Antagonism of Tetherin Restriction of HIV-1 Release by Vpu Involves Binding and Sequestration of the Restriction Factor in a Perinuclear Compartment

Abstract

The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed β-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit β-TrCP, suggesting that this activity is taking place independently from β-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by β-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin.

Endogenous CD317/Tetherin Limits Replication of HIV-1 and Murine Leukemia Virus in Rodent Cells and Is Resistant to Antagonists from Primate Viruses

“…For the prototypic CD317 antagonist Vpu, downregulation of hCD317 from the cell surface and accelerated degradation have been proposed as key mechanisms (10,15,53). Recent reports (7,12,24) and the current study demonstrate that a reduction of cell-associated CD317 levels is neither sufficient ( Fig. 2A and B) nor required ( Fig.…”

The Restriction Factors of Human Immunodeficiency Virus

“…Vpu-mediated targeting of tetherin to lysosomes also requires Rab7A (52), a small GTPase essential for the maturation of late endosomes and lysosomal fusion. Other studies have revealed a ␤-TrCP2-independent mechanism of tetherin antagonism by Vpu, which leads to the sequestration of tetherin in a perinuclear compartment without degradation (48,53,54). This could occur either by trapping newly synthesized tetherin or by blocking the recycling of tetherin to the plasma membrane (55,56).…”

Membrane Anchoring by a C-terminal Tryptophan Enables HIV-1 Vpu to Displace Bone Marrow Stromal Antigen 2 (BST2) from Sites of Viral Assembly

“…BST2 entraps lipid enveloped virions as they bud from the plasma membrane, preventing their release. Vpu, a single-pass type I transmembrane protein (3), binds directly to BST2, a single-pass type II transmembrane protein with a glycosylphosphatidylinositol anchor (4), via an anti-parallel interaction between the proteins' transmembrane domains (5)(6)(7)(8)(9). This interaction enables Vpu to decrease the concentration of BST2 on the plasma membrane and induce the degradation of BST2 by an endolysosomal mechanism (7, 8, 10 -13).…”