Species-Specific Activity of HIV-1 Vpu and Positive Selection of Tetherin Transmembrane Domain Variants

Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins

Species-Specific Activity of HIV-1 Vpu and Positive Selection of Tetherin Transmembrane Domain Variants

Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins

SIV Nef Proteins Recruit the AP-2 Complex to Antagonize Tetherin and Facilitate Virion Release

Lentiviral Nef proteins have multiple functions and are important for viral pathogenesis. Recently, Nef proteins from many simian immunodefiency viruses were shown to antagonize a cellular antiviral protein, named Tetherin, that blocks release of viral particles from the cell surface. However, the mechanism by which Nef antagonizes Tetherin is unknown. Here, using related Nef proteins that differ in their ability to antagonize Tetherin, we identify three amino-acids in the C-terminal domain of Nef that are critical specifically for its ability to antagonize Tetherin. Additionally, divergent Nef proteins bind to the AP-2 clathrin adaptor complex, and we show that residues important for this interaction are required for Tetherin antagonism, downregulation of Tetherin from the cell surface and removal of Tetherin from sites of particle assembly. Accordingly, depletion of AP-2 using RNA interference impairs the ability of Nef to antagonize Tetherin, demonstrating that AP-2 recruitment is required for Nef proteins to counteract this antiviral protein

Vpu Binds Directly to Tetherin and Displaces It from Nascent Virions

<div><p>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.</p></div

Mapping Vpu and Tetherin TMD residues that mediate interaction using a cysteine cross-linking assay.

<p>Each individual TMD cysteine-mutant Vpu protein was co-expressed with either Tetherin^CC/SS-HA (“WT”), or with each of five individual TMD cysteine-mutant Tetherin^CC/SS-HA proteins. The positions of the amino acids that were mutated to cysteine in the Vpu and Tetherin proteins are indicated above the blots. Transfected cells were treated with oxidizer and the cell lysates were analyzed using Western blots, probed with α-HA and α-FLAG antibodies. The migration of markers of the indicated molecular weights (kDa) is indicated, and bracket labeled “XL” indicate presumptive cross-linked Tetherin-Vpu products.</p

Multiple Vpu CTD elements are required for optimal Tetherin downregulation and degradation.

<p>(<b>A</b>) 293T cells stably expressing Tetherin-HA were transduced with VSV-G pseudotyped HIV-1 based vector encoding GFP and various mutant Vpu proteins. Cells transduced at an MOI of 0.5 were surface-stained using an α-HA antibody and analyzed by FACS to determine the relative level of Tetherin-HA expression. The FACS plots show examples of this assay. (<b>B</b>) Cells transduced with the same vectors used in (<b>A</b>), but at an MOI of 3, were lysed and analyzed using quantitative fluorescence-based Western blotting. Blots were probed with antibodies specific for Vpu, GFP, tubulin, capsid and HA, as indicated. (<b>C</b>) Chart summarizing data from assays carried out as described in (<b>A</b>) and (<b>B</b>). For FACS analysis, the geometric mean fluorescence after gating on GFP-positive cells was quantified (gray bars). For Western blot analysis band intensities were quantified using a LI-COR scanner (red bars). The mean and standard deviation of the relative amounts of Vpu expression (from four independent experiments) is plotted, with the amount of Tetherin expression observed following transduction with the ΔVpu vector set to 100%.</p

Models depicting the effect of Vpu on Tetherin.

<p>(<b>A</b>) Model of how Vpu acts to remove Tetherin from site of virion assembly: In the absence of Vpu, Tetherin is able to tether the virions at the cell surface by the partitioning of membrane anchors between cell and virion membranes. In the presence of Vpu, Tetherin and Vpu interact via their TMDs and Vpu removes Tetherin from sites of viral assembly, gray portion of the membrane. Vpu CTD H1, acts to cause intracellular retention while H2 displaces Tetherin from sites of virion assembly at the cell surface. (<b>B</b>) Recapitulation of these effects by fusing Tetherin CTD elements to the Tetherin protein.</p

Effect of cysteine substitutions on Vpu activity as determined by Tetherin downregulation and degradation assays.

<p>(<b>A</b>) 293T cells stably expressing Tetherin-HA were transduced with VSV-G pseudotyped HIV-1 based vectors encoding GFP and various mutant Vpu proteins. Cells transduced at an MOI of 0.5 were surface-stained using an α-Tetherin antibody and analyzed by FACS to determine the relative level of Tetherin-HA expression. (<b>B</b>) Cells transduced with the same vectors used in (<b>A</b>), but at an MOI of 3, were lysed and analyzed using quantitative fluorescence-based Western blotting. Blots were probed with antibodies specific for Vpu, GFP, tubulin, and HA (Tetherin), as indicated. (<b>C</b>) Chart summarizing data from assays carried out as described in (<b>A</b>) and (<b>B</b>). For FACS analysis, the geometric mean fluorescence after gating on GFP-positive cells was quantified (gray bars). For Western blot analysis band intensities were quantified using a Li-COR scanner (red bars). The mean and standard deviation of the relative amounts of Vpu expression (from four independent experiments) is plotted, with the amount of Tetherin expression observed following transduction with the ΔVpu vector set to 100%.</p

Localization of Lck-Vpu-Tetherin-HA fusion proteins.

<p>(<b>A</b>–<b>E</b>) Single cell clones of 293T cells stably expressing the indicated proteins namely: Lck-Tetherin (<b>A</b>), Lck-CTD^2/6-Tetherin (<b>B</b>), Lck-H1-Tetherin (<b>C</b>), Lck-H2-Tetherin (<b>D</b>) or WT Tetherin (<b>E</b>) were fixed, permeabilized and stained using α-HA antibodies (Alexa-594, red) and with DAPI to reveal nuclei (blue). Scale bar represents 5 µm. (<b>F</b>) Single cell clones stably expressing proteins from (<b>A–E</b>) were cell surface stained using an antibody against Tetherin directly conjugated to the flourochrome APC. Living and singlet cells were then gated and the Tetherin signal was reported as the median fluorescent intensity. (<b>G</b>) Western blot analysis of cell lysates from single cell clones expressing various forms of Tetherin as described in (<b>A–E</b>).</p

Summary of crosslinking and other evidence for Vpu and Tetherin TMD interactions.

<p>References: a <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003299#ppat.1003299-Vigan1" target="_blank">[43]</a>, b <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003299#ppat.1003299-Kobayashi1" target="_blank">[42]</a>, c <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003299#ppat.1003299-Skasko2" target="_blank">[44]</a>, d <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003299#ppat.1003299-McNatt1" target="_blank">[38]</a>, e <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003299#ppat.1003299-Gupta1" target="_blank">[37]</a>, f <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003299#ppat.1003299-Rong1" target="_blank">[40]</a>.</p><p>Superscript numbers indicate combinations of mutations that were necessary to affect Vpu's Tetherin antagonist function or Tetherin's sensitivity to Vpu.</p><p>NMR = Nuclear Magnetic Resonance, BiFC = Bimolecular Fluorescence complementation, IP = Immunoprecipitation.</p