Determinants of Tetherin Antagonism in the Transmembrane Domain of the Human Immunodeficiency Virus Type 1 Vpu Protein

Abstract

Tetherin (BST2/CD317) potently restricts the particle release of human immunodeficiency virus type 1 (HIV-1) mutants defective in the accessory gene vpu. Vpu antagonizes tetherin activity and induces its cell surface downregulation and degradation in a manner dependent on the transmembrane (TM) domains of both proteins. We have carried out extensive mutagenesis of the HIV-1 NL4.3 Vpu TM domain to identify three amino acid positions, A14, W22, and, to a lesser extent, A18, that are required for tetherin antagonism. Despite the mutants localizing indistinguishably from the wild-type (wt) protein and maintaining the ability to multimerize, mutation of these positions rendered Vpu incapable of coimmunoprecipitating tetherin or mediating its cell surface downregulation. Interestingly, these amino acid positions are predicted to form one face of the Vpu transmembrane alpha helix and therefore potentially contribute to an interacting surface with the transmembrane domain of tetherin either directly or by modulating the conformation of Vpu oligomers. While the equivalent of W22 is invariant in HIV-1/SIVcpz Vpu proteins, the positions of A14 and A18 are highly conserved among Vpu alleles from HIV-1 groups M and N, but not those from group O or SIVcpz that lack human tetherin (huTetherin)-antagonizing activity, suggesting that they may have contributed to the adaption of HIV-1 to human tetherin.Tetherin (CD317/BST2) is an interferon-induced type II membrane glycoprotein of unusual topology (4, 25, 44) that potently restricts the release of diverse mammalian enveloped viral particles from infected cells (24,33,42,45,49,52,63). The protein consists of a short 21-amino-acid cytoplasmic tail, a transmembrane (TM) domain, a predominantly helical extracellular domain containing three cysteine residues that mediate tetherin dimerization (1,44,48) and an extended parallel coiled-coil (18), and a C-terminal glycophosphatidylinositol anchor that links it back to the cellular membrane (25). These structural features are key to the mode of tetherin activity (48). Tetherin is localized to the plasma membrane (PM) and constitutively recycles through intracellular compartments (25,50). It is incorporated into budding virions and acts as a physical tether, cross-linking the virion and cellular membranes, thereby preventing virus particle release from the host cell (11,15,16,48). Strong evidence suggests that the dual membrane anchor of tetherin allows it to form parallel dimers with one terminal in the virion membrane and the other in the cell (48). The result of this is that mature viral particles are retained on cell surfaces by protease-sensitive linkages that contain tetherin, and virions can then be endocytosed and accumulate in endosomal compartments (41,42,48). This relatively nonspe-

Tetherin antagonism by Vpu protects HIV-infected cells from antibody-dependent cell-mediated cytotoxicity

“…3B). Although the effects of these mutants on tetherin down-modulation are less than previously observed by other assays (28) (Fig. 3B).…”

“…Whereas W22A and A14L directly impair Vpu binding to tetherin (28), changes in the subcellular distribution of the A18H mutant may also account for its inability to interact with tetherin (29). Notably, the A14L substitution does not affect CD4 down-regulation (28).…”

“…transmembrane domain of Vpu that impair binding to tetherin result in partial (W22A) to nearly complete (A14L and A18H) loss of anti-tetherin activity (28,29). Whereas W22A and A14L directly impair Vpu binding to tetherin (28), changes in the subcellular distribution of the A18H mutant may also account for its inability to interact with tetherin (29).…”

“…Whereas W22A and A14L directly impair Vpu binding to tetherin (28), changes in the subcellular distribution of the A18H mutant may also account for its inability to interact with tetherin (29). Notably, the A14L substitution does not affect CD4 down-regulation (28). Tetherin antagonism by Vpu also depends in part on the recruitment of βTrCP-2, an adaptor protein for an E3 ubiquitin ligase involved in targeting tetherin for degradation (24,25,30,31).…”

Innate Barriers to Viral Infection

“…Thus, HIV-1 Vpu is a transmembrane protein that promotes the release of virions from the cell but can only target human, chimpanzee and gorilla tetherin sequences. It does so through Vpu transmembrane domain sequences [8]. However, for most primate lentiviruses, the accessory protein Nef targets tetherin through a sequence that is absent in the human protein.…”