Conserved residues within the HIV-1 Vpu transmembrane-proximal hinge region modulate BST2 binding and antagonism

Sabelo Lukhele, Éric A. Cohen

BST2 inhibits HIV-1 release by tethering nascent virions to the surface of infected cells. HIV-1 Vpu overcomes this restriction by removing BST2 from viral budding sites via BST2 intracellular trapping and sequestration, surface downregulation and/or displacement mechanisms. Vpu is composed of a short luminal tail, a transmembrane domain (TMD) and a cytoplasmic hinge region that is followed by two helices. BST2 counteraction relies on the ability of Vpu to physically bind BST2 through TMD interactions and recruit the clathrin-dependent trafficking machinery via a canonical acidic di-leucine signalling motif within the helix-2 of Vpu. The highly conserved Vpu transmembrane-proximal hinge region encompasses residues that resemble an acidic leucine-based trafficking motif, whose functional roles are currently ill-defined. In this study, we investigated the contribution of these residues towards Vpu-mediated BST2 antagonism. We show that while these conserved residues have no intrinsic activity on the cellular distribution of Vpu in the absence of BST2, they regulate the ability of Vpu to bind to BST2 and, consequently, govern both BST2-dependent trafficking properties of the protein as well as its co-localization with BST2. Moreover, these residues, particularly a glutamic acid residue positioned immediately following the TMD, are a determinant not only for efficient targeting of BST2, but also binding and degradation of CD4, another host membrane protein targeted by Vpu. Mechanistically, our data are consistent with a role of these residues in the maintenance of the Vpu TMD conformational configuration such that interactions with membrane-associated host targets are favoured. Altogether, this work demonstrates an important regulatory role of the transmembrane-proximal Vpu hinge region residues towards enabling the protein to efficiently engage its target host proteins. Thus, this highly conserved, cytosolic Vpu hinge region may represent an attractive target for the development of anti-Vpu inhibitors.