Mutation of a Single Residue Renders Human Tetherin Resistant to HIV-1 Vpu-Mediated Depletion

Abstract

The recently identified restriction factor tetherin/BST-2/CD317 is an interferon-inducible trans-membrane protein that restricts HIV-1 particle release in the absence of the HIV-1 countermeasure viral protein U (Vpu). It is known that Tantalus monkey CV1 cells can be rendered non-permissive to HIV-1 release upon stimulation with type 1 interferon, despite the presence of Vpu, suggesting species-specific sensitivity of tetherin proteins to viral countermeasures such as Vpu. Here we demonstrate that Tantalus monkey tetherin restricts HIV-1 by nearly two orders of magnitude, but in contrast to human tetherin the Tantalus protein is insensitive to HIV-1 Vpu. We have investigated tetherin's sensitivity to Vpu using positive selection analyses, seeking evidence for evolutionary conflict between tetherin and viral countermeasures. We provide evidence that tetherin has undergone positive selection during primate evolution. Mutation of a single amino acid (showing evidence of positive selection) in the trans-membrane cap of human tetherin to that in Tantalus monkey (T45I) substantially impacts on sensitivity to HIV-1 Vpu, but not on antiviral activity. Finally, we provide evidence that cellular steady state levels of tetherin are substantially reduced by Vpu, and that the T45I mutation abrogates this effect. This study provides evidence that tetherin is important in protecting mammals against viral infection, and that the HIV-1 Vpu–mediated countermeasure is specifically adapted to act against human tetherin. It also emphasizes the power of selection analyses to illuminate the molecular details of host–virus interactions. This work suggests that tetherin binding agents might protect it from viral encoded countermeasures and thus make powerful antivirals.

Simian immunodeficiency virus envelope glycoprotein counteracts tetherin/BST-2/CD317 by intracellular sequestration

“…We co-transfected fixed amounts of plasmids encoding Tantalus monkey (TanTHN), rhesus monkey (RhTHN), human (HuTHN), and sooty mangabey (SmTHN) tetherins with HIV-1 vector plasmids and a titration of SIVtan envelope encoding plasmid into 293T cells. Expression of primate tetherin proteins strongly inhibited virus release as previously described (7,8), and expression of SIVtan Env was able to partially counteract Tantalus tetherin antiviral activity (Fig. 1A).…”

“…Tetherin's sensitivity to HIV-1 Vpu is species-specific and determined by the sequence of its transmembrane (TM) domain (7,8). To map residues important for sensitivity to antagonism by SIVtan Env, we sought a tetherin molecule able to restrict HIV-1 but insensitive to antagonism by either Vpu or SIVtan Env.…”

“…We previously used positive selection analysis of tetherin to identify a key determinant of sensitivity to HIV-1 Vpu in the TM region (7). To identify determinants of sensitivity to SIVtan Env, we mutated human tetherin amino acids with evidence for positive selection to the homologous residues in the porcine protein.…”

“…It is proposed to form a protein tether linking assembled virions to infected cells, leading to their endocytosis and degradation in lysosomes (1,6). All tetherin variants tested thus far inhibit the release of enveloped viral particles including retroviruses, filoviruses, and arena viruses (1,2,(7)(8)(9)(10). Many simian immunodeficiency variants do not encode a Vpu protein, suggesting the existence of an alternative means of overcoming tetherin restriction.…”

Oligomerization state and supramolecular structure of the HIV‐1 Vpu protein transmembrane segment in phospholipid bilayers

“…39,40 Sequence variations in the TM domain of tetherin that affect sensitivity to Vpu have been identified. 41,42 Formation of Vpu/tetherin hetero-oligomers through direct interactions between their TM domains may therefore be essential for Vpu function, and these hetero-oligomers may be structurally related to Vpu homo-oligomers. A proposed therapeutic strategy is to use ''decoy'' peptides to sequester Vpu through formation of unproductive hetero-oligomers, allowing tetherin to inhibit viral release from infected cells even in the presence of Vpu.…”