a; The Veterans Affairs San Diego Healthcare System, San Diego, California, USA b BST2 (HM1.24; CD317; tetherin) is an interferon-inducible transmembrane protein that restricts the release of several enveloped viruses, including HIV, from infected cells. Before its activity as an antiviral factor was described, BST2 was identified as an inducer of NF-B activity. Here we show that human BST2 induces NF-B in a dose-dependent manner. This activity is separable from the restriction of virus release: a YxY sequence in the cytoplasmic domain of BST2 is required for the induction of NF-B but is dispensable for restriction, whereas the glycosylphosphatidylinositol (GPI) addition site in the protein's ectodomain is required for restriction but is largely dispensable for the induction of NF-B. Mutations predicted to disrupt the coiledcoil structure of the BST2 ectodomain impaired both signaling and restriction, but disruption of the tetramerization interface differentially affected signaling BST2 (bone marrow stromal cell antigen 2) (also known as tetherin) is an interferon (IFN)-inducible transmembrane and glycosylphosphatidylinositol (GPI)-anchored protein that restricts the release of several enveloped viruses from infected cells (1, 2). Viruses susceptible to BST2 include all retroviruses so far tested as well as members of the Rhabdoviridae, Paramyxoviridae, Filoviridae, and Herpesviridae families (reviewed in reference 3). Most of these viruses encode BST2 antagonists, which degrade the protein or remove it from the cell surface; the prototype BST2 antagonist is the HIV-1 accessory protein Vpu (1, 2).Although the release of cell-free virions and cell-free infectivity can be dramatically inhibited by BST2, restriction of the cell-tocell spread of virus is less effective (4, 5). This observation calls into question whether restriction of enveloped viruses is the sole function of this protein. Consistent with the possibility of additional functions, BST2 reportedly serves as the ligand for ILT7, a receptor on plasmacytoid dendritic cells that negatively regulates the expression of type I interferon (6). Moreover, BST2 reportedly stimulates the activity of the NF-B family of transcription factors (7), although the determinants of this activity in BST2 and its consequences have until very recently been unknown.Here we confirm that BST2 induces NF-B activity. We show that this activity is genetically separable from the restriction of virion release, yet it requires conserved features of the protein. These features include a YxY motif in the cytoplasmic domain (CD) of BST2 that directs the interaction with a TAK1-and TAB1-containing signaling complex. The BST2 antagonist encoded by HIV-1, Vpu, inhibits the activation of NF-B by BST2 in a manner dependent on its ability to bind the cellular ␤-TrCP-containing, cullin-1-based E3 ubiquitin ligase complex. In the absence of Vpu, however, the expression of HIV-1 augments the activation of NF-B. This suggests that BST2, like the restriction factor Trim5␣ (8), serves not only as ...

BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins.DOI: http://dx.doi.org/10.7554/eLife.02362.001

Background: HIV-1 Vpu counteracts the cellular antiviral factor BST-2 via an interaction that maps to the transmembrane domains of each protein.Results: This interaction is detectable by NMR spectroscopy and involves conserved faces of each helix. Conclusion: HIV-1 avoids an innate host defense via a lipid-embedded helix-helix interface. Significance: Intermolecular interactions within the lipid bilayer can be highly specific and shape the host-pathogen relationship.

The HIV-1 protein Vpu counteracts the antiviral activity of the innate restriction factor BST-2/tetherin by a mechanism that partly depends on its interaction with ␤-TrCP, a substrate adaptor for an SCF (Skp-Cullin 1-F box) E3 ubiquitin ligase complex. This suggests that Vpu stimulates the ubiquitination of BST-2 and that this underlies the relief of restriction. Here, we show that Vpu stimulates ubiquitination of BST-2. Mutation of all potential ubiquitination sites in the cytoplasmic domain of BST-2, including lysines, cysteines, serines, and threonines, abrogates Vpu-mediated ubiquitination. However, a serine-threonine-serine sequence specifically mediates the downregulation of BST-2 from the cell surface and the optimal relief of restricted virion release. Serine-threonine ubiquitination of BST-2 is likely part of the mechanism by which Vpu counteracts innate defenses.

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.

Recent evidence suggests that transmembrane domain (TMD) interactions are essential for HIV-1 Vpu-mediated antagonism of the restriction factor BST-2/tetherin. We made Vpu TMD mutants to study the mechanism of BST-2 antagonism. Vpu-I17A, -A18F, - W22L, and -S23L co-localized with BST-2 within endosomal membranes while effectively enhancing virion release and down-regulating surface BST-2. However, Vpu-A18H was confined to an endoplasmic reticulum (ER)-like distribution, resulting in impaired down-regulation of BST-2 and reduced virion release. Brefeldin A confined wild type Vpu to the ER, resulting in a similarly impaired phenotype, as did the addition of a C-terminal ER-retention signal to Vpu. We determined the half-life of cell-surface BST-2 to be ~8 hours, whereas Vpu mediated an ~80% reduction of surface BST-2 within 6 hours, suggesting that TMD interactions between Vpu and BST-2 occur within post-ER membranes to directly and rapidly remove BST-2 from the cell surface and relieve restricted virion release.

has received a speaker fee from Gilead Sciences. Nicolas Chomont has served on the scientific advisory board of Theravectys. Jintanat Ananworanich has participated in advisory meetings for ViiV Healthcare, Merck, AbbVie, Gilead, and Roche. The remaining authors report no relevant conflicts of interest. DATA SHARING STATEMENT The Henry M. Jackson Foundation for the Advancement of Military Medicine (HJF) and the U.S. Department of the Army are committed to safeguarding the privacy of research participants. De-identified participant-level data and accompanying research resources are available upon request. Distribution of data will require compliance with all applicable regulatory and ethical processes, including establishment and approval of an appropriate data-sharing agreement. The research protocol, informed consent documents, and instructions for submitting data requests can be found at https://www.hivresearch.org/RV397_Protocol.

The HIV-1 accessory protein Nef is well known for its manipulation of host cell endosomal trafficking. By linking transmembrane proteins to endosomal coats, Nef removes them from the surface of infected cells. Modulation of MHC proteins leads to viral evasion of cellular adaptive immunity, whereas modulation of receptors for the HIV envelope glycoprotein, including CD4, enhances viral infectivity. The other HIV-1 accessory proteins, Vif, Vpr and Vpu, share a mechanism of action distinct from Nef in that each interacts with a multi-subunit ubiquitin ligase complex to target cellular proteins for proteosomal degradation. However, newly uncovered functions and mechanistic aspects of Vpu likely involve endosomal trafficking: these include counteraction of the innate antiviral activity of the cellular transmembrane protein BST-2 (tetherin), as well as the removal of the lipid-antigen presenting protein CD1d and the natural killer cell ligand NTB-A from the cell surface. This review focuses on how Nef and Vpu interfere with normal intracellular membrane trafficking to facilitate the spread and virulence of HIV-1.