Antiviral activity of the interferon-induced cellular protein BST-2/tetherin

Abstract

Pathogenic microorganisms encode proteins that antagonize specific aspects of innate or adaptive immunity. Just as the study of the HIV-1 accessory protein Vif led to the identification of cellular cytidine deaminases as host defense proteins, the study of HIV-1 Vpu recently led to the discovery of the interferon-induced transmembrane protein BST-2 (CD317; tetherin) as a novel component of the innate defense against enveloped viruses. BST-2 is an unusually structured protein that restricts the release of fully formed progeny virions from infected cells, presumably by a direct retention mechanism that is independent of any viral protein target. Its spectrum of activity includes at least four virus families: retroviruses, filoviruses, arenaviruses, and herpesviruses. Viral antagonists of BST-2 include HIV-1 Vpu, HIV-2 and SIV Env, SIV Nef, the Ebola envelope glycoprotein, and the K5 protein of KSHV. The mechanisms of antagonism are diverse and currently include viral cooption of cellular endosomal trafficking and protein degradation pathways, including those mediated by ubiquitination. Orthologs of human BST-2 are present in mammals. Primate BST-2 proteins are differentially sensitive to antagonism by lentiviral Vpu and Nef proteins, suggesting that BST-2 has subjected lentiviruses to evolutionary pressure and presents barriers to cross-species transmission. BST-2 functions not only as an effector of the interferon-induced antiviral response but also as a negative feedback regulator of interferon production by plasmacytoid dendritic cells. Future work will focus on the role and regulation of BST-2 during the innate response to viral infection, on the mechanisms of restriction and of antagonism by viral gene products, and on the role of BST-2 in primate lentiviral evolution. The augmentation of BST-2 activity and the inhibition of virally encoded antagonists, in particular Vpu, represent new approaches to the prevention and treatment of HIV-1 infection.

FIG. 1.

Domain structure of BST-2. BST-2 is a type II (N-terminus in the cytoplasm) single-pass transmembrane protein whose C-terminus is attached to the lipid bilayer via a glycosylphosphatidylinositol (GPI) anchor. The cytoplasmic domain contains several conserved features including a YxY motif that binds the plasma membrane clathrin adaptor AP-2, a DDIWK sequence found in nonhuman primates that is required for response to Nef, and lysines (K18 and K21) that are targets for ubiquitination by the K5 protein of KSHV. The extracellular domain contains two α-helical regions (blue) that are predicted to form dimeric coiled-coils. Cysteines that contribute to disulfide-linked homodimerization and N-linked glycosylation sites are indicated.

FIG. 2.

BST-2 and HIV-1 Gag colocalize along the cell surface and in endosomes. Cells (HeLa), which express BST-2 constitutively, were transfected to express an HIV-1 genome lacking the vpu gene, as well as GFP (green in the color image) as a marker of the transfected cells, then stained for the structural HIV-1 protein Gag (p17/p55; left panel; blue in the color image) and BST-2 (middle panel; red in the color image). In the color (merge) image, the overlap of Gag and BST-2 appears purple.

FIG. 3.

Evidence for direct retention of nascent virions on the cell surface by BST-2 and topological models of restriction. (A) Electron microscopic evidence of direct retention and virion incorporation. HeLa cells expressing an HIV-1 genome lacking vpu were stained on their surface for BST-2 using an antibody to the protein's ectodomain, followed by a secondary labeling system using electron-dense cadmium selenide/zinc sulfide nanocrystals, which appear as small black dots in these transmission electron microscopic images. BST-2 is associated with virions and located between virions and the cell surface, supporting a direct tethering mechanism. (B) Schematic representations of direct tethering models. The left schematic depicts “ectodomain self-interaction,” whereas the middle and right depict “membrane spanning” mechanisms in which BST-2 homodimers are arranged in antiparallel or parallel orientation. (C) Predicted structure of the coiled-coil ectodomain. The structure of the monomeric ectodomain was predicted using the SAMT06 server and rendered using DeepView.

FIG. 4.

Cellular pathways potentially involved in the antagonism of BST-2 via removal from the cell surface by viral proteins. Upon synthesis at the endoplasmic reticulum (ER), BST-2 is normally delivered to the plasma membrane (PM), internalized from the PM via AP-2-dependent endocytosis, and presumably recycled from endosomes to the PM. The viral proteins that decrease the expression of BST-2 at the cell surface (HIV-I Vpu, HIV-2 and SIV Env, KSHV K5, and SIV Nef ) and that decrease the total cellular expression of BST-2 (Vpu and K5) may act at various steps. Certain mechanisms would block newly synthesized BST-2 from reaching the PM. For example, during exocytosis, BST-2 could be rerouted from the ER to the proteosome for degradation (possibly for Vpu) or rerouted from the Golgi to endosomes and lysosomes. BST-2 could also be sequestered after synthesis in the trans-Golgi network (possibly for Vpu and HIV-2 and SIV Env). Alternative mechanisms would directly remove BST-2 from the plasma membrane. For example, the rate of endocytosis of BST-2 could be enhanced (likely for SIV Nef and possibly for HIV-2 and SIV Env). Alternatively or in addition, BST-2 could be rerouted after endocytosis to late endosomes/multivesicular bodies (MVB) with further trafficking to the lysosome (likely for Vpu and K5). The latter pathway would be at the expense of recycling to the cell surface. Mechanisms involving the direct removal of BST-2 from the PM are appealing mechanistically. They are potentially the most rapid, because, unlike mechanisms in which newly synthesized BST-2 is prevented from reaching the PM, they do not rely on the endogenous rate of turnover at the cell surface to clear BST-2 from its site of action as a tethering factor. See text for references.

FIG. 5.

Vpu recruits a multisubunit ubiquitin ligase complex to BST-2. Vpu interacts with BST-2 via its transmembrane domain, while recruiting an SCF-E3 multisubunit ubiquitin ligase complex via a conserved DSGxxS motif in its cytoplasmic domain (green dots). The DSGxxS sequence binds β-TrCP, the substrate adaptor for the complex, via its WD domain. The presumed consequence of this interaction is the ubiquitination of BST-2, which may lead to endocytosis, endosomal sequestration, lysosomal degradation, and/or proteasomal degradation. Any of these mechanisms would remove BST-2 from the cell surface, it site of activity as a tethering factor, and so counteract restriction virion release. See text for references.

FIG. 6.

Residues of the BST-2 TMD potentially involved in an interaction with Vpu. Both genetic studies mapping residues under positive selection and functional studies have indicated that residues in the transmembrane domain (TMD) of human BST-2 are required for responsiveness to Vpu. Changes in these residues found in the BST-2 orthologs of nonhuman primates relative to the human protein account for the inactivity of HIV-1 Vpu against these proteins (see text for details). Remarkably, these residues are predominantly on one face of the TMD α-helix (highlighted in yellow), suggesting an interaction along that face with the TMD of Vpu. Image drawn using PyMOL.