Vpu Binds Directly to Tetherin and Displaces It from Nascent Virions

Abstract

Tetherin (Bst2/CD317/HM1.24) is an interferon-induced antiviral host protein that inhibits the release of many enveloped viruses by tethering virions to the cell surface. The HIV-1 accessory protein, Vpu, antagonizes Tetherin through a variety of proposed mechanisms, including surface downregulation and degradation. Previous studies have demonstrated that mutation of the transmembrane domains (TMD) of both Vpu and Tetherin affect antagonism, but it is not known whether Vpu and Tetherin bind directly to each other. Here, we use cysteine-scanning mutagenesis coupled with oxidation-induced cross-linking to demonstrate that Vpu and Tetherin TMDs bind directly to each other in the membranes of living cells and to map TMD residues that contact each other. We also reveal a property of Vpu, namely the ability to displace Tetherin from sites of viral assembly, which enables Vpu to exhibit residual Tetherin antagonist activity in the absence of surface downregulation or degradation. Elements in the cytoplasmic tail domain (CTD) of Vpu mediate this displacement activity, as shown by experiments in which Vpu CTD fragments were directly attached to Tetherin in the absence of the TMD. In particular, the C-terminal α-helix (H2) of Vpu CTD is sufficient to remove Tetherin from sites of viral assembly and is necessary for full Tetherin antagonist activity. Overall, these data demonstrate that Vpu and Tetherin interact directly via their transmembrane domains enabling activities present in the CTD of Vpu to remove Tetherin from sites of viral assembly.

Author Summary

At the cell surface, HIV-1 particles are assembled and then released to infect new cells. However, an anti-viral host restriction factor, Tetherin, can tether outgoing virions to the infected cell surface, preventing their dissemination. HIV-1 overcomes this block through the expression of the viral accessory protein Vpu, which antagonizes Tetherin. In this study, we demonstrate that the domains of Vpu and Tetherin that are embedded in the outer cell membrane bind directly to each other within the membrane, and we identify amino acids that participate directly in the interaction between these two proteins. After binding to Tetherin, Vpu can induce its removal from the cell surface and degradation. However, a mutant Vpu lacking these activities retains some capacity to antagonize Tetherin. We show that this residual activity requires a particular portion of the intracellular domain of Vpu and is manifested as an ability to displace Tetherin from sites of viral assembly, without affecting the overall level of Tetherin at the cell surface. These data indicate that Vpu directly binds to Tetherin and then employs multiple mechanisms, including displacement, to counteract Tetherin's ability to restrict virus particle release.