including two membrane anchors, a transmembrane domain near the N terminus, and a glycophosphatidylinositol (GPI)-linked anchor at the C terminus. The ectodomain of the protein features three cysteine residues that may mediate homodimerization, two N-linked glycosylation sites, and a coiled-coil domain that has yet to be fully functionally defined (see Figure 1A). The investigators introduced mutations into the different domains of tetherin, singly and in combination, and then assessed the ability of the mutated protein to prevent release of particles from the surface of 293T cells. They demonstrate that disulfide bonds indeed are required for dimerization of tetherin and are necessary for potent tethering of viral particles. Elimination of most of the coiled-coil domain also diminished tethering, although the underlying mechanism is unclear. Importantly, elimination of either the N-terminal transmembrane or the C-terminal GPI anchor eliminated the ability of the molecule to tether virions. The mutagenesis work thus suggested that both membrane anchors (transmembrane and GPI) and homodimerization are essential for tetherin activity, but did not fully address the fundamental issue: is tetherin itself a physical tether holding viral particles at the membrane? The investigators next took an elegant approach to determine whether tetherin acts directly or indirectly. They replaced the major domains of tetherin—transmembrane, coiled-coil, and GPI anchor—with those from three unrelated proteins, in each case creating a functional chimeric protein that restricted viral release. In the most startling result of the study, Perez-Caballero and coworkers show that an entirely artificial form of tetherin, made up solely of domains from three proteins that were “tetherin-like” in size, topology, and posttranslational modifications but shared no sequence homology with tetherin inhibited particle release in a manner remarkably similar to tetherin. The similarities of the artificial tetherin to the real protein extended to scanning electron microscopy analysis, where both wild-type and artificial tetherin induced large accumulations of HIV-1 particles on the plasma membrane. Vpu, however, was unable to inhibit the activity of the artificial tetherin protein, in agreement with previous genetic studies indicating an important and specific role for the tetherin transmembrane domain in the interaction with Vpu. The fact that an entirely artificial tetherin of unrelated sequence can restrict the release of virions argues strongly against models in which tetherin acts to retain virions through cofactor interaction, signaling, or other indirect mechanisms. In a final and convincing set of experiments, the authors demonstrate that tetherin does act directly on virions. Using both biochemical techniques and electron microscopy, they show that tetherin is incorporated into viral particles and argue that incorporation may occur through either its GPI anchor or its transmembrane domain. Moreover, they present evidence that tetherin is likely incorporated into virions as a disulfide-linked parallel dimer (Figure 1). Together, their findings argue eloquently for a simple model: tetherin is a direct physical tether, holding virions to the plasma membrane and linking virions to each other. In other words, Perez-Caballero and colleagues chose the name for BST-2/CD317 quite appropriately in their previous work; tetherin is as tetherin does. Is the story complete now for tetherin? Although this report clearly establishes direct physical tethering by tetherin, perhaps it is still too early to discount completely the influence of other cellular factors. A number of key questions remain. The function of the coiled-coil domains of the tetherin dimer remains incompletely defined. The coiled-coil motif could, for example, interact with other coiled-coil proteins or with additional dimers of tetherin, creating more complex oligomers that play a role in tethering particles to the plasma membrane. Cytoskeletal elements may also play a role, as suggested by a recent report showing that the proteins RICH2, EBP50, and ezrin create a link between tetherin/CD317 and the apical actin cytoskeleton (Rollason et al., 2009Rollason R. Korolchuk V. Hamilton C. Jepson M. Banting G. J. Cell Biol. 2009; 184: 721-736Crossref PubMed Scopus (102) Google Scholar). Does tetherin and its link to both the actin cytoskeleton and to lipid rafts (through its GPI anchor) help to define the viral particle budding site? What cellular factors influence the enrichment of tetherin on the budding particle, given the known absence of a specific interaction with viral structural proteins? Detailed studies of the role of endogenous tetherin in HIV replication in macrophages, where large collections of HIV particles appear to assemble in intracellular compartments rather than at the plasma membrane, could reveal additional roles for tetherin in HIV biology. Furthermore, the mechanism by which Vpu overcomes the action of tetherin remains under considerable debate. Although it is too early to say that the entire picture has been painted, the new report by Perez-Caballero and coworkers offers a clear view of tetherin as the direct, physical link responsible for retention of viral particles at the plasma membrane and provides a unique insight into an important innate host defense mechanism against attack by pathogenic viruses. Tetherin Inhibits HIV-1 Release by Directly Tethering Virions to CellsPerez-Caballero et al.CellOctober 30, 2009In BriefTetherin is an interferon-induced protein whose expression blocks the release of HIV-1 and other enveloped viral particles. The underlying mechanism by which tetherin functions and whether it directly or indirectly causes virion retention are unknown. Here, we elucidate the mechanism by which tetherin exerts its antiviral activity. We demonstrate, through mutational analyses and domain replacement experiments, that tetherin configuration rather than primary sequence is critical for antiviral activity. 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