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

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.

HIV-1辅助蛋白Vpu通过阻止tetherin（BST-2/CD317）整合进入病毒粒子、降低其表面表达水平，并最终促进其降解，从而拮抗该限制因子的抗病毒作用。本研究对B亚型Vpu胞质尾第二个α螺旋中一段推定的转运基序EXXXLV进行了表征，证实其是有效拮抗tetherin所必需的。该基序的突变会阻断tetherin/Vpu复合物的内体分选复合物（ESCRT）依赖型降解以及tetherin的细胞表面下调，但不会影响其与Vpu的物理结合。尤为关键的是，该基序是从CD4+ T细胞中有效释放无细胞病毒粒子所必需的，尤其是在CD4+ T细胞暴露于1型干扰素之后，这表明在病毒体内可能遭遇的生理环境中，降低tetherin表面水平并促进其降解的能力，对于拮抗该限制因子的作用至关重要。携带EXXXLV突变的Vpu突变体与tetherin共同聚集于细胞表面及内体区室，但仍能结合β-TrCP2与HRS，这表明该基序对于结合后的转运事件是必需的——该事件使tetherin经由ESCRT依赖途径降解，并阻止其转运至细胞膜与病毒出芽区域。我们进一步发现，尽管Vpu的功能依赖于网格蛋白，且Vpu胞质尾的整个第二个α螺旋可由源自HIV-1 Nef的网格蛋白衔接蛋白结合肽在功能上进行互补，但该过程并不依赖于经典的网格蛋白衔接蛋白或retromer复合物。最终我们证实，Vpu EXXXLV突变体的残留活性需要tetherin中完整的内吞基序，这表明Vpu与tetherin在其循环过程中的物理结合，在一定程度上足以削弱tetherin的活性。