Vpu Antagonizes BST-2–Mediated Restriction of HIV-1 Release via β-TrCP and Endo-Lysosomal Trafficking

Abstract

The interferon-induced transmembrane protein BST-2/CD317 (tetherin) restricts the release of diverse enveloped viruses from infected cells. The HIV-1 accessory protein Vpu antagonizes this restriction by an unknown mechanism that likely involves the down-regulation of BST-2 from the cell surface. Here, we show that the optimal removal of BST-2 from the plasma membrane by Vpu requires the cellular protein β-TrCP, a substrate adaptor for a multi-subunit SCF E3 ubiquitin ligase complex and a known Vpu-interacting protein. β-TrCP is also required for the optimal enhancement of virion-release by Vpu. Mutations in the DSGxxS β-TrCP binding-motif of Vpu impair both the down-regulation of BST-2 and the enhancement of virion-release. Such mutations also confer dominant-negative activity, consistent with a model in which Vpu links BST-2 to β-TrCP. Optimal down-regulation of BST-2 from the cell surface by Vpu also requires the endocytic clathrin adaptor AP-2, although the rate of endocytosis is not increased; these data suggest that Vpu induces post-endocytic membrane trafficking events whose net effect is the removal of BST-2 from the cell surface. In addition to its marked effect on cell-surface levels, Vpu modestly decreases the total cellular levels of BST-2. The decreases in cell-surface and intracellular BST-2 are inhibited by bafilomycin A1, an inhibitor of endosomal acidification; these data suggest that Vpu induces late endosomal targeting and partial degradation of BST-2 in lysosomes. The Vpu-mediated decrease in surface expression is associated with reduced co-localization of BST-2 and the virion protein Gag along the plasma membrane. Together, the data support a model in which Vpu co-opts the β-TrCP/SCF E3 ubiquitin ligase complex to induce endosomal trafficking events that remove BST-2 from its site of action as a virion-tethering factor.

Ubiquitination of BST-2 Protein by HIV-1 Vpu Protein Does Not Require Lysine, Serine, or Threonine Residues within the BST-2 Cytoplasmic Domain

“…2E). In general, our results corroborate those reported in several recent manuscripts (27,32,35), which have each found that the two cytosolic lysines have no phenotype with respect to viral egress or surface down-regulation by Vpu. However, as described above, disagreement remains regarding the contribution that these lysines make toward Vpu-dependent ubiquitination.…”

Simian immunodeficiency virus envelope glycoprotein counteracts tetherin/BST-2/CD317 by intracellular sequestration

“…The mechanism of tetherin inhibition remains incompletely characterized but again, there are clearly significant mechanistic differences between the various viral antagonists. HIV-1 Vpu appears to lead to a reduction of steady-state tetherin levels (6,7,9) and exclusion from sites of particle assembly at the plasma membrane (2,26). Recent data suggest that Nef may remove tetherin from the cell surface (3) as it does other cell surface molecules (27).…”

“…Several recent studies have suggested that Vpu expression leads to tetherin degradation (6,7,9). We therefore examined the impact of SIVtan envelope expression on tetherin levels.…”

“…Tetherin is an IFNinducible dimeric transmembrane protein with an extra-cellular coiled-coil and a predicted GPI anchor (5). It is proposed to form a protein tether linking assembled virions to infected cells, leading to their endocytosis and degradation in lysosomes (1,6). All tetherin variants tested thus far inhibit the release of enveloped viral particles including retroviruses, filoviruses, and arena viruses (1,2,(7)(8)(9)(10).…”

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

“…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). Hence, substitutions in a conserved DSGxxS motif in the cytoplasmic tail of Vpu (S52,56N) that prevent the recruitment of βTrCP-2 partially impair tetherin antagonism by preventing its degradation, but not its endosomal sequestration (25,(30)(31)(32)(33). These substitutions are also known to abrogate CD4 down-regulation, but do not affect Vpu-mediated down-modulation of NTB-A (23).…”