- Open Access
Determinants in HIV-2 Env and tetherin required for functional interaction
© Exline et al. 2015
- Received: 5 March 2015
- Accepted: 23 July 2015
- Published: 7 August 2015
The interferon-inducible factor BST-2/tetherin blocks the release of nascent virions from the surface of infected cells for certain enveloped virus families. The primate lentiviruses have evolved several counteracting mechanisms which, in the case of HIV-2, is a function of its Env protein. We sought to further understand the features of the Env protein and tetherin that are important for this interaction, and to evaluate the selective pressure on HIV-2 to maintain such an activity.
By examining Env mutants with changes in the ectodomain of the protein (virus ROD14) or the cytoplasmic tail (substitution Y707A) that render the proteins unable to counteract tetherin, we determined that an interaction between Env and tetherin is important for this activity. Furthermore, this Env-tetherin interaction required an alanine face in the tetherin ectodomain, although insertion of this domain into an artificial tetherin-like protein was not sufficient to confer sensitivity to the HIV-2 Env. The replication of virus carrying the ROD14 substitutions was significantly slower than the matched wild-type virus, but it acquired second-site mutations during passaging in the cytoplasmic tail of Env which restored the ability of the protein to both bind to and counteract tetherin.
These results shed light on the interaction between HIV-2 and tetherin, suggesting a physical interaction that maps to the ectodomains of both proteins and indicating a strong selection pressure to maintain an anti-tetherin activity in the HIV-2 Env.
- Cytoplasmic Tail
- Direct Physical Interaction
- Human Tetherin
- Aspartic Acid Substitution
- Tetherin Antagonism
Tetherin/BST-2 is a multi-functional cellular protein that plays roles in cell membrane organization, as well as contributing to both the sensing and inhibition of enveloped virus replication [reviewed in 1]. Depending on the cell type, tetherin can be constitutively expressed or stimulated by interferon [2–5]. Tetherin localizes to lipid raft membrane microdomains, where it links to the actin cytoskeleton and helps to stabilize the apical actin network and microvilli in polarized cells [6, 7]. Tetherin also has antiviral properties, that were first described against HIV-1 [8, 9]. In HIV-1 infected cells, tetherin retains newly assembled virions at the cell surface which both reduces the production of cell-free virus [8, 10] and also promotes natural killer cell mediated antibody-dependent killing of infected cells [11–13]. Additionally, the human form of tetherin, and to a lesser extent chimpanzee tetherin, can act as pattern recognition receptors, since cross-linking of the protein by tethered virions or antibodies activates the NF-κB pathway and promotes entry into an antiviral state [14, 15].
Structurally, tetherin is a type 2 transmembrane glycoprotein, with a short cytoplasmic tail and membrane-spanning domain at its N-terminus, and a GPI anchor at its C-terminus . These membrane anchors flank an extracellular coiled-coil domain that mediates tetherin–tetherin interactions and promotes the formation of parallel homodimers, which can be further organized into tetramers [16, 17]. Tetherin retains budding virions at the cell surface in an axial conformation, with the GPI anchors preferentially incorporated into virions and the transmembrane domains anchored in cellular membranes . All three of the major structural features of the protein are required for its ability to inhibit virus release [8, 19, 20], although the actual sequences are not essential, and its function can be recapitulated in a wholly artificial tetherin construct .
Since tetherin presents a barrier to virus replication at multiple levels, it is not surprising that the primate lentiviruses have evolved several strategies to counteract its actions. Most SIVs use the Nef protein to block tetherin [21–25], in a mechanism based on intracellular sequestration via a direct physical interaction between Nef and tetherin’s cytoplasmic tail . Alternatively, some SIVs such as SIVgsn use Vpu to counteract tetherin, and Vpu persists as the viral anti-tetherin factor in present day group M HIV-1 [8, 9, 23]. Here the mechanism is also predominantly through intracellular sequestration, combined with ubiquitination and endolysosomal degradation [27–32]. A direct physical interaction between Vpu and tetherin has also been reported, that maps to the trans-membrane domains of each protein [33, 34].
In HIV-2, which does not encode Vpu, the anti-tetherin factor is the Env protein [35–37]. HIV-2 Env has been reported to both interact with tetherin  and to remove it from the cell surface, leading to its concentration in a perinuclear compartment [29, 37, 38]. This interaction appears to be mediated by the extracellular domains of the two proteins since a chimeric Env comprising the extracellular domain of HIV-2 Env linked to the transmembrane and cytoplasmic domains of the non-functional HIV-1 Env is still able to antagonize tetherin . Conversely HIV-2 Env can counteract a tetherin derivative substituted with the transmembrane and cytoplasmic domains of the transferrin receptor, but retaining the extracellular domain and GPI anchor of native tetherin . In addition to a requirement for the extracellular domain of HIV-2 Env, a tyrosine based sorting motif in the cytoplasmic tail has also been shown to be required for anti-tetherin activity [37, 39].
In the present study, we sought to more fully map the determinants in tetherin and the HIV-2 Env that allow their interaction, and to investigate the impact of the loss of anti-tetherin activity on HIV-2 replication. Specifically, we asked whether there was a selective pressure for a virus that had lost the ability to antagonize tetherin following mutation of Env to re-acquire this function, and whether this would once again map to the Env protein.
Interaction of HIV-2 Env and tetherin is required for tetherin antagonism
To examine whether the two mutants Envs were still capable of binding to tetherin, we created GFP-tagged versions of all three Env proteins to facilitate co-immunoprecipitation assays using anti-GFP antibodies. As expected, no tetherin was immunoprecipitated when it was co-expressed with GFP alone, or when an untagged ROD10 Env was used. However co-expression of tetherin with a GFP-tagged ROD10 Env allowed its pull-down (Fig. 1b). In contrast, the ROD14 Env mutant did not interact with tetherin. Interestingly, despite its complete lack of tetherin antagonism, the ROD10 EnvY707A mutant was still able to immunoprecipitate some tetherin, and when the lower cellular levels of the ROD10 EnvY707A protein were taken into consideration, it was found to be 67% (n = 3 experiments) as efficient at immunoprecipitating tetherin as the WT ROD10 Env (data not shown). These differences may be sufficient to account for its lack of anti-tetherin activity or, alternatively, this may result from some other characteristic of the Y707A mutant, such as being present in a different cellular localization than the WT Env, or because the loss of its endocytosis signal makes it unable to remove tetherin from the cell surface, as we and others have previously reported [29, 37].
The extracellular domain of HIV-2 Env is not sufficient for anti-tetherin activity
An alanine motif in tetherin’s coiled-coil domain controls sensitivity to HIV-2 Env
We next asked whether the HIV-2-resistant phenotype of tetherin A100D was a result of disrupting the interaction between the two proteins. As described earlier, a GFP-tagged ROD10 Env can specifically immunoprecipitate the wild-type tetherin. However, the A100D mutant was not immunoprecipitated by the HIV-2 Env (Fig. 3b), suggesting that this substitution directly impacted the interaction between the two proteins, and accounts for the insensitivity of the mutant tetherin to HIV-2 Env.
Investigation of the sequence surrounding the A100 residue in tetherin revealed the presence of additional alanines at positions 97, 100, 104, and 107 (Fig. 3c), which are also highly conserved among primate tetherins, but absent in porcine tetherin. When mapped onto the crystal structure of a dimer of tetherin’s coiled-coil domain , these alanines were seen to line up on a single face, opposite to the dimerization interface, suggesting that they could be accessible to other protein partners such as HIV-2 Env. To test the hypothesis that the alanine face contributed to the interaction with HIV-2 Env, we introduced single aspartic acid substitutions at each of the four positions and tested the resulting tetherin mutants for their ability to inhibit HIV-1 VLP release, and to be counteracted by the ROD10 Env (Fig. 3d). We found that while each mutant tetherin retained the ability to restrict VLP release, and remained sensitive to Vpu, substitution of any of the alanines rendered the mutant tetherins completely resistant to ROD10 Env. These results therefore identify an alanine face on tetherin as being necessary for both the interaction with HIV-2 Env and the resulting ability of the protein to counter tetherin restriction.
The conserved alanine motif does not render an artificial tetherin-like molecule sensitive to HIV-2 Env
Artificial tetherin (art-tetherin) contains the same structural features as native tetherin, but without the conservation of any primary sequence . It is able to restrict HIV-1 release, but is resistant to both Vpu and HIV-2 Env, suggesting a sequence specific interaction between tetherin and these antagonists. However, it can be overcome by co-expression of the Ebola GP, which appears to use a different mechanism of action against tetherin [38, 43].
Rates of HIV-2 replication in the presence of ROD10 and ROD14 Envs
The late rise in virus replication seen for the mutant virus suggested the possibility of the emergence of a revertant virus with increased fitness. To test this hypothesis, we took clarified supernatants from the day 25 cultures for both viruses and used them to begin new infections in fresh JLTRG cells. We observed that the passaged ROD10(14 Env) stock was now able to replicate with similar kinetics as the ROD10(WT Env) virus (Fig. 5b), suggesting that changes had occurred that increased the replicative fitness of this mutant virus, either by restoring anti-tetherin activity or through an alternate compensatory mechanism such as enhanced cell-to-cell spread.
Revertant ROD14 Env mutants have acquired the ability to counteract tetherin
To determine whether these mutations had produced Env proteins with anti-tetherin activity, we performed VLP release assays using expression plasmids for each of the Rev A–C Envs (Fig. 6b). We found that the first mutant in the series, Rev A, was not able to counteract tetherin. However each of the remaining proteins had acquired some activity, albeit to a lesser extent than the wild-type ROD10 Env. The ability of the mutations in Rev B to restore this activity demonstrates that mutations in the ectodomain of the ROD14 Env that prevent anti-tetherin activity can be compensated for by alterations in the cytoplasmic tail alone.
We next assessed if the anti-tetherin phenotype of this series of Envs matched their ability to interact with tetherin in a co-immunoprecipitation assay. Mirroring the virus release assay results, we found that the Rev A Env did not immunoprecipitate tetherin any more than the background levels observed with the non-functional ROD14 parent, while the Rev B, C1, and C2 Envs had all re-acquired some ability to interact with tetherin (Fig. 6c). These findings further support the idea that a direct physical interaction with tetherin is required for antagonism by HIV-2 Env and, additionally, that tetherin imposes an evolutionary pressure on HIV-2 to evolve such a tetherin counteraction strategy.
Finally, we evaluated which of the three common cytoplasmic tail mutations present in functional Rev B, C1 and C2 variants were necessary for this phenotype by evaluating single and double combinations. The lack of activity of mutant Rev A had implicated D830G as essential, but this further analysis revealed that both of the substitutions D830G and K796R were required (Fig. 6c).
Rev B Env cytoplasmic tail changes are not sufficient to confer anti-tetherin activity
The cell surface protein BST-2/tetherin exhibits several antiviral activities that derive from its ability to retain newly assembled virions at the surface [8–10]. Such tethered viruses result in a reduction in the production of cell-free virus, enhance presentation to the immune system [11–13], and lead to the induction of an antiviral state [14, 15]. To combat these activities, a range of approaches have evolved in the primate lentiviruses through adaptations in the Vpu, Nef or Env proteins of specific viruses. These antagonists act to reduce the amount of tetherin at the cell surface following intracellular sequestration [27–29, 37], displacement from the sites of viral budding [34, 45], and/or enhanced degradation [30–32]. These mechanisms appear to all involve direct or possibly indirect interactions between tetherin and the viral antagonists, since tetherin can be immunoprecipitated by each viral protein. To do this, multiple domains in tetherin are targeted, including the cytoplasmic tail by SIV Nef , the transmembrane region by HIV-1 Vpu [30, 46–48] and the extracellular domain by HIV-2 Env.
We confirmed a physical interaction between tetherin and HIV-2 Env by co-immunoprecipitation, and further determined that previously identified mutations in the ROD14 Env that abolished anti-tetherin activity also prevented this interaction. Furthermore, we identified an alanine face on the extracellular helical domain of tetherin as being necessary for the interaction, with substitutions at any one of four alanine residues both preventing co-immunoprecipitation and rendering human tetherin resistant to the HIV-2 Env. However inserting the alanine motif into an artificial tetherin-like molecule was not sufficient to convert the molecule to a form that could co-immunoprecipitate with the HIV-2 Env, or that was now sensitive to its antagonism, suggesting that the alanine motif is necessary, but not sufficient, for the tetherin-Env interaction.
Interestingly, an alanine face has also been implicated in the interaction between Vpu and tetherin [33, 47–49], occurring in the trans-membrane region of Vpu. Alanine faces have also been implicated in other protein–protein interactions, promoting homodimerization in other transmembrane domains [50, 51] and in receptor-agonist interactions [52, 53]. Although it was originally speculated that the alanine face in Vpu represented a direct tetherin interaction face, cysteine-scanning mutagenesis and crosslinking experiments have instead pointed to a role for the motif in maintaining the overall structure of Vpu in a form that is competent to target tetherin . Therefore, while we cannot rule out that the lack of HIV-2 Env recognition of the art-tetherin molecules substituted with the alanine motif was caused by a less than optimal presentation in the context of this chimeric construct, it is also possible that the motif is not a direct interaction face, and is instead involved in maintaining the overall structure and organization of the protein’s ectodomain.
Although all of the major groups of the primate lentiviruses express anti-tetherin factors [8, 9, 21–23, 37], and their activity is easy to observe in one-round virus release assays, the importance of these activities has been less consistent in studies of virus replication. For example, studies of wild-type and Vpu-deficient HIV-1 have reported either less efficient replication for the mutant viruses [54–56], or no effect . Using a similar in vitro replication assay for HIV-2 expressing the ROD10 or ROD14 Envs, we were able to observe a distinct difference in replication rates. Furthermore, the ROD14 mutant acquired wild-type replication kinetics after passage in culture, consistent with a strong selection pressure to restore, or compensate for, the loss of this activity.
Analysis of the passaged viruses revealed that they had not undergone a simple reversion back to the WT (ROD10) Env sequence. Instead, we observed both retention of the original deleterious mutations (K422R and A598T) and the acquisition of additional mutations throughout the protein. Interestingly, we found that the minimal changes required to restore anti-tetherin activity mapped to the cytoplasmic domain of the protein, specifically K796R, and D830G. Further analysis revealed that substitutions in the cytoplasmic tail also restored the ability to interact with tetherin in a co-immunoprecipitation assay, despite the presence of the ROD14 ectodomain mutations, and further suggesting that direct interactions between the two proteins is an essential part of tetherin antagonism by the HIV-2 Env.
We have previously mapped the anti-tetherin function of the HIV-2 Env to the ectodomain of the protein . These findings of compensatory changes in the cytoplasmic tail suggested that either the mutations had created an additional tetherin-interacting domain in this region, or that the changes in the tail were having long-range effects on the conformation of the ectodomain and thereby restoring activity. Support for the former hypothesis comes from the characterization of Nef-deleted SIV variants that were used to infect macaques and which eventually acquired a new tetherin-binding domain in the cytoplasmic tail of Env . However, chimeras created between the HIV-1 Env and these mutant cytoplasmic domains were unable to block tetherin restriction, ruling out this potential explanation. Instead, we favor a model where these mutations in the cytoplasmic tail have an ‘inside-out’ influence on the conformation of the ectodomian, and allow more permissive interactions. Long-range impacts of a cytoplasmic domain on the structure and function of a protein’s ectodomain have previously been described [58–62]. Finally, it is also possible that these cytoplasmic tail mutations could be having a more indirect effect by altering the sub-cellular localization or cell surface stability of the HIV2 Env, and thereby enhancing the activity of a less potent tetherin antagonist.
BST-2/tetherin inhibits the release of budding lentiviruses. To prevent this action, HIV-2 Env sequesters tetherin in an intracellular location, in a mechanism that requires an interaction between the two proteins and which involves their ectodomains. We have mapped an alanine face in tetherin that is required, and shown that residues in both the ectodomain and cytoplasmic tail of Env can influence this interaction. HIV-2 viruses with at least one class of mutant Env protein (ROD14) reacquire this ability when passaged in culture due to second site mutations in the cytoplasmic tail, illustrating the importance of this anti-tetherin activity for viral fitness.
293T cells were obtained from the American Type Culture Collection; 293A cells were obtained from Qbiogene/MP Biomedicals (Irvine, CA, USA) and JLTRG cells  were obtained from the AIDS Research, Reference, and Reagent Program (ARRRP). 293A and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Herndon, VA, USA) supplemented with 10% fetal bovine serum (FBS) (Denville, Metuchen, NJ, USA) and JLTRG cells maintained in RPMI-1640 (Mediatech) supplemented with 10% FBS and 1% penicillin/streptomycin (JR Scientific, Woodland, CA, USA).
Plasmid pHIV-1-pack expresses HIV-1 Gag-Pol and Rev and produces HIV-1 virus-like particles (VLPs) . Plasmid pcDNA-Vphu (Vpu) encodes a human codon-optimized form of Vpu from HIV-1 isolate NL4-3 . Plasmid pEboGP expresses Ebola Zaire GP-8A, the full-length form of the Ebola virus glycoprotein . The HIV-2 Env expression plasmids pROD10 Env, pROD14 Env, and pROD10Y707A Env have previously been described [35, 39]. C-terminal eGFP tagged versions of all Env clones were generated by 2-step PCR using plasmid pAcEGFP-N1 (Clonetech, Moutainview, CA, USA) as an eGFP template. A GPI anchored version of the extracellular domain of ROD10 Env was created by 2-step PCR to fuse residue Trp673 of Env to the GPI domain (codons 303–335) from the urokinase-type plasminogen activator receptor (uPAR) . Expression plasmids for tetherin/BST-2, an eGFP-tagged tetherin/BST-2, and an artificial tetherin (art-tetherin) have been previously described [20, 29, 38]. Art-tetherin mutants containing insertions of tetherin residues 96–108 were generated by 2-step PCR. The infectious ROD10 proviral clone [36, 66] was kindly provided by Klaus Strebel (NIH). Derivatives were created containing either the ROD14 or the ROD10Y707A Envs in the ROD10 backbone, using restriction sites BstAPI and BsmI at positions 8582 and 9437 in the genome. Chimeric proteins containing the transmembrane and extracellular domains of HIV-1 Env and the cytoplasmic domain of HIV-2 Env were created by 2-step PCR using the HIV-1BH10 proviral clone and the HIV-2ROD10 expression plasmids as templates, as previously described , and using a reverse primer that added a FLAG tag at the carboxyl terminus of the Env protein.
Production and analysis of HIV-1 VLPs
HIV-1 VLPs were generated from 293A cells by transient transfection of pHIV-1-pack using TurboFect transfection reagent (Thermo Scientific, Glen Burnie, MD, USA), as previously described . The following amounts of plasmid DNA were used per 10-cm plate of cells: 2 μg of Vpu, Ebola GP and all HIV-2 Env constructs; 100 ng of tetherin and derivatives; 500 ng of art-tetherin; 1 μg of art-tetherin mutants. Cell lysates and viral particles were collected at 24 h post-transfection and the levels of p24 proteins in both lysates and supernatants analyzed by Western blot, as previously described [29, 35, 38, 39]. The intensity of p24-reacting bands on Western blots was measured and calculated as the ratio of the signal in VLPs:lysates, normalized to the ratio for the pHIV-1-pack only control. Specific proteins were detected by Western blotting using the following antibodies: rabbit ant-HIV-1SF2 p24 at 1:3,000 dilution, rabbit anti-HIV-2ST SU at 1:3,000 dilution, rabbit anti-tetherin at 1:10,000 dilution, and rabbit anti-HIV-1 Vpu at 1:3,000 dilution (all from ARRRP), as well as rabbit anti-GFP at 1:3,000 dilution (Abcam, Cambridge, MA, USA) and mouse anti-FLAG at 1:1,000 (Roche Applied Science, Indianapolis, IN, USA). The secondary antibodies used were HRP-conjugated goat anti-rabbit IgG at a 1:10,000 (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and goat anti-mouse IgG (1:10,000) (Sigma-Aldrich, St. Louis, MO, USA). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test from GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA).
293T cells in 10-cm dishes were co-transfected by TurboFect using 200 ng of tetherin plasmid and 2 μg of the indicated eGFP-tagged HIV-2 Env plasmids or 200 ng of eGFP-tagged tetherin and either 2 μg of untagged ROD10 Env or the indicated amount of ROD10 Envgpi. Cell lysates were collected at 24 h post transfection and GFP pull-down assays performed using the μMACS GFP isolation kit (Miltenyi Biotech Inc., Auburn, CA, USA). Initial cell lysates (1% input) and immunoprecipitates were analyzed by Western blotting.
Flow cytometry for Env surface expression
293A cells were in 10-cm dishes were co-transfected by Turbofect with 100 ng of an eGFP expression vector and either 2 μg of ROD10 Env or 2–8 μg amounts of ROD10 Envgpi. Twenty-four hours later, cells were washed 3× with PBS then blocked in 10% FBS for 30 min. Cells were then stained with HIV-2ST Su antibody 1410 at a 1:300 dilution for 15 min at 4C. Cells were washed 3× with PBS then counterstained with goat anti-rabbit IgG conjugated to Alexa Fluor 647 (Invitrogen) at a 1:300 dilution for 20 min for an additional 15 min. GFP + cells (10,000 events) were analyzed on a BD FACS Canto II (BD Biosciences, San Jose, CA, USA)., and collected data was analyzed using FlowJo 6.2 software (Tree Star, Ashland, OR, USA). The mean fluorescence intensity (MFI) was determined within the software and compared to cells stained with secondary antibody alone.
Viruses and infections
HIV-2 stocks were produced in 293T cells by transient transfection using TurboFect and 10 μg of proviral plasmids, followed by harvesting and filtration of supernatants 48 h later. Stocks were quantitated using a HIV-2 p27 ELISA kit (Zeptometrix, Buffalo, NY, USA). Infections were performed by incubating 5 × 106 JLTRG cells with the equivalent of 3 μg of p27 in a total of 0.5 ml RPMI for 4 h, followed by replacement of the media with 5 ml fresh media. Every 3 days, cells were analyzed for GFP expression by flow cytometry using a FACS Canto II, with uninfected cells used to set the negative population. At each data point, 20,000 cells were collected and the data analyzed using FlowJo 6.2 software. 0.45 μm filter clarified supernatants from infected JLTRG cultures were equilibrated and added to fresh JLTRG cells for 4 h to initiate second round infections that were then tracked by flow cytometry analysis every 3 days.
Viral sequences from infected cells were obtained by isolating genomic DNA (Qiagen, Valencia, CA, USA), followed by PCR amplification using the Accuprime Taq DNA Polymerase system (Invitrogen, Carlsbad, CA, USA). The HIV-2 Env primers used were forward (GGCTTTGCACCCAACTGTTCTAAAGTAGTAGC) and reverse (CTCACTTATCGTCGTCATCCTTGTAATCCAGGAGGGCGATTTCTGCTCC), which added a FLAG tag to the cytoplasmic tail. PCR products were ligated into a TOPO-TA cloning vector (Invitrogen) and single clones selected and sequenced.
CME participated in the design of the study, performed most of the experiments, and wrote the draft manuscript. SY, KGH, SR, LAL, MED, and ES contributed to the experiments and review of the manuscript. PMC conceived and coordinated the study, and wrote the final manuscript. All authors read and approved the final manuscript.
This work was funded by NIH grant AI068546, and by the California HIV/AIDS Research Program award ID10-USC-066 and fellowship F10-USC-207 (to CME). We also gratefully acknowledge the James B. Pendleton Charitable Trust for its support.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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