Functional Antagonism of Rhesus Macaque and Chimpanzee BST-2 by HIV-1 Vpu Is Mediated by Cytoplasmic Domain Interactions

Abstract

bHuman immunodeficiency virus type 1 (HIV-1) Vpu enhances the release of viral particles from infected cells by interfering with the function of BST-2/tetherin, a cellular protein inhibiting virus release. The Vpu protein encoded by NL4-3, a widely used HIV-1 laboratory strain, antagonizes human BST-2 but not monkey or murine BST-2, leading to the conclusion that BST-2 antagonism by Vpu is species specific. In contrast, we recently identified several primary Vpu isolates, such as Vpu of HIV-1 DH12 , capable of antagonizing both human and rhesus BST-2. Here we report that while Vpu interacts with human BST-2 primarily through their respective transmembrane domains, antagonism of rhesus BST-2 by Vpu involved an interaction of their cytoplasmic domains. Importantly, a Vpu mutant carrying two mutations in its transmembrane domain (A 14 L and W 22 A), rendering it incompetent for interaction with human BST-2, was able to interact with human BST-2 carrying the rhesus BST-2 cytoplasmic domain and partially neutralized the ability of this BST-2 variant to inhibit viral release. Bimolecular fluorescence complementation analysis to detect Vpu-BST-2 interactions suggested that the physical interaction of Vpu with rhesus or chimpanzee BST-2 involves a 5-residue motif in the cytoplasmic domain of BST-2 previously identified as important for the antagonism of monkey and great ape BST-2 by simian immunodeficiency virus (SIV) Nef. Thus, our study identifies a novel mechanism of antagonism of monkey and great ape BST-2 by Vpu that targets the same motif in BST-2 used by SIV Nef and might explain the expanded host range observed for Vpu isolates in our previous study.

Vpu of a Simian Immunodeficiency Virus Isolated from Greater Spot-Nosed Monkey Antagonizes Human BST-2 via Two AxxxxxxxW Motifs

“…These mKGC and mKGN fragments were fused to the human or monkey BST-2 and Vpu, respectively (Fig. 1A), and the mKG fragments were able to form an active structure and emit fluorescence only when these proteins were bound to each other as shown previously (8,9,44). The fluorescence of reconstituted mKG proteins in live cells was measured by flow cytometry, and the geometric mean (Geo Mean) of fluorescence represents the relative binding efficiency.…”

“…In the absence of Vpu, BST-2 tethers nascent virions at the surface of infected cells (6,7). HIV-1 Vpu physically interacts with human BST-2 via its transmembrane (TM) domains (8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and downregulates human BST-2 from the plasma membrane (16)(17)(18)(19)(20)(21)(22). A mutational analysis predicted that residues A14, W22, and A18 in the TM domain of HIV-1 NL4-3 Vpu form one face of the Vpu TM alpha helix and mediate its binding to human BST-2 (14).…”

“…Indeed, SIVcpz Vpu is unable to antagonize chimpanzee or human BST-2 but is able to induce CD4 degradation (32,43). In addition, Vpu from SIVcpzMB897, which is thought to be phylogenetically close to HIV-1 group M, cannot bind to chimpanzee BST-2 (8) or human BST-2 (44). It was reported previously that W22 is invariant in HIV-1/SIVcpz Vpu proteins whereas A14 and A18 are highly conserved among Vpu alleles from HIV-1 groups M and N but not among those from group O or SIVcpz, which lack the ability to antagonize human BST-2 (14,45).…”

A Review of Functional Motifs Utilized by Viruses

“…The nucleoprotein of influenza B virus (BNP) harbors a conserved 44 KRxR 47 motif, and a mutation on the K or R residue results in the disruption or failure of nuclear import and localization, suggesting that the motif is a NLS sequence [93,94]. Enhance virion-release, anti-tetherin activity DSGxxS [112,113] Helix-Helix Interactions AxxxAxxxAxxxW and VxxxIxxLxxxL [114,115] Heparan sulfate-binding motif, post-internalization steps of adenovirus KKTK, or bbxb and bbbxxb [48][49][50][51] HIV neutralization by human antibodies GPG[RQ] [43][44][45][46][47] HIV 1 Degenerate residues are bracketed, braces refer to the excluded residues (i.e., any residues except those between braces), "x" means any residue, b refers to basic residues (H, K or R), "ϕ" denotes large hydrophobic residues (F, I, L or V), and the number of recurrence is indicated after residues.…”

“…This transmembrane interaction is required for Vpu interactions with APs [163]. Two other domains in Vpu (Yxxϕ and DSGxxS) could mediate anti-tetherin activity [112], whereas the [GD]DIWK motif in monkey BST2, but not in human, is required for interaction with HIV-1 Vpu [113].…”