A Cytoplasmic Tail Determinant in HIV-1 Vpu Mediates Targeting of Tetherin for Endosomal Degradation and Counteracts Interferon-Induced Restriction

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Abstract
The HIV-1 accessory protein Vpu counteracts tetherin (BST-2/CD317) by preventing its incorporation into virions, reducing its surface expression, and ultimately promoting its degradation. Here we characterize a putative trafficking motif, EXXXLV, in the second alpha helix of the subtype-B Vpu cytoplasmic tail as being required for efficient tetherin antagonism. Mutation of this motif prevents ESCRT-dependent degradation of tetherin/Vpu complexes, tetherin cell surface downregulation, but not its physical interaction with Vpu. Importantly, this motif is required for efficient cell-free virion release from CD4+ T cells, particularly after their exposure to type-1 interferon, indicating that the ability to reduce surface tetherin levels and promote its degradation is important to counteract restriction under conditions that the virus likely encounters in vivo. Vpu EXXXLV mutants accumulate with tetherin at the cell surface and in endosomal compartments, but retain the ability to bind both β-TrCP2 and HRS, indicating that this motif is required for a post-binding trafficking event that commits tetherin for ESCRT-dependent degradation and prevents its transit to the plasma membrane and viral budding zones. We further found that while Vpu function is dependent on clathrin, and the entire second alpha helix of the Vpu tail can be functionally complemented by a clathrin adaptor binding peptide derived from HIV-1 Nef, none of the canonical clathrin adaptors nor retromer are required for this process. Finally we show that residual activity of Vpu EXXXLV mutants requires an intact endocytic motif in tetherin, suggesting that physical association of Vpu with tetherin during its recycling may be sufficient to compromise tetherin activity to some degree. Tetherin inhibits the release of several diverse enveloped viruses from infected cells and is counteracted by the HIV-1 accessory gene Vpu. Vpu prevents tetherin's incorporation into nascent viral particles, promotes its downregulation from the cell surface and targets tetherin for degradation. Here we identify a determinant that resembles an acidic-dileucine-based sorting sequence in the Vpu cytoplasmic tail that is required for efficient counteraction of tetherin activity, particularly in CD4+ T cells treated with type-1 interferon. Mutation of this motif prevents cell-surface downregulation and degradation of Vpu/tetherin complexes but does not affect their interaction. Rather, in its absence, Vpu accumulates in early endosomes and at the cell surface where it becomes incorporated into assembling virions with tetherin, indicating that this motif modulates sub-cellular trafficking of tetherin. Furthermore Vpu activity is clathrin-dependent and can be reconstituted by replacing a portion of the cytoplasmic tail encompassing this motif with one derived from HIV-1 Nef that is known to bind several clathrin adaptors. Finally, we demonstrate that residual function of the mutant Vpu requires a trafficking motif in tetherin, suggesting that physical interaction of tetherin with Vpu during its recycling to the cell-surface can interfere with its function to a variable extent.