The Transmembrane Domain of HIV-1 gp41 Inhibits T-Cell Activation

Feb 1, 2016 - E-mail: [email protected]. Cite this:Biochemistry 55, 7, 1049-1057. Abstract. Abstract Image. To successfully infect and persi...
1 downloads 3 Views 2MB Size
Download PDF
Article pubs.acs.org/biochemistry

The Transmembrane Domain of HIV‑1 gp41 Inhibits T‑Cell Activation by Targeting Multiple T‑Cell Receptor Complex Components through Its GxxxG Motif Etai Rotem, Eliran Moshe Reuven,† Yoel A. Klug, and Yechiel Shai* Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel S Supporting Information *

ABSTRACT: To successfully infect and persist within its host, HIV-1 utilizes several immunosuppressive motifs within its gp41 envelope glycoprotein to manipulate and evade the immune system. The transmembrane domain (TMD) of gp41 downregulates T-cell receptor (TCR) signaling through a hitherto unknown mechanism. Interactions between TMDs within the membrane milieu have been shown to be typically mediated by particular amino acids, such as interactions between basic and acidic residues and dimerization motifs as GxxxG. The HIV-1 TMD exhibits both a polar arginine (Arg696) residue and a GxxxG motif, making them ideal candidates for mediators of TMD−TCR interaction. Using a primary T-cell activation assay and biochemical and biophysical methods, we demonstrate that the gp41 TMD directly interacts with TMDs of the TCR and the CD3 coreceptors (δ, γ, and ε) within the membrane, presumably leading to impairment of complex assembly. Additionally, we reveal that although Arg696 does not affect TMD immunosuppression, the GxxxG motif is crucial in mediating gp41’s TMD interaction with the CD3 coreceptors of the TCR. These findings suggest that compared with other gp41 immunosuppressive motifs, the gp41 TMD has multiple targets within the TCR complex, suggesting less susceptibility to evolutionary pressure and consequently being advantageous for the virus over the host immune response. Furthermore, as the GxxxG motif mediates interactions of the gp41 TMD with multiple receptors, it emerges as an attractive drug target. This multitarget inhibitory mechanism might be a strategy utilized by HIV to interfere with the function of additional host receptors.

T

activity through several membrane-associating segments of gp41.13,26,27 One of these segments is the gp41 transmembrane domain (TMD) that was previously shown to inhibit T-cell activation by disrupting TCR complex signaling.28 The TCR complex is composed of TCR α and β that are responsible for antigen recognition and the CD3 coreceptor dimers (δ−ε, γ−ε, and ζ−ζ) that drive signal propagation. The CD3 coreceptors contain two acidic transmembrane residues, which interact with a basic residue within the TCRα TMD.29,30 gp41 has been shown to interfere with these interactions;27,31 however, the inhibitory mechanism and the manner in which the gp41 TMD targets the TCR complex are unknown. It was previously reported that HIV-1 gp41 and the TCRα TMD possibly interact with one another via a shared nineamino acid motif.28 The TMD, which is a highly conserved region of the gp41 sequence,32 contains a single charged arginine residue (Arg696) and a GxxxG motif, allowing intramembrane interactions between basic and acidic residues30 and driving interactions between membrane-embedded helices, respectively.33−39 Moreover, it was recently shown that the GxxxG motif within the gp41 TMD contributes to the interaction between gp41 and TLR2 TMDs leading to inhibition of the TLR2-mediated immune response.40 Interest-

he evolutionary pressure applied on viruses by our immune system has driven them to develop various strategies, such as antigen presentation antagonism, glycan shielding, and immunosuppressive motifs, to modify cellular processes and evade the immune response.1−4 Among these are the hepatitis C virus,5 Epstein−Barr virus,6 respiratory syncytial virus,7 human T-cell leukemia virus,8 and human immunodeficiency virus (HIV).9,10 HIV employs many of its proteins, such as Nef and Vpu, to manipulate the antiviral immune response by downregulating MHC class 1 from the cell surface and by internalization and degradation of CD4 in infected cells.11,12 However, these cellular manipulations can be utilized only after the virus has entered its host cell, yet the virus can employ different immunosuppressive elements within its envelope (ENV) fusion protein to interfere with T-cell receptor (TCR) activity during the membrane fusion process.13 HIV enters the host cell by membrane fusion,14 either through cell−cell transmission or directly as a free virion.15−17 The HIV ENV that mediates membrane fusion is composed of two noncovalently bound subunits, gp120 and gp41, which bind cellular receptors and initiate fusion, respectively.18−22 gp41 is composed of the cytoplasmic, transmembrane, and extracellular domains, and the latter contains the C-terminal heptad repeat, loop, N-terminal heptad repeat, and fusion peptide (Figure 1A).23,24 CD4 is the main receptor of HIV and is adjacent to the T-cell receptor (TCR) and CD3 coreceptors.25 HIV-1 exploits this proximity to inhibit TCR © 2016 American Chemical Society

Received: December 4, 2015 Revised: February 1, 2016 Published: February 1, 2016 1049

DOI: 10.1021/acs.biochem.5b01307 Biochemistry 2016, 55, 1049−1057

Article

Biochemistry

Peptide Synthesis and Fluorescent Labeling. Peptides were synthesized using the Fmoc solid phase method on Rink amide resin (0.65 mmol/g), as previously described.42 The peptides were purified by reverse phase high-performance liquid chromatography (RP-HPLC) to >95% homogeneity on a C4 or C2 column using a linear gradient of 20 to 70% acetonitrile in 0.1% trifluoroacetic acid (TFA) for 45 min. The peptides were subjected to ESI-MS (electrospray ionization mass spectrometry) analysis to confirm their composition. Two lysine residues were added to the C-termini of the peptides to confer water solubility to the hydrophobic TM domains. It was previously shown that hydrophobic peptides conjugated to lysine tags were correctly oligomerized and inserted into the membrane.43−45 For NBD-F fluorescent labeling, resin-bound peptides were treated with NBD-F (2-fold excess) dissolved in dimethylformamide (DMF), leading to the formation of resinbound N-terminal NBD peptides. After 24 h, the resins were washed thoroughly with DMF and then with dichloromethane (DCM) and dried under a nitrogen flow. For Rho-N fluorescent labeling, the Fmoc protecting group was removed from the N-terminus of the resin-bound peptides by incubation with piperidine for 12 min, whereas all the other reactive amine groups of the attached peptides were kept protected. The resinbound peptides were washed twice with DMF and then treated with rhodamine-N-hydroxysuccinimide (2-fold excess), in anhydrous DMF containing 2% DIEA, leading to the formation of a resin-bound N-terminal rhodamine peptide. After 24 h, the resin was washed thoroughly with DMF and then with DCM and dried under a nitrogen flow. The labeled peptides were purified on a RP-HPLC C4 or C2 column as described above. Unless stated otherwise, stock solutions of concentrated peptides were maintained in DMSO to prevent aggregation of the peptides prior to use. In Vitro T-Cell Proliferative Response. Primary CD4 Tcells specific to MOG p35−55 were plated onto round 96-well plates in medium containing RPMI-1640 supplemented with 2.5% fetal calf serum (FCS), 100 units/mL penicillin, 100 μg/ mL streptomycin, 50 μM β-mercaptoethanol, and 2 mM Lglutamine. Each of the 96 wells had a final volume of 200 μL and contained 10 × 103 T-cells, 5 × 105 irradiated (25 gray) spleen cells, as antigen-presenting cells (APC), and 5 μg/mL MOG p35−55. In addition, the relevant peptide was added. Each treatment was made with eight repeats. To exclude interaction between the examined peptides and the MOG p35−55 antigen, we initially added the MOG p35−55 antigen to the APC in a test tube, and to a second test tube, we added the examined peptides to the T-cells. After 1 h, we mixed the APC with the T-cells and incubated them for 48 h in a 96-well round-bottom plate. Then T-cells were pulsed with 1 μCi of [H3]thymidine, with a specific activity of 5.0 Ci/mmol, for 24 h, and [H3]thymidine incorporation was measured using a 96-well plate β-counter. The mean counts per minute ± the standard deviation (SD) was calculated for each quadruplicate. The results are shown as the percentage of T-cell proliferation inhibition triggered by the antigen in the absence of any peptide. In several experiments, cells were activated with 50 ng/ mL PMA (phorbol 12-myristate 13-acetate) together with 1 μM ionomycin (Sigma Chemical Co., Rehovot, Israel). Determination of IFN-γ Secretion. Cells were activated with precoated CD3 and CD28 antibodies (LEAFTM purified anti-mouse clones 145-2-C11 and 37.51, respectively, from Biolegend) at final concentration of 2 μg/mL, in the presence of gp41-derived peptides. Each treatment was made with three

Figure 1. Schematic representation of HIV-1 (HXB2) gp41 and the WT and GGG peptides derived from the gp41 TMD. (A) Scheme showing the functional regions of HIV-1 gp41. Abbreviations: CHR, C-terminal heptad repeat; NHR, N-terminal heptad repeat; CT, cytoplasmic tail. (B) Helical wheel representation of the amino acid positions within the trimeric gp41 TMD and GGG mutant. Marked in red circles are the glycine residues within the GxxxG motif.

ingly, apart from containing acidic residues that can potentially bind Arg696, the TMDs of the CD3 coreceptors contain GxxxGlike motifs and, therefore, might serve as gp41 TMD-interacting partners. In this study, we investigated the mechanism underlying the ability of the HIV gp41 TMD to downregulate T-cell activation. Utilizing wild-type and mutant TMDs, T-cell proliferation assays, and biochemical and biophysical methods, we reveal that the gp41 TMD exerts its inhibitory effect on T-cells by interacting with several TCR complex components within the membrane. These interactions are mediated by the GxxxG motif, which is responsible for the interaction of the gp41 TMD with the CD3 coreceptors. Furthermore, we show that Arg696 does not affect the TMD’s inhibitory ability. These results suggest that in addition to the facilitation of fusion, the gp41 TMD can bind multiple targets within the TCR complex, mediated by its conserved GxxxG interaction motif, thus inhibiting complex activation.

■

EXPERIMENTAL PROCEDURES Mice. C57Bl/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were 2−3 months old when used in the experiments. The Institutional Animal Care and Use Committee of the Weizmann Institute has approved the experiments (Permit 02370413-2), which were performed in accordance with its relevant guidelines and regulations. Cell Lines. Antigen-specific T-cell lines were selected in vitro41 from primed lymph node cells derived from C57Bl/6J mice that had been immunized 9 days before with antigen [100 μg of myelin peptide, MOG(35−55)] emulsified in complete Freund’s adjuvant (CFA) containing 150 μg of Mycobacterium tuberculosis (Mt) H37Ra (Difco Laboratories, Detroit, MI). All T-cell lines were maintained in vitro in medium containing interleukin-2 (IL-2), with alternate stimulation with the antigen every 14 days. 1050

DOI: 10.1021/acs.biochem.5b01307 Biochemistry 2016, 55, 1049−1057

Article

Biochemistry

Statistical Analysis. A one-way analysis of variance test was used where appropriate. P < 0.05 was considered significant (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Results are displayed as means ± SEM.

repetitions. Analysis of IFN-γ secretion was performed with an enzyme-linked immunosorbent assay (ELISA) 24 h after cell activation according to standard protocols from R&D Systems. The data are presented as the mean inhibition of IFN-γ secretion. Preparation of Large Unilamellar Vesicles (LUVs). Thin 9:1 phosphatidylcholine/cholesterol (PC/Chol) films were generated after dissolving the lipids in a 2:1 (v/v) CHCl3/ MeOH mixture and drying them under a stream of nitrogen gas while rotating them. The films were lyophilized overnight, and the samples were sealed with argon gas to prevent oxidation of the lipids. Before the experiments, films were suspended in PBS and vortexed for 1.5 min. The lipid suspension underwent five cycles of freezing−thawing and extrusion through polycarbonate membranes with 1 and 0.1 μm diameter pores to create LUVs. Förster Resonance Energy Transfer (FRET) Measurements. FRET experiments were performed using NBD- and Rho-labeled peptides. Fluorescence spectra were recorded at room temperature via the Cytation 5 plate reader from Lumitron Ltd., with excitation set at 465 nm (10 nm slit) and emission scanned from 500 to 600 nm (10 nm slits). In a typical experiment, a NBD-labeled peptide was added first from a stock solution in DMSO [final concentration of 0.1 μM, lipid:peptide ratio of 1:10−3, and a maximum of 0.25% (v/v) DMSO] to a dispersion of 9:1 PC/Chol LUVs (100 μM) in PBS. This was followed by the addition of the Rho-labeled peptide in several sequential doses ranging from 0.0025 to 0.01 μM, generating lipid:peptide ratios ranging from 1:2.5 × 10−5 to 1:10−4 (stock in DMSO). Fluorescence spectra were recorded before and after addition of the Rho-labeled peptide. The effect of sample dilution on signal intensity was calculated and subtracted by measuring the changes in NBD fluorescence upon the addition of each acceptor as an unlabeled peptide in increasing amounts (from 0.0025 to 0.01 μM). The fluorescence values were corrected by subtracting the corresponding blank (buffer with the same vesicle concentration). FRET efficiencies were calculated by the relative reduction in NBD’s maximal emission (525−530 nm) upon the addition of Rho-labeled peptides. Circular Dichroism (CD) Spectroscopy. CD spectra were recorded using an Applied Photophysics spectropolarimeter. The spectra were scanned using a thermostatic quartz cuvette with a path length of 1 mm. Wavelength scans were performed at 25 °C; the average recording time was 7 s, in 1 nm steps and over a wavelength range of 190−260 nm, and recordings were conducted in triplicate. All CD spectra were recorded in HEPES buffer (5 mM, pH 7.4) with 1% lysophosphatidylcholine (LPC) for a membrane mimetic environment at a peptide concentration of 25 μM. Cytotoxicity Assay (XTT proliferation). Aliquots of 2.5 × 104 cells were distributed onto a 96-well plate in the presence of 1.25−40 μM gp41 TMD-derived peptides for 18 h. Wells in the last two columns served as a blank (medium only) and a 100% survival control (cells and medium only). Following incubation, the XTT reaction solution (benzenesulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate, mixed in a proportion of 50:1) was added for an additional 2 h. The optical density was read at a wavelength of 450 nm in an enzyme-linked immunosorbent assay plate reader. The percentage of toxicity was calculated relative to the control, 2.5 × 104 cells in medium with no peptide added.

■

RESULTS The GxxxG Motif Mediates gp41’s TMD Inhibitory Activity on T-Cells via the CD3 Coreceptors. gp41 has been shown to modulate T-cell proliferation via an unknown mechanism.28 To elucidate this mechanism, we utilized a wildtype gp41 TMD-derived peptide together with an unrelated control peptide originating from the cytoplasmic domain of gp41 and two TMD mutant peptides, termed GGG and R/L, each mutated in a single conserved element, GxxxG and Arg696, respectively (Table 1). Whereas R/L removes a possibly crucial Table 1. Designations, Sequences, Toxicities, and Origins of Peptides Used in This Study designation gp41 TMD (WT) GGG R/L control peptide CD3-δ CD3-γ CD3-ε TCR-α TAR-1

sequencea

toxicityb (μM)

origin

KKIVGGLVGLRIVFAVLS

≥20

HIV-1

KKIVGGGLVLRIVFAVLS KKIVGGLVGLLIVFAVLS EALKYWWNLLQY

≥20 ≥20 nd

HIV-1 HIV-1 HIV-1

KKGIIVTDVIATLLLALG KKGFLFAEIVSIFVLAVG KKVMSVATIVIVDICITG KKVIGFRILLLKVAGFNL KKMVLGVFALLSLISGSLKK

nd nd nd nd nd

human human human human Escherichia coli

a

All peptides were synthesized using the Fmoc solid phase method and purified by reverse phase HPLC. Two Lys residues were added to the N-terminus of all TMD-derived peptides to enhance solubility. The gp41 sequences are derived from the HXB2 isolate of HIV-1. The TMDs of CD3-δ, -ε, and -γ and TCR-α are human-based. The TAR-1 sequence is derived from the E. coli aspartate receptor TMD. bToxicity concentrations represent a minimum 15% toxicity. nd, not determined.

basic residue, the GGG peptide disrupts the GxxxG motif (Figure 1B) and, thus, the intrinsic ability of the TMD to assemble into its trimeric form.46,47 Next, we determined the proliferative responses of C57BL/6J mMOG(35−55)-specific primary T-cells in the presence or absence of these gp41derived peptides (10 μM) upon activation by APC through the TCR. Peptides were not toxic to T-cells at concentrations used in this study (Figure S1). Interestingly, the GxxxG mutant inhibited T-cell proliferation with a potency higher than that of the wild-type gp41 TMD peptide. In contrast, mutation of the arginine residue did not affect the activity of the wild-type TMD (Figure 2A). To evaluate the specificity of the gp41 TMD for the TCR complex, mMOG(35−55) T-cells were activated either through the TCR using MOG(35−55) presenting splenocytes or downstream of the TCR using PMA and ionomycin (Figure 2B). The wild-type gp41 TMD exclusively inhibited T-cells that were activated through the TCR and not through downstream activation (Figure 2A,B). The gp41 TMD was previously shown to inhibit CD3- and CD28-based T-cell stimulation28 bypassing the TCR. Therefore, we aimed to evaluate the role of the GxxxG motif and Arg696 on this inhibition. We evaluated the activity of our 1051

DOI: 10.1021/acs.biochem.5b01307 Biochemistry 2016, 55, 1049−1057

Article

Biochemistry

effect on IFN-γ secretion compared to that of the wild type (Figure 2C). This is surprising as one would expect a trend similar to that of the proliferative response, let alone an opposing one. These findings demonstrate that the GxxxG motif is crucial in mediating gp41’s TMD inhibitory effect on T-cell activation through the CD3 coreceptors. The Secondary Structure of the gp41 TMD Does Not Rely on the GxxxG Motif. It is possible that the reason for the change in biological activity is due to a secondary structure shift in the GGG mutant as GxxxG is known to aid in helix− helix packing in membranes.33−39 Therefore, the gp41 TMD and GGG peptides were examined by circular dichroism spectroscopy in a membrane mimetic environment. Both the gp41 TMD and the GGG peptides exhibited α-helical structure (Figure 3). The peptides were subjected to secondary structure analysis and found to have similar proportions of secondary structure fractions (Table 2). This analysis emphasizes that the differences found between the wild-type peptide and the GxxxG mutant do not result from structural changes. Disruption of the GxxxG Motif Weakens the Interaction of the gp41 TMD with TCR Complex Components As Revealed by Förster Resonance Energy Transfer (FRET). In light of the proliferation and IFN-γ results, we assessed the interaction of the wild-type gp41 TMD and the GGG mutant with TCR complex components via FRET analysis. The assay was performed in a model lipid environment of LUVs composed of phosphatidylcholine (PC) and cholesterol (Chol) (9:1). We labeled the TMDs of the TCR complex components with nitrobenzoxadiazole (NBD) and the gp41 TMD-derived peptides with rhodamine (Rho). The maximal emission wavelength of NBD is 525−530 nm, and it overlaps with the excitation spectrum of Rho, allowing energy transfer between the two fluorophores. Rhodamine’s maximal emission wavelength is 575−580 nm. FRET is observed by a reduction in NBD emission, and FRET efficiency represents the relative reduction in NBD fluorescence upon addition of Rho-labeled peptides. The wild-type gp41 TMD exhibited interactions with TCR complex components significantly stronger than those of the GGG mutant (Figure 4A−D). The differences in FRET efficiencies between the GGG and wildtype peptides were most significant with the CD3 coreceptors (Figure 4B−D), while the difference observed with TCR-α was markedly smaller (Figure 4A). The control peptide TAR-1, derived from the TMD of the aspartate receptor of Escherichia coli, exhibited no FRET with the gp41 TMD or the GGG peptide (Figure 4E). This analysis demonstrates that the GxxxG motif is crucial for the ability of the gp41 TMD to interact with TCR complex components, especially with the CD3 coreceptors. These findings are in line with T-cell activation results demonstrating that when T-cells are activated directly through their CD3 coreceptors the wild type significantly inhibits activation whereas the GGG mutant does not.

Figure 2. Inhibition of T-cell activation by gp41 TMD and its mutants. Assays were performed in the presence of gp41-derived peptides at 10 μM. (A and B) The proliferative responses were assessed by the [3H]thymidine proliferation assay and normalized to the proliferation of nonactivated T-cells. The data are presented as the mean inhibition of proliferation. The changes between activated T-cell proliferative responses and nonactivated T-cell proliferative response were (i) 39.67 ± 9.99 (SEM) for irradiated MOG(35−55) presenting APC activation and (ii) 26.17 ± 4.36 (SEM) for PMA and ionomycin activation. Error bars represent ±SEM (n ≥ 12). ***P < 0.001. (A) Following APC and MOG(35−55) activation, the GGG peptide inhibits T-cell proliferation significantly more strongly than the WT peptide does. (B) The WT gp41 TMD inhibits T-cell proliferation specifically through the TCR. (C) IFN-γ secretion was measured with an ELISA following activation with antibodies against CD3 and CD28. The WT peptide inhibits IFN-γ secretion significantly more strongly than the GGG peptide does. The data are presented as the mean inhibition of IFN-γ secretion. Error bars represent ±SEM (n = 9). *P < 0.05.

■

DISCUSSION HIV-1 inhibits T-cell activation using various regions of the gp41 fusion protein.13,26,27 Although reported to possess inhibitory activity,28 the gp41 TMD mode of action has yet to be characterized. In this study, we aimed to elucidate this matter by examining the effect of mutations in the GxxxG motif and Arg696 on the function of the gp41 TMD and its interactions with the TMDs of the TCR complex proteins. Using primary T-cell proliferation assays coupled with ELISA and biophysical methods, we revealed that the gp41 TMD

peptides by measuring interferon-γ (IFN-γ) secretion using an ELISA as when activated via CD3 and CD28, the T-cell proliferative response is at background levels (Figure S2). IFNγ is produced by CD4 Th1 and cytotoxic T-cells and is one of the primary cytokines produced upon T-cell stimulation.48,49 In accordance with activation through the TCR, the R/L and wildtype gp41 TMD peptides did not differ in their inhibitory effects. However, the GGG peptide exerted a weaker inhibitory 1052

DOI: 10.1021/acs.biochem.5b01307 Biochemistry 2016, 55, 1049−1057

Article

Biochemistry

Figure 3. Secondary structures of the WT and GGG peptides derived from the gp41 TMD as revealed by CD spectroscopy. CD spectra were measured at 25 °C in a 0.1 cm path-length cuvette at a peptide concentration of 25 μM. Both CD spectra were measured in 5 mM HEPES buffer (N2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) (5 mM, pH 7.4) with 1% lysophosphatidylcholine (LPC). Both peptides exhibit a typical αhelical curve.

CD28 T-cell activation the GGG mutant would remain highly active, yet a weak inhibitory effect is observed. However, our FRET results reveal that although the GGG mutant retains relatively strong FRET with the TCRα TMD, a weak FRET with the TMDs of the CD3 coreceptors is observed compared to that of the wild-type peptide. Therefore, despite a possible higher active concentration and notable inhibition via the TCR, the GGG peptide cannot impede T-cell activation via CD3 components due to limited interactions. Because the GGG mutant exhibited reduced inhibitory activity on T-cells, we further examined the role of the GxxxG motif in gp41’s TMD suppressive activity in T-cells. The gp41 TMD sequence contains three glycines within its GxxxG motif that creates a bend in the TMD helix.50 Glycine residues add structural flexibility to proteins and specifically to TMDs and FPs of viral envelope proteins.51 Altering the position of the glycines in the GGG mutant might result in a structural change. Therefore, we tested whether the wild-type and mutant gp41 TMD differ in activity because of structural changes. However, despite marked differences in their suppressive activity, both the gp41 TMD and GGG peptides were found to possess similar secondary structures, indicating that the observed differences are not due to changes in secondary structure. As shown in this study, the gp41 TMD inhibits T-cell activation specifically through the TCR complex. Therefore, we aimed to determine whether it can interact with the TCRα and the CD3 coreceptors. By employing FRET, we revealed that the gp41 TMD has the capacity to interact with all of the examined TCR complex components. This demonstrates that using its gp41 TMD the virus can bind multiple targets within the TCR complex, thus hindering their activity. This lack of restriction to a single target within the same receptor complex can be highly advantageous for the virus over the immune system. During pathogen−host co-evolution, one might assume that the immune system adopts mutations in its components to avoid detrimental interactions with viral proteins. This multiple-target strategy utilized by the virus requires numerous alterations in the host’s immune components, thus creating a high hurdle for the immune system to overcome. This strategy has been shown to be utilized by other viruses, such as the Vaccinia virus.52,53 Importantly, disruption of the GxxxG motif weakened the interactions with TCR complex components and most significantly impaired the ability of the wild-type gp41 TMD to interact with the CD3 coreceptors. Because the CD3 coreceptors contain GxxxG-like motifs in their TMDs, it is reasonable that HIV-1 exploits the GxxxG motif within its

Table 2. CD Spectral Analysis of the gp41 TMD and Its GxxxG Mutant by the CDNN Secondary Structure Analysis Programa WT GGG

α-helix

antiparallel

parallel

β-turn

random coil

23.9 29.0

14.9 9.1

9.5 9.5

16.8 16.5

34.9 35.9

a

Peptides were dissolved in solution, and secondary structure was obtained by circular dichroism spectroscopy (190−260 nm) and analyzed via CDNN (Applied Photophysics Ltd.). Values represent the relative amount of structure out of 100%.

exerts its inhibitory effect on T-cells by interacting with several TCR complex components within the membrane. Specifically, we demonstrate that its GxxxG motif is essential for targeting the CD3 coreceptors leading to successful inhibition. Through this mechanism, the virus possibly targets multiple components of the TCR complex with a single segment of its envelope using a highly conserved interaction motif. Activation of T-cells can be induced in vitro by antigen presentation through the TCR, or downstream from the TCR using CD3 and CD28 antibodies or PMA and ionomycin.27,28 Mutation of Arg696 did not affect the immunosuppressive activity of the wild-type gp41 TMD on T-cells, when activated both through the TCR and using CD3 and CD28 antibodies. Arg696 is highly conserved, yet it does not play a role in immunosuppression by the gp41 TMD. The high degree of conservation may be explained by gp41’s TMD role in membrane fusion, a crucial stage of HIV infection. During HIV-1 virus−cell fusion, gp41’s TMD interacts with its FP, promoted by Arg696, thus facilitating lipid mixing and merging of the two membranes.47 Moreover, when T-cells were activated using antibodies against CD3 and CD28, the GGG mutant exerted an inhibitory effect weaker than that of the wildtype gp41 TMD. This demonstrates the importance of the GxxxG motif for the ability of the gp41 TMD to inhibit CD3 coreceptor activity. In contrast, when we activated T-cells through the TCR, the GxxxG mutant had an inhibitory effect that was stronger than that of the wild-type gp41 TMD on their proliferation. Because the GxxxG motif is known for its capacity to drive dimerization of the gp41 TMD, it is reasonable to believe that because of its monomeric nature the GGG mutant has an effective concentration higher than that of the wild type, resulting in a stronger inhibitory effect.47 This might explain the high activity observed with the GGG peptide in activation of Tcells through the TCR. One would expect that in CD3 and 1053

DOI: 10.1021/acs.biochem.5b01307 Biochemistry 2016, 55, 1049−1057

Article

Biochemistry

Figure 4. Fluorescence resonance energy transfer measurements of the interactions between the gp41 TMD-derived peptides and TCR complex component TMDs. Fluorescence spectral excitation was set at 467 nm, and an emission scan was conducted at 500−600 nm. The NBD-labeled transmembrane peptide of either (i) TCRα (A), (ii) CD3-δ (B), (iii) CD3-ε (C), (iv) CD3-γ (D), or (v) TAR-1 (E) as a control was first added (0.1 μM) to a dispersion of PC/Chol (9:1) LUVs in PBS (100 μM), resulting in a lipid:peptide ratio of 1:10−3, presented as a dashed line. This was followed by the addition of either rhodamine-labeled WT or GGG peptides in several sequential doses ranging from 0.0025 to 0.01 μM, generating lipid:peptide ratios ranging from 1:2.5 × 10−5 to 1:10−4 and Rho:NBD ratios of 1:40, 1:20, and 1:10, presented from top to bottom, respectively. FRET efficiencies were calculated for each FRET pair and are presented from the lowest concentration to the highest (from black to gray, respectively). The WT gp41 TMD demonstrates FRET efficiencies significantly higher than those of the GGG. The data are presented as the mean FRET efficiency. Error bars represent ±SEM (n = 4). **P < 0.01. 1054

DOI: 10.1021/acs.biochem.5b01307 Biochemistry 2016, 55, 1049−1057

Biochemistry

■ ■

TMD to interact with them and inhibit their activity. A recent study of the structure of the gp41 TMD50 indicated a change in the orientation of the helix axis at position 690GGLV693 within the GxxxG motif. The bending at this position results from higher flexibility around this region, creating a hinge, which might be crucial for the proper function and interactions of the gp41 with its targets. The weakened interactions of the GxxxG mutant, harboring the 690GGGL693 sequence, with TCR complex components might be attributed to higher flexibility due to its three consecutive glycines, resulting in a more pliable peptide that fails to promote strong interactions. Taken together, this experimental evidence suggests that the gp41 TMD GxxxG motif might mediate the interaction with TMDs of several TCR complex components in vitro, resulting in inhibition of T-cell activation. In summary, this study shows that the immunosuppressive effect of the gp41 TMD is achieved by interaction with the TMDs of the TCR and its CD3 coreceptors. As activation of the TCR complex is dependent upon interaction between those TMDs,30 by targeting them, the gp41 TMD presumably disrupts complex assembly. Although Arg696 does not play a role in immunosuppression by gp41’s TMD, the GxxxG motif is crucial for the binding of the TMD to the CD3 coreceptors. The ability of HIV to bind and suppress multiple targets within the TCR complex through a single segment of gp41 can be highly beneficial for the virus, because avoiding the virally induced suppression requires the cell to undergo mutations in multiple proteins, posing a high hurdle for the cell to overcome. As the gp41 TMD is involved in membrane fusion,47 immunosuppression via multiple targets by the TMD highlights how HIV may influence different cellular processes, although using a limited repertoire of proteins, by reducing complexity and allowing their multifunctional activity.

■

ACKNOWLEDGMENTS

The authors thank Roland Schwarzer for his valuable input.

ABBREVIATIONS APC, antigen-presenting cells; Arg, arginine; CD, circular dichroism; CFA, complete Freund’s adjuvant; Chol, cholesterol; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; ENV, envelope; FCS, fetal calf serum; FP, fusion peptide; FRET, Förster resonance energy transfer; GP, glycoprotein; HEPES, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; HIV, human immunodeficiency virus; LPC, lysophosphatidylcholine; LUV, large unilamellar vesicle; MOG, myelin oligodendrocyte glycoprotein; NBD, 7-nitrobenzo-2-oxa-1,3-diazole; TFA, trifluoroacetic acid; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PMA, phorbol myristate acetate; Rho, rhodamine; SEM, standard error of the mean; TCR, T-cell receptor; TLR, Tolllike receptor; TMD, transmembrane domain; WT, wild type.

■

REFERENCES

(1) Alcami, A., and Koszinowski, U. H. (2000) Viral mechanisms of immune evasion. Immunol Today 21, 447−455. (2) Iannello, A., Debbeche, O., Martin, E., Attalah, L. H., Samarani, S., and Ahmad, A. (2005) Viral strategies for evading antiviral cellular immune responses of the host. J. Leukocyte Biol. 79, 16−35. (3) Jerome, K. R. (2008) Viral modulation of T-cell receptor signaling. J. Virol 82, 4194−4204. (4) Cook, J. D., and Lee, J. E. (2013) The secret life of viral entry glycoproteins: moonlighting in immune evasion. PLoS Pathog. 9, e1003258. (5) Crotta, S., Stilla, A., Wack, A., D’Andrea, A., Nuti, S., D’Oro, U., Mosca, M., Filliponi, F., Brunetto, R. M., Bonino, F., Abrignani, S., and Valiante, N. M. (2002) Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J. Exp. Med. 195, 35−41. (6) Ressing, M. E., van Leeuwen, D., Verreck, F. A., Keating, S., Gomez, R., Franken, K. L., Ottenhoff, T. H., Spriggs, M., Schumacher, T. N., Hutt-Fletcher, L. M., Rowe, M., and Wiertz, E. J. (2005) Epstein-Barr virus gp42 is posttranslationally modified to produce soluble gp42 that mediates HLA class II immune evasion. J. Virol 79, 841−852. (7) Bukreyev, A., Yang, L., Fricke, J., Cheng, L., Ward, J. M., Murphy, B. R., and Collins, P. L. (2008) The secreted form of respiratory syncytial virus G glycoprotein helps the virus evade antibody-mediated restriction of replication by acting as an antigen decoy and through effects on Fc receptor-bearing leukocytes. J. Virol 82, 12191−12204. (8) Schlecht-Louf, G., Renard, M., Mangeney, M., Letzelter, C., Richaud, A., Ducos, B., Bouallaga, I., and Heidmann, T. (2010) Retroviral infection in vivo requires an immune escape virulence factor encrypted in the envelope protein of oncoretroviruses. Proc. Natl. Acad. Sci. U. S. A. 107, 3782−3787. (9) Schnittman, S. M., Greenhouse, J. J., Psallidopoulos, M. C., Baseler, M., Salzman, N. P., Fauci, A. S., and Lane, H. C. (1990) Increasing viral burden in CD4+ T cells from patients with human immunodeficiency virus (HIV) infection reflects rapidly progressive immunosuppression and clinical disease. Ann. Intern. Med. 113, 438− 443. (10) Palefsky, J. M., and Holly, E. A. (2003) Chapter 6: Immunosuppression and co-infection with HIV. J. Natl. Cancer Inst. Monogr. 2003, 41−46. (11) Johnson, W. E., and Desrosiers, R. C. (2002) Viral persistence: HIV’s strategies of immune system evasion. Annu. Rev. Med. 53, 499− 518.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01307. Effect of gp41 TMD-derived peptides on MOG(35−55) antigen-specific T-cell viability at various concentrations (Figure S1) and comparison of the proliferative responses and IFN-γ secretion fold changes between Tcells activated with antibodies against CD3 and CD28 and nonactivated T-cells (Figure S2) (PDF)

■

Article

AUTHOR INFORMATION

Corresponding Author

*Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. Telephone: 972-8-9342711. Fax: 972-8-9344112. E-mail: [email protected]. Present Address †

E.M.R.: Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel. Funding

This study was supported by the Israel Science Foundation and Yeda-Sela Center. Y.S. is the incumbent of the Harold S. and Harriet B. Brady Professorial Chair in Cancer Research. Notes

The authors declare no competing financial interest. 1055

DOI: 10.1021/acs.biochem.5b01307 Biochemistry 2016, 55, 1049−1057

Article

Biochemistry (12) Malim, M. H., and Emerman, M. (2008) HIV-1 accessory proteins–ensuring viral survival in a hostile environment. Cell Host Microbe 3, 388−398. (13) Ashkenazi, A., Faingold, O., and Shai, Y. (2013) HIV-1 fusion protein exerts complex immunosuppressive effects. Trends Biochem. Sci. 38, 345. (14) White, J. M. (1990) Viral and cellular membrane fusion proteins. Annu. Rev. Physiol. 52, 675−697. (15) Jolly, C., and Sattentau, Q. J. (2004) Retroviral spread by induction of virological synapses. Traffic 5, 643−650. (16) Haller, C., and Fackler, O. T. (2008) HIV-1 at the immunological and T-lymphocytic virological synapse. Biol. Chem. 389, 1253−1260. (17) Alvarez, R. A., Barria, M. I., and Chen, B. K. (2014) Unique features of HIV-1 spread through T cell virological synapses. PLoS Pathog. 10, e1004513. (18) Berger, E. A., Murphy, P. M., and Farber, J. M. (1999) Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev. Immunol. 17, 657−700. (19) Eckert, D. M., and Kim, P. S. (2001) Mechanisms of viral membrane fusion and its inhibition. Annu. Rev. Biochem. 70, 777−810. (20) Gallo, S. A., Finnegan, C. M., Viard, M., Raviv, Y., Dimitrov, A., Rawat, S. S., Puri, A., Durell, S., and Blumenthal, R. (2003) The HIV Env-mediated fusion reaction. Biochim. Biophys. Acta, Biomembr. 1614, 36−50. (21) Finzi, A., Xiang, S. H., Pacheco, B., Wang, L., Haight, J., Kassa, A., Danek, B., Pancera, M., Kwong, P. D., and Sodroski, J. (2010) Topological layers in the HIV-1 gp120 inner domain regulate gp41 interaction and CD4-triggered conformational transitions. Mol. Cell 37, 656−667. (22) Blumenthal, R., Durell, S., and Viard, M. (2012) HIV entry and envelope glycoprotein-mediated fusion. J. Biol. Chem. 287, 40841− 40849. (23) Chan, D. C., Fass, D., Berger, J. M., and Kim, P. S. (1997) Core structure of gp41 from the HIV envelope glycoprotein. Cell 89, 263− 273. (24) Chan, D. C., and Kim, P. S. (1998) HIV entry and its inhibition. Cell 93, 681−684. (25) Brownlie, R. J., and Zamoyska, R. (2013) T cell receptor signalling networks: branched, diversified and bounded. Nat. Rev. Immunol. 13, 257−269. (26) Quintana, F. J., Gerber, D., Kent, S. C., Cohen, I. R., and Shai, Y. (2005) HIV-1 fusion peptide targets the TCR and inhibits antigenspecific T cell activation. J. Clin. Invest. 115, 2149−2158. (27) Ashkenazi, A., Faingold, O., Kaushansky, N., Ben-Nun, A., and Shai, Y. (2013) A highly conserved sequence associated with the HIV gp41 loop region is an immunomodulator of antigen-specific T cells in mice. Blood 121, 2244−2252. (28) Cohen, T., Cohen, S. J., Antonovsky, N., Cohen, I. R., and Shai, Y. (2010) HIV-1 gp41 and TCRalpha trans-membrane domains share a motif exploited by the HIV virus to modulate T-cell proliferation. PLoS Pathog. 6, e1001085. (29) Cosson, P., Lankford, S. P., Bonifacino, J. S., and Klausner, R. D. (1991) Membrane protein association by potential intramembrane charge pairs. Nature 351, 414−416. (30) Call, M. E., Pyrdol, J., Wiedmann, M., and Wucherpfennig, K. W. (2002) The organizing principle in the formation of the T cell receptor-CD3 complex. Cell 111, 967−979. (31) Bloch, I., Quintana, F. J., Gerber, D., Cohen, T., Cohen, I. R., and Shai, Y. (2007) T-cell inactivation and immunosuppressive activity induced by HIV gp41 via novel interacting motif. FASEB J. 21, 393− 401. (32) Shang, L., Yue, L., and Hunter, E. (2008) Role of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein in cell-cell fusion and virus infection. J. Virol. 82, 5417−5428. (33) Russ, W. P., and Engelman, D. M. (2000) The GxxxG motif: a framework for transmembrane helix-helix association. J. Mol. Biol. 296, 911−919.

(34) Senes, A., Gerstein, M., and Engelman, D. M. (2000) Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions. J. Mol. Biol. 296, 921−936. (35) Curran, A. R., and Engelman, D. M. (2003) Sequence motifs, polar interactions and conformational changes in helical membrane proteins. Curr. Opin. Struct. Biol. 13, 412−417. (36) McClain, M. S., Iwamoto, H., Cao, P., Vinion-Dubiel, A. D., Li, Y., Szabo, G., Shao, Z., and Cover, T. L. (2003) Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori vacuolating toxin. J. Biol. Chem. 278, 12101−12108. (37) Munter, L. M., Voigt, P., Harmeier, A., Kaden, D., Gottschalk, K. E., Weise, C., Pipkorn, R., Schaefer, M., Langosch, D., and Multhaup, G. (2007) GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Abeta42. EMBO J. 26, 1702−1712. (38) Fink, A., Sal-Man, N., Gerber, D., and Shai, Y. (2012) Transmembrane domains interactions within the membrane milieu: principles, advances and challenges. Biochim. Biophys. Acta, Biomembr. 1818, 974−983. (39) Bronnimann, M. P., Chapman, J. A., Park, C. K., and Campos, S. K. (2013) A transmembrane domain and GxxxG motifs within L2 are essential for papillomavirus infection. J. Virol 87, 464−473. (40) Reuven, E. M., Ali, M., Rotem, E., Schwarzter, R., Gramatica, A., Futerman, A. H., and Shai, Y. (2014) The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses. PLoS Pathog. 10, e1004248. (41) Ben-Nun, A., and Lando, Z. (1983) Detection of autoimmune cells proliferating to myelin basic protein and selection of T cell lines that mediate experimental autoimmune encephalomyelitis (EAE) in mice. J. Immunol 130, 1205−1209. (42) Merrifield, R. B., Vizioli, L. D., and Boman, H. G. (1982) Synthesis of the antibacterial peptide cecropin A (1−33). Biochemistry 21, 5020−5031. (43) Rapaport, D., Peled, R., Nir, S., and Shai, Y. (1996) Reversible surface aggregation in pore formation by pardaxin. Biophys. J. 70, 2502−2512. (44) Melnyk, R. A., Partridge, A. W., and Deber, C. M. (2001) Retention of native-like oligomerization states in transmembrane segment peptides: application to the Escherichia coli aspartate receptor. Biochemistry 40, 11106−11113. (45) Liu, F., Lewis, R. N., Hodges, R. S., and McElhaney, R. N. (2004) Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylethanolamine Bilayers. Biophys. J. 87, 2470−2482. (46) Kim, J. H., Hartley, T. L., Curran, A. R., and Engelman, D. M. (2009) Molecular dynamics studies of the transmembrane domain of gp41 from HIV-1. Biochim. Biophys. Acta, Biomembr. 1788, 1804−1812. (47) Reuven, E. M., Dadon, Y., Viard, M., Manukovsky, N., Blumenthal, R., and Shai, Y. (2012) HIV-1 gp41 transmembrane domain interacts with the fusion peptide: implication in lipid mixing and inhibition of virus-cell fusion. Biochemistry 51, 2867−2878. (48) Thompson, C. B., Lindsten, T., Ledbetter, J. A., Kunkel, S. L., Young, H. A., Emerson, S. G., Leiden, J. M., and June, C. H. (1989) CD28 activation pathway regulates the production of multiple T-cellderived lymphokines/cytokines. Proc. Natl. Acad. Sci. U. S. A. 86, 1333−1337. (49) Sen, G. C. (2001) Viruses and interferons. Annu. Rev. Microbiol. 55, 255−281. (50) Apellaniz, B., Rujas, E., Serrano, S., Morante, K., Tsumoto, K., Caaveiro, J. M., Jimenez, M. A., and Nieva, J. L. (2015) The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region. J. Biol. Chem. 290, 12999−13015. (51) Weise, K., and Reed, J. (2008) Fusion peptides and transmembrane domains of fusion proteins are characterized by different but specific structural properties. ChemBioChem 9, 934−943. 1056

DOI: 10.1021/acs.biochem.5b01307 Biochemistry 2016, 55, 1049−1057

Article

Biochemistry (52) DiPerna, G., Stack, J., Bowie, A. G., Boyd, A., Kotwal, G., Zhang, Z., Arvikar, S., Latz, E., Fitzgerald, K. A., and Marshall, W. L. (2004) Poxvirus protein N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by toll-like receptors. J. Biol. Chem. 279, 36570−36578. (53) Stack, J., Haga, I. R., Schroder, M., Bartlett, N. W., Maloney, G., Reading, P. C., Fitzgerald, K. A., Smith, G. L., and Bowie, A. G. (2005) Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence. J. Exp. Med. 201, 1007−1018.

1057

DOI: 10.1021/acs.biochem.5b01307 Biochemistry 2016, 55, 1049−1057