The HIV gp41 Fusion Protein Inhibits T-Cell Activation through the

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The HIV gp41 fusion protein inhibits T cell activation through the lentiviral lytic peptide 2 motif Yoel Alexander Klug, Roland Schwarzer, Etai Rotem, Meital Charni, Alon Nudelman, Andrea Gramatica, Batya Zarmi, Varda ROTTER, and Yechiel Shai Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01175 • Publication Date (Web): 02 Jan 2019 Downloaded from http://pubs.acs.org on January 5, 2019

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The HIV gp41 fusion protein inhibits T cell activation through the lentiviral lytic peptide 2 motif Yoel A. Klug*1, Roland Schwarzer*3, Etai Rotem1, Meital Charni2, Alon Nudelman1, Andrea Gramatica3, Batya Zarmi1, Varda Rotter2 and Yechiel Shai1** From the 1Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel; 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 7610001, Israel;3Gladstone Institute for Virology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA

Running title: Gp41 cytoplasmic tail inhibition of T-cell activation * Equal contribution ** To whom correspondence should be addressed: Yechiel Shai: Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel; [email protected]; Tel (+972) 089342715

Key words: Human immuno deficiency virus, host-pathogen interaction, T-cell, immunosuppression, membrane protein, cytoplasmic tail, gp41

Abstract The Human Immunodeficiency Virus (HIV) enters its host cells by membrane fusion, initiated by the gp41 subunit of its envelope protein. Gp41 has also been shown to bind T-cell receptor (TCR) complex components, interfering with TCR signaling leading to reduced T-cell activation. This immunoinhibitory activity is suggested to occur during the membrane fusion process and is attributed to various membranotropic regions of the gp41 ectodomain and to the transmembrane domain. Although extensively studied, the cytosolic region of gp41, termed the cytoplasmic tail (CT), has not been examined in the context of immune suppression. Here we investigated whether the CT inhibits T-cell activation in different T-cell models by utilizing gp41 derived peptides and expressed full gp41 proteins. We found that a conserved region of the CT, termed lentiviral lytic peptide 2 (LLP2), specifically inhibits the activation of mouse, Jurkat and human primary T-cells. This inhibition resulted in reduced T-cell proliferation, gene expression, cytokine secretion and

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cell surface expression of CD69. Differential activation of the TCR signaling cascade revealed that CT based immune suppression occurs downstream of the TCR complex. Moreover, LLP2 peptide treatment of Jurkat and primary human T-cells impaired Akt but not NFκB and ERK1/2 activation, suggesting that immune suppression occurs through the Akt pathway. These findings identify a novel gp41 T-cell suppressive element with a unique inhibitory mechanism able to take place post membrane fusion.

Introduction HIV infection begins with viral entry into host cells (1) promoted by the trimeric envelope glycoprotein gp160, comprising the two subunits gp120 and gp41 (2-5). The gp41 fusion protein consists of the fusion peptide (FP), N- and C-terminal heptad repeats, loop, transmembrane domain (TMD) and cytoplasmic tail (6,7) (Figure 1A) and is responsible for mediating membrane fusion (3,5). Gp41 also inhibits T-cell activation by interfering with T-cell receptor (TCR) complex assembly via its fusion peptide (8), loop (9) and TMD (10,11). Additionally, the loop has also been shown to downregulate T-cell activation by inhibiting protein kinase C (12). The CT of gp41 has 4 distinguishable regions; the Kennedy sequence, followed by the LLPs designated 1, 2 and 3 (Figure 1A) and is involved in many aspects of the viral life cycle. During budding, the CT has been shown to bind the Gag polyprotein that facilitates recruitment of gp41 to the virion budding sites (13-16) by preventing recycling of gp41 from the plasma membrane (17-19). LLP1 truncation has been attributed to diminished envelope incorporation into budding virions and to a lack of infectivity thereof (20-22). In addition, successful membrane fusion is suggested to depend on LLP2 and LLP3 as well. Large truncations, namely up to LLP3 or even the whole cytoplasmic tail, have shown that LLP2 and LLP3 bind the plasma membrane and different sections of the envelope itself, possibly facilitating fusion (22-26). A well-defined role of cytoplasmic tail functionality is to promote clathrin-mediated endocytosis of the envelope. Two distinct motifs, in two separate domains were found to facilitate endocytosis after trafficking to the plasma membrane (27,28). Disruption of the individual motifs affected viral load and pathogenesis and showed strong synergy when both were mutated (27,29). Various CT interacting partners have been described such as calmodulin (30), α-catenin (31), guanine nucleotide exchange factor p115- RhoGEF (32), prenylated Rab acceptor PRA1 (33), transcription factor luman (34), and prohibitin 1 and 2 (35). However, for the most part, the 2 ACS Paragon Plus Environment

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physiological role of these interactions remains unclear. Recently LLP2 has been shown to activate T-cells by inducing nuclear factor κB (NFκB) activation in the cytosol by binding TGF-bActivated Kinase 1, promoting viral replication (36). Yet, no other immune modulating functions of the CT have been reported. T cell activation is a highly-regulated process that integrates different environmental triggers as well as a multitude of intracellular signaling cascades. The mechanistic target of rapamycin (mTOR) is s a key element of this complex system, regulating and coordinating metabolic programs, cell proliferation and anergy (37). mTOR signaling is closely intertwined with the activity of the RAC-alpha serine/threonine-protein kinase (Akt) also termed protein kinase B. Akt activates the mTOR complex by phsophorlyation of the inhibitory Akt1 Substrate 1 (PRAS40) and tuberous sclerosis complex 1 and 2 (TSC-1 and TSC-2) (37). The mTOR protein is an integral and crucial element of two protein complexes, mTORC1 and mTORC2, with distinct regulatory functions. Interestingly, Akt is also a downstream target of the mTORC2 complex thus serving as both, substrate and regulator of mTOR signaling (37).Recently, mTOR was also identified as an important factor of HIV-1 transcription in CD4 T cells (38). In this study we assessed the ability of the HIV gp41 CT to inhibit T-cell activation. We reveal that the LLP2 motif, situated in the CT, suppresses mouse and human T-cell activation. Utilizing gp41 CT derived peptides in T-cell proliferation assays, we show that LLP2 inhibits T-cell activation downstream of the TCR complex resulting in inhibition of cytokine expression and secretion. Furthermore, we show that LLP2 specifically inhibits the phosphorylation of Akt mTOR pathway in human Jurkat and primary peripheral T-cells. For a broader biological relevance we show that when expressed in human T cells, gp41 lacking the LLP2 motif loses much of its inhibitory activity. Overall, our findings suggest that the LLP2 motif of the gp41 CT suppresses T cell activation by inhibiting the Akt pathway. These findings reveal a previously unknown gp41 immune suppressive segment with a distinct inhibitory mechanism.

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Figure 1: Immune suppression by a conserved region of the gp41 CT. A) Scheme of the gp41 domains including the fusion peptide (FP), N and C terminal repeats (NHR and CHR respectively), loop, transmembrane domain (TMD) and cytoplasmic tail (CT) depicting the three lentiviral lytic peptides (LLP1-3). B) A conserved region spanning LLP2. The y-axis depicts the probability of finding the annotated amino acid at that position. X-axis depicts the position relative to the first depicted amino acid. Below, a schematic representation of LLP2-3 (HXB2 strain 762-802 ). LLP2 is underlined and LLP3 is in red lettering. Depicted below are the derived peptides. C-F) MOG35-55–specific line T cells were cultured with irradiated splenocytes as APCs and MOG35-55 in the presence or absence of several HIV peptides. Their proliferative response was measured in an H3-thymidine proliferation assay. The fold change between uninhibited T-cell proliferative responses and background proliferation in the absence of antigen was 28.8 ± 11.2 for B&C and 42± 15 for D. Results presented are the mean percentage inhibition of the proliferative response to MOG35-55 peptide relative to the control (in the absence of HIV peptides). C) The graph shows the average inhibition of proliferation by 10µM of HIV peptides. n≥12. D) Dose dependent inhibition of proliferation of the three active peptides with increasing concentrations of 2.5 µM, 5 µM, and 10 µM (light 4 ACS Paragon Plus Environment

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gray, gray and black, respectively). n= 8. E) Average inhibition of proliferation by 10µM LLP2 and equal length control peptides each comprising half of LLP2 and running either up or downstream of it. n=24. F) Average inhibition of proliferation by 10µM LLP2 and LLP2 – scrambled. n=12 *P< 0.05, **P 95% homogeneity. The molecular weight of the peptides was confirmed by platform LCA electrospray mass spectrometry. The day before administration of peptides into biological assays, peptides were dissolved in 95% TFA that was then evaporated by N2. Next peptides were dissolved in 50% acetonitrile and lyophilized overnight. Before administration of peptides into in-vitro and ex-vivo cell culture experiments, peptides were dissolved in DMSO. Cells were cultured in medium not exceeding 0.5% DMSO concentrations.

In vitro T-cell proliferation and activation Antigen-specific T-cell lines (mouse) were selected in vitro (46) from primed lymph node cells derived from C57Bl/6J mice that had been immunized 9 days before with antigen (100 µg myelin peptide, MOG35-55) emulsified in CFA containing 150 µg Mycobacterium tuberculosis (Mt) H37Ra (Difco Laboratories, Detroit, MI). All T-cell lines were maintained in vitro in medium containing IL-2 with alternate stimulation with the antigen, every 10–14 days. T cells specific to MOG35-55 (mMOG35-55 T cells) were plated onto round 96-well plates in medium containing RPMI-1640 supplemented with 2.5% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM β-mercaptoethanol, and 2mM L-glutamine. Each of the 96 wells had a final volume of 200 μl and contained 20x103 T cells, 5x105 irradiated (25 gray) syngeneic spleen cells as APCs, without or with 5 μg/ml of MOG35-55. In addition, the relevant peptide was added. In


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order to exclude interaction between the examined peptides and the MOG35-55 peptide, initially the MOG35-55 peptide was added to the APCs in a test tube (2 hour incubation), and in a second test tube the examined peptides were added to the T cells (30 min incubation). The same procedure was carried out for the following inhibitors: BAY 11-7082, SP600125 and SB 203580, Santa Cruz Biotechnology, Inc., Wortmannin, mTOR inhibitor XI Torin1, Sigma-Aldrich Inc. and SC66, Biovision Inc. After 2 hours, the APCs were mixed with the T cells and were co-incubated for 48h in a 96-well flat bottom plate. Then the T cells were pulsed with 1μCi (H3) thymidine, with a specific activity of 5.0 Ci/mmol, and after overnight incubation, the (H3) thymidine incorporation was measured using a Matrix 96 Direct Beta Counter (Packard Instrument, Meriden, CT). Additionally, cells were activated with pre-coated CD3 and CD28 antibodies (LEAFTM purified anti mouse clones 145-2-C11 and 37.51, respectively from Biolegend) at a final concentration of 2 µg/ml, or activated with 50 ng/mL of phorbol 12-myristate 13-acetate (PMA) together with 1 μM of ionomycin (Sigma Chemical Co, Israel). In-vitro T-cell activation experiments involving animals were conducted under the approval of the IACUC of the Weizmann Institute, permit numbers: 26980516-3. The facility where this research was conducted is accredited by AAALAC and has an approved Office of Laboratory Animal Welfare (OLAW) Assurance (#A5005-01). The facility operates according to the guide for the care and use of laboratory animals 8th edition by the national research council. All procedures were conducted by trained personnel under the supervision of veterinarians.

In-vitro cytokine secretion Antigen-specific T-cells were plated in 96-well plates in T cell effector medium as used for proliferation assays. Each of the 96 wells had a final volume of 200μl and contained 10x104 Tcells and either 5x105 irradiated spleen cells and 5μg/ml of MOG35-55 peptide or CD3&CD28 antibodies previously bound to the plate. In addition, the relevant peptide was added. Each treatment was made with triplicates. Analysis of IFN-γ (UniProtKB - P01580 mouse and P01579 human) and TNFα (UniProtKB – P06804 mouse and P01375 human) secretion was performed by ELISA 24 hours after cell activation according to standard protocols from R&D systems. RAW264.7 cells (mouse) purchased from ATCC (ATCC®-TIB71™) were cultured overnight in DMEM supplemented with 10% FBS, L-glutamine, sodium pyruvate, non-essential amino acids, and antibiotics in a 96-well plate (105 cells/ well). The following day, media was replaced with 6 ACS Paragon Plus Environment

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fresh DMEM including all the supplements. Cells were incubated with the peptide for 2 hours, washed and incubated with fresh media containing Pam3CSK4 for 5 hours and then analyzed for TNFα secretion according to standard protocols from R&D systems.

Confocal analysis of peptide localization Jurkat E6 cells were incubated with 1μM of the rhodamine peptides for 2 hours. Next, nuclear staining was performed by with by adding 0.5μl Hoechst 33342 H3570, life technologies, per 1ml of 1X10^6 cells for 10 min followed by two washing steps. Cells were fixed in 4% paraformaldehyde for 15 min. Next 10μl cells were seeded with vectashield H-1000, vector laboratories, on a poly-L- lysin coated glass cover slips and closed with a cover slip. Images were acquired via the Olympus FV1000 confocal microscope as optical sections with a ΔZ resolution of 3 micron and the optical section was 1.2 micron.

RNA isolation and quantitative real time PCR (qRT-PCR) Total RNA from cells was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany) a 2μg aliquot of the total RNA was reverse transcribed into cDNA using Bio-RT (BioLab, Jerusalem, Israel), dNTPs and random hexamer primers. qRT-PCR was performed on Step One Plus, ABI instrument (Applied Biosystems, Grand Island, NY, USA) using SYBR Green PCR Master Mix (Quanta BioSciences, Gaithersburg, MD, USA). The values for the specific genes were normalized to α-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (human) or Rpl13a (mouse) as housekeeping controls and the data are described in arbitrary units. PCR reactions were performed in duplicate. The specific primers used for qRT-PCR are available on request. NFκB activation in 5A8 cells. A sub-clone of the J Lat cell line 5A8 (human) that features a stable expression of kB-DsRed2 (56) was used to assess NFκB (UniProtKB - P19838activation). J Lat cell lines 5A8 were generated in the lab of Warner C Greene, Gladstone Institute of Virology and Immunology, San Francisco, CA 94158, USA, from Jurkat cells obtained from American Type Culture Collection. 0.5x106 cells were plated in conical bottom 96 well plates in 0.1 ml media and activated with CD3&CD28 beads. Peptides were added in parallel at a concentration of 10 μM. After 24 h, cells were fixed and subjected to flow cytometry. A detailed gating strategy is described in the SI. The percentages of 7 ACS Paragon Plus Environment

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RFP positive cells from peptide treated, activated samples were normalized to the immunosuppression negative control sample, treated with CD3&CD28 beads and the solvent DMSO.

Western Blot analysis Cell pellets were lysed in RIPA buffer as previously described (9) together with protease and phosphatase inhibitors (Sigma Chemical Co., Israel). Supernatants were analyzed for protein concentration using Pierce BCA reagent kit. 10µg of protein were boiled and loaded onto a 12% SDS-polyacrylamide gel. Gels were transferred to Polyvinylidene difluoride (PVDF) membranes using wet transfer 30v overnight. The immunoblot was incubated overnight with blocking solution (2% BSA, 0.05% Tween-20 in Tris-buffered saline TBST for detection of phosphorylated proteins or Dulbecco’s phosphate buffered saline PBST for detection of non-phosphorylated proteins). The following primary antibodies were used: α-phospho extracellular signal–regulated kinases 1/2 (ERK1/2 - UniProtKB - P27361/ P28482) #4370 cell signaling, α-phospho protein kinase B/Akt (UniProtKB - P31749) #4051 cell signaling, α-phospho S6 (UniProtKB - P62753) #2215 cell signaling, α-ERK1/2 sigma M5670, α-Akt p160 sigma, α-S6 #2217 cell signaling, α-tubulin A11126 ThermoFisher Scientific, α-GAPDH MAB374 Millipore. The protein–antibody complexes were detected using goat anti-mouse or goat anti-rabbit horseradish peroxidaseconjugated secondary antibodies (Jackson Immuno Research Laboratories INC.) that were applied for 2h at room temperature. The blots treated were developed on an enhanced chemiluminescence (ECL) detection system using LuminataTM, Merck.

Purification of primary human peripheral T-cells Human peripheral T lymphocytes were isolated from whole blood of healthy adult donors that provided written informed consent under the regulations and authorization of the Weizmann Institutional Review Board, Project 247-2. T lymphocytes were isolated by dextran sedimentation and Ficoll (Sigma) gradient separation followed by depletion of B cells using nylon wool column (Unisorb). Cells were incubated in a complete RPMI growth medium for a minimum of 2 h, and then the non-adherent cells T-cells were harvested and transferred to a new plate with the T-cell growth media: 450 ml RPMI-1640, 10% FCS, 2 mM L-Glutamine, 1 mM final Na pyruvate,


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50,000 units penicillin and 50mg streptomycin. Activated T-cells were grown in filtered T cell media supplemented with 2mM β mercaptoethanol.

XTT cytotoxicity assay E6-1 Jurkat T –cells (human) were provided by the NIH AIDS Research and Reference Reagent Program (cat 177, lot 10040153). Aliquots of 10 x 104 cells of were distributed onto a 96-well plate in the presence of peptides for 4 hours. Wells in the last two columns served as blank (medium only), and 100% survival controls (cells and medium only). After incubation, the XTT reaction solution (benzene sulfonic acid hydrate and N-methyl dibenzopyrazine methyl sulfate, mixed in a proportion of 50:1), was added for 2 more hours. Optical density was read at a 450-nm wavelength in an enzyme-linked immunoabsorbent assay plate reader. For mMOG35-55 T cells and primary peripheral human T-cells, incubation with the peptides was for 16 hours, then XTT reaction solution was added and optical density was read every hour for a period of 12 hours. The time point with the biggest difference between blank and 100% survival control was chosen for analysis. The percentage of toxicity was calculated relative to the control; 2.5 x 104 cells in medium with no peptide added.

Construct preparation YFP-gp41 variants Fluorescently labeled protein constructs were generate by molecular cloning as previously described (74). Briefly, gp41 (UniProtKB - P04578) was amplified via preparative PCR from an HXB2 envelope expression plasmid obtained from the NIH AIDS reagent program, originally provided by Dr. Kathleen Page and Dr. Dan Littman (AIDS reagent catalogue number: 1069). All gp41 variants were inserted by restriction-based cloning into an in-house pmYFP-N1 vector, containing an ER-targeting signal peptide at the N-terminus of the monomeric (A206K) yellow fluorescent protein (YFP- UniProtKB - A0A059PIR9). The protein of interest was introduced at the C-terminus of the YFP, yielding gp41 constructs that are fluorescently tagged at the protein’s ectodomain. A gp41-CD8 hybrid protein, in which the physiological gp41 transmembrane domain (TMD) was replaced with a CD8 TMD, was generated by ligation-free cloning (Quickchange). In addition, the loss-of-function mutation Y712A2 was introduced in the gp41 internalization motif 712YSPL715 by ligation-free cloning, in order to obtain gp41 constructs with a significantly elevated surface expression. Truncation variants of hybrid gp41 constructs were generated by 9 ACS Paragon Plus Environment

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restriction-based cloning, using suited forward and reverse primers. The original pmYFP-N1 vector without a membrane-targeting signal peptide was used as a negative control for fluorescence-based experiments. Plasmids and sequences are available upon request.

YFP-gp41 expression and effect on CD69 expression Jurkat T-cells were harvested and suspended in in Optimem (Thermo: 31985070) 3X106per 100µl medium and mixed with 30µg DNA. Cells were then electroporated in cuvettes using the NEPA21 super electroporator (NEPAGENE). Impedance by was assessed before each electroporation and continued if units were between 30 to50. The poring pulse was 150V, 5 ms length, 50 ms intervals, 2 pulses , 10D rate and + polarity. The transfer pulse was 20V, 50 ms length, 50 ms intervals, 5 pulses, 40D rate and +/- polarity. Following electroporation 1ml new medium was added into the cuvette, mixed and transferred to 24 well plates. Cells were incubated 24h at 37°C for recovery. Next, Cells were activated by anti CD3 and CD28 antibodies. After 20h the cells were harvested and fixated in 4% paraformaldehyde then stained with PE-Cyanine7 (PE-Cy7) labeled anti CD69 (molecular probes A16361). CD69 expression was assessed via FACS analysis. To assess gp41 surface expression, YFP-gp41 expressing live cells were stained with mouse α-YFP MA5-15256 pierce, and αmouse Cy5. YFP and Cy5 mean fluorescence intensity was assessed via FACS.

Statistical analysis Differences between group means were tested using one-way analysis of variance (ANOVA) followed by a Tukey post hoc test unless stated otherwise. P< 0.05 was considered significant. Analyses were done using GraphPad Prism (data analysis software) version 6.05. (*P≤0.05, **P≤0.01, ***P≤0.001). Results are displayed as mean ±SEM.

Results The gp41 cytoplasmic tail contains a conserved immunosuppressive sequence The CT influences multiple functions of gp41, yet T-cell immune suppression has only been reported to be facilitated by the gp41 ectodomain and TMD (reviewed in (39)). Here we examined whether the CT plays a role in T-cell immune suppression. To identify regions of interest we utilized the Los Alamos HIV Sequence Database (http://www.hiv.lanl.gov/) AnalyzeAlign tool with the HIV-1/SIVcpz filtered web database. The results show a high degree of conservation in 10 ACS Paragon Plus Environment

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the CT including the kennedy sequence, LLP2, LLP3 and LLP1 (Figure S1A). As gp41 immune suppression was reported to occur in the membrane (8,11,40), we focused on membranotropic LLP regions (17-19,41), specifically on the LLP2-3 region previously shown to bind the membrane (22-25). To this aim we evaluated LLP2-3 conservation in validated HIV-1 gp41 CT sequences acquired via PERL from Uniprot (42). Next, the sequences were run through MUSCLE EMBLEB (43) for sequence alignment and run through WebLogo3 for conservation analysis (44,45) (Figure S1C). A conserved motif overlapping the LLP2 to LLP3 regions emerged as highly conserved with minimal insertions (Figure 1B and S1). Subsequently, overlapping peptides derived from the LLP2-3 region (HXB2 strain) (1 and Figure 1B) were synthesized and examined for immune modulatory activity (Figure 1C). To this aim, proliferation was evaluated on a MOG(35–55)-specific murine T-cell line [mMOG-(35–55) T-cells] (46). This T-cell line enables T-cell activation by antigen presentation through the TCR, consequently resulting in proliferation as previously described (9).

We found that segments LLP2 and LLP2-3 strongly suppressed T-cell proliferation (Figure 1C) and were not toxic at inhibitory levels (Figure S2). The peptides LLP2t and LLP3N, each pertaining half of LLP2, did not show strong suppression of T-cell proliferation. When mixed together, T-cell suppression was once again obtained (Figure 1C). Furthermore, LLP2-3, LLP2 and LLP2t+LLP3N all exhibited dose dependent inhibition of T-cell proliferation (Figure 1D). It is possible that the two LLP2 halves did not immune suppress alone due to their shorter length relative to LLP2. Therefore two peptides pertaining half of LLP2 but with equal length and spanning either upstream (LLP2N) or downstream (LLP2C) were synthesized and evaluated for immune suppressive properties. Both peptides suppressed T-cell proliferation, though significantly lower than LLP2 (Figure 1E). To further assess LLP2 sequence specificity we utilized a scrambled LLP2 peptide, consisting of the same amino acid length and composition, yet differing from the WT LLP2 in sequence (Table 1). The scrambled LLP2 exhibited a drastically reduced immune suppressive activity (Figure 1F). Overall these results suggest that the full LLP2 region is required to permit immune suppressive activity.


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Table 1: Peptide designation and sequence

a b

Peptide designation

Sequencea, b

LLP2-3 LLP2 LLP2N LLP2C LLP2t LLP3N LLP3C Control Peptide T-20 LLP2-scram LLP2allD

YHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQY YHRLRDLLLIVTRIVELL SLCLFSYHRLRDLLLIVT LLLIVTRIVELLGRRGWE YHRLRDLLLIVT RIVELLGRRGWE EALKYWWNLLQY SNKSLEQIWNHTTWMEWD YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF ILRELTVILYLRDVLHLR YHRLRDLLLIVTRIVELL

Sequences are derived from the HXB2 strain underlined amino acids denote D-enantiomers

LLP2 inhibits T-cell proliferation downstream of the TCR complex To further examine LLP2-3’s mode of action we assessed at what level of the TCR signaling cascade the peptide operates. Apart from antigen presenting cell (APC) based activation, T cells can be activated in-vitro either through CD3ε and CD28 (CD3&CD28) antibodies or the PKC activator phorbol 12-myristate 13-acetate (PMA) together with ionomycin, a Ca2+ ionophore. AntiCD3&CD28 only bypasses the TCRα and β subunits and PMA/ionomycin bypasses the entire TCR complex (9,11). However, stimulation by anti- CD3&CD28 is weaker than APC based stimulation. This results in low levels of proliferation, making this type of readout insufficient (9). This is overcome by assessing cytokine secretion levels, such as IFNγ; a hallmark of Th1 activation (reviewed in (47)). LLP2-3 significantly reduced IFNγ secretion levels upon CD3&CD28 activation (Figure 2A). This result suggests that the peptide does not act at the TCR level. To test whether LLP2-3 operates downstream of the TCR complex level, T-cells were activated with PMA/ionomycin, treated with LLP2-3 and their proliferation was determined as described above. LLP2-3 strongly inhibited the proliferation of PMA/ionomycin activated T-cells (Figure 2B). We further examined LLP2 and found that it retains the ability of LLP2-3 to inhibit PMA/ionomycin activated T cells (Figure 2C) suggesting that immune suppression by LLP2 occurs downstream of the TCR complex.


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Biochemistry

Figure 2: LLP2-3 and LLP2 inhibit T cells that are activated downstream of the TCR complex. A) LLP2-3 significantly reduced IFNγ secretion levels in MOG35-55 T cells following activation via antibodies against CD3&CD28. IFNγ levels were assessed via an ELISA following treatment of 10µM LLP2-3.n=9. ***P≤0.001. Results are displayed as mean ±SEM. B&C) LLP2-3 (B) and LLP2 (C) inhibit the proliferation of T-cells treated with PMA/ionomycin . MOG35-55–specific line T cells were treated with PMA/ionomycin or incubated with APCs and MOG and the extent of proliferation was determined via H3 thymidine uptake as described above. The fold change between uninhibited T-cell proliferative responses and background proliferation for LLP2-3 experiments was 563.2 ± 34 for PMA/ionomycin and 21.6 ± 4 for APCs and MOG and for LLP2 experiments 53.7 ± 8.7 for PMA/ionomycin and 58.4 ± 13.2 for APCs and MOG. Results presented are the mean percentage inhibition of the proliferative response to either MOG35-55 peptide or PMA/ionomycin relative to the control (in the absence of HIV peptides). n≥8. The LLP2 peptide penetrates cells and does not function in the membrane The gp41 CT is primarily situated either in the cytosol or bound to the inner leaflet of the plasma membrane (reviewed in (48)). As the LLP2 peptide is added exogenically, we evaluated peptide localization in Jurkat T-cells by confocal microscopy. We utilized rhodamine labeled LLP2 (rhoLLP2), and LLP2N (rho-LLP2N) as a control. Both peptides were observed primarily inside the cell (Figure 3A and B and Figure S3) showing that the peptides can enter and possibly exert their


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activity in the cytoplasm. Rhodamine did not affect peptide activity (Figure 3C) and rhodamine labeled peptides were not toxic at inhibitory levels (Figure S2). To evaluate whether the LLP2 operates in either the membrane or aqueous environment, we utilized an all D-enantiomer LLP2 (LLP2allD) (Table 1) as interactions of peptides and proteins in the membrane have been shown to be chirality independent (49-51). LLP2allD could not induce immune suppression in activated mMOG-(35–55) T-cells similarly to LLP2N (Figure 3D). This finding suggests that LLP2 exerts its activity in an aqueous environment.


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Biochemistry

Figure 3: LLP2 exerts its activity from within the cell. A and B) Representative images of rhodamine labeled LLP2N (A) and LLP2 (B) localization in Jurkat T-cells. LLP2N (A) and LLP2 (B) both penetrate the cells. Hoechst 33342 was used as a nuclear stain. Images were taken with the Olympus FV1000 confocal microscope. Upper panels are an enlargement of the marked region in the bottom panels. C and D) MOG3555–specific line T cells were cultured with irradiated splenocytes as APCs and MOG35-55 in the presence or absence of several HIV peptides. Their proliferative response was measured in an H3-thymidine proliferation assay. The fold change between uninhibited T-cell proliferative responses and background proliferation in the absence of antigen was 12.5 ± 1.2 for C and 83.1 ± 4.2 for D. Results presented are the mean percentage inhibition of the proliferative response to MOG35-55 peptide relative to the control (in the absence of HIV peptides). 10µM of HIV peptides were used. n≥12. C) Average inhibition of proliferation by rhodamine labeled LLP2 and LLP2N. Rho-LLP2 retains its activity. n= 12. D) Average inhibition of proliferation by LLP2allD, LLP2 and LLP2N. LLP2allD does not inhibit T-cell proliferation. n=8. ***P< 0.001. Results are displayed as mean ±SEM.

Gene expression and cytokine secretion is inhibited by LLP2 We continued our studies on the shortest active segment, i.e. LLP2, and assessed the extent of inhibition by measuring cytokine expression and secretion. We focused on relative mRNA expression via qRT-PCR of Th1 and Th2 genes as well as common genes expressed during T-cell activation (52,53) following LLP2 treatment. The genes tested 18 hours after activation were i) IFNG, TNFA and lymphotoxin alpha (LTA), all are elevated in Th1 activation (47), ii) signal transducer and activator of transcription 4 (STAT4), a transcription factor responsible for IFNγ expression (54), iii) interleukin-4 (IL4) and interleukin-10 (IL10), both elevated in Th2 activation and iv) interleukin-16 (IL16), a chemokine common to T-cells but found to be expressed in Th2 activation (52,53). The results show that LLP2 treatment reduces gene expression of Th1 related genes (Figure 4A). We further assessed whether the changes at the transcription level were also apparent at the protein level. We tested secretion of the Th1 cytokines TNFα and IFNγ and found that both were significantly reduced following LLP2 treatment (Figure 4B and C), supporting the qRT-PCR analysis. To assess specificity of LLP2, we tested its inhibitory effect on cultured RAW264.7 macrophages activated by the TLR1/2 ligand PAM3CSK. LLP2 had no effect on TNFα secretion by RAW264.7 cells (Figure 4D). Taken together, these results show that inhibition of T-cells by LLP2 results in a reduction of TCR complex induced gene expression and cytokine secretion. 15 ACS Paragon Plus Environment

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Figure 4: LLP2 inhibits cytokine expression and secretion. A) LLP2 inhibits mRNA expression of Th1 cytokine genes following TRC activation. qRT-PCR analysis of IFNG, TNFA, STAT4, LTA, IL4, IL10 and IL16 following treatment of 10µM LLP2. n=4. *P< 0.05, **P≤0.01. Results are displayed as mean ±SEM. B,C and D) LLP2 specifically inhibits cytokine secretion in activated T-cells. Cytokine levels were assessed via ELISA following treatment of 10µM LLP2. N=3, n=3. *P< 0.05, **P≤0.01. Results are displayed as mean ±SEM. B and C) LLP2 significantly reduced TNFα (B) and IFNγ (C) secretion levels in T cells. MOG35-55–specific line T cells were cultured with irradiated splenocytes as APCs and MOG35-55 in the presence or absence of LLP2. D) PAM3CSK activated RAW264.7 cells were not affected by LLP2 as seen by no change in TNFα secretion

LLP2 inhibits AKT activation in Jurkat and primary human T-cells Our previous results suggest that LLP2 inhibits antigen specific TCR activation downstream of the TCR complex. Activation via the TCR complex results in the initiation of multiple signal transduction pathways (reviewed in (55)). In order to assess what pathways might be inhibited by 16 ACS Paragon Plus Environment

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Biochemistry

LLP2 we initially observed T-cell gene expression of transcription factors whose expression is elevated under TCR activation. As HIV is a human pathogen, to achieve higher biological relevance this was performed on the human Jurkat E6 T-cell line. Jurkat E6 T-cells were activated via CD3&CD28 antibodies for 4 hours and gene expression was evaluated via qRT-PCR (Figure 5A). Significant reduction of gene expression following LLP2 administration was observed for nuclear factor of activated T-cells (NFAT) C1 transcription factor. JUN was reduced, though not significantly, while ATF2, NFATC2 and C-fos were not affected. LLP2 was not toxic at inhibitory levels (Figure S4). Overall, no clear pattern pertaining to a specific pathway was observed. Next, we treated activated mMOG-(35–55) T-cells with inhibitors of pathways induced by the TCR and followed cell proliferation (Figure 5B). Inhibition of phosphoinositide 3-kinase (PI3K) by wortmannin as well as specific inhibition of the PI3K target Akt resulted in a dose dependent reduced proliferation (Figure S5A), as did the specific inhibition of Akt- mTOR and NFκB. JNK1/2 inhibition had no effect and p38 inhibition resulted in a dose dependent increase of proliferation (Figure 5B and Figure S5A). We further investigated proliferation induction by the p38 inhibitor by combining it with either NFκB or mTOR inhibitors. We found that both NFκB and mTOR inhibition sufficiently blocks proliferation induced by the p38 inhibitor (Figure S5B). Compounds were not toxic at inhibitory levels (Figure S6).


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Figure 5: The Akt pathway is inhibited by LLP2. A) LLP2 inhibits mRNA expression of the NFTC1 transcription factor following CD3&CD28 activation in human Jurkat cells. qRT-PCR analysis of NFATC1, NFATC2, ATF2, C-fos and C-jun following treatment of 10µM LLP2. n=3. *P< 0.05. Results are displayed as mean ±SEM. B) Inhibition of PI3K-AKT-mTOR and NFκB pathways results in attenuated Tcell proliferation. Results represent highest does used of each inhibitor. Detailed does dependency and inhibitor designation can be found in Figure S. MOG35-55–specific line T cells were cultured with irradiated splenocytes as APCs and MOG35-55 in the presence or absence of specific inhibitors Their proliferative response was measured in an H3-thymidine proliferation assay. The fold change between uninhibited T-cell proliferative responses and background proliferation in the absence of antigen was 76 ± 5.8. Results presented are the mean percentage inhibition of the proliferative response to MOG35-55 peptide relative to the control (in the absence of inhibitors and peptide). n≥8. Results are displayed as mean 18 ACS Paragon Plus Environment

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Biochemistry

±SEM. C) Inhibition of the NF𝛋B pathway was probed with 5A8 cells, transduced with kB-DsRed2. Cells were activated with CD3/CD28 beads in presence and absence of different peptides and the percentage of RFP positive cells was assessed by flow cytometry. n=5. **P< 0.01 Results are displayed as mean ±SEM D) Phosphorylation inhibition by LLP2 of Akt and S6 but not ERK1/2 in Jurkat T-cells. IκBα was not affected by either CD3&CD28 activation or LLP2. Representative western blots following treatment of either 10µM LLP2 or LLP2N. Either GAPDH, α-tubulin or S6 were used as loading controls for the phosphorylated proteins. Peptides were added to Jurkat T-cells and they were immediately activated by antibodies against CD3 and CD28 for the indicated times. n≥2.

These results suggest that NFκB and Akt/mTOR might be the pathways targeted by LLP2. To investigate this possibility, we evaluated the effect of LLP2 on NFκB activation via FACS analysis using Jurkat-derived sub-cell line 5A8 that expresses a red fluorescent kB reporter controlled by a tandem NFkB consensus sequences when activated (56) (Figure S7). As controls we used LLP2N and a control peptide derived from gp41 previously shown to not inhibit T-cells activated by CD3&CD28 antibodies (9) as well as T-20, a drug used in the clinic, shown to not be immune suppressive (57). Reduction in RFP expression was slightly elevated following treatment by LLP2 when compared to the control peptide (Figure 5C). The results suggest that LLP2 does not inhibit NFκB activation. To assess Akt-mTOR we activated Jurkat E6 T-cells via CD3&CD28 and evaluated the phosphorylated state of Akt and S6, a small ribosomal subunit that is directly downstream to mTOR (58-60), following the administration of LLP2 and LLP2N (Figure 5D). CD3&CD28 activation induces an increase in phosphorylated Akt and S6 observed at five minutes, though intrinsic phosphorylation is apparent without activation (time 0). LLP2 administration resulted in a decrease of Akt and S6 phosphorylation. LLP2N did not inhibit phosphorylation of both proteins to the extent of LLP2. To evaluate the specificity of the LLP2 effect on Akt and S6 we checked the phosphorylation of ERK1/2, both directly upstream of c-fos whose expression was not affected by LLP2 (Figure 5A). ERK1/2 phosphorylation was not affected by either LLP2 or LLP2N (Figure 5D). In addition, following peptide treatment we evaluated the degradation of IκBα, an inhibitor of NFkB that is degraded upon activation (Figure 5D). No degradation was observed following activation. Therefore, we assessed the phosphorylation of IκBα revealing that IκBα is intrinsically phosphorylated in Jurkat T-cells. Taken together, the results reveal that the activation of the Akt pathway is inhibited by LLP2 and that IκBα cannot be assessed in Jurkat T-cells. 19 ACS Paragon Plus Environment

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For further biological relevance we administered LLP2 and LLP2N to anti CD3&CD28 activated human primary peripheral T-cells and assessed cytokine secretion as well as Akt phosphorylation and IκBα degradation (Figure 6). LLP2 treatment resulted in a strong reduction in IFNγ and TNFα secretion in comparison to LLP2N that only slightly reduced TNFα levels significantly (Figure 6A and B). LLP2 was not toxic at inhibitory levels (Figure S8). Western blotting revealed that Akt phosphorylation was reduced upon LLP2 administration compared to non-treated and LLP2N, similarly to Jurkat T-cells (Figure 6C). In contrast, IκBα degradation was not affected by either LLP2 or LLP2N (Figure 6C) in line with the results obtained using the red fluorescent kB reporter system (Figure 5C). Overall, the results suggest that LLP2 specifically inhibits the Akt pathway in both human Jurkat and primary peripheral T-cells.


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Biochemistry

Figure 6: LLP2 inhibits activation of primary human T-cells. A, B and C) Human peripheral T lymphocytes were isolated from whole blood and activated using CD3&CD28 antibodies, in the presence of LLP2 and LLP2N at 10μM. A and B) LLP2 specifically inhibits IFNγ and TNFα secretion. IFNγ (A) and TNFα (B) levels were assessed via ELISA following peptide treatment. 3 donors, 4 repeats per donor. *P< 0.05, ***P≤0.001. Results are displayed as mean ±SEM. C) LLP2 inhibits phosphorylation of Akt but not degradation of IκBα. Representative western blots of T-cells that were activated by antibodies against CD3 and CD28 for the indicated times. GAPDH was used as a loading control. n≥2.

Gp41 constructs reveal that the LLP2 motif specifically inhibits T-cell activation To examine the physiological relevance of inhibition by the LLP2 peptide, full gp41 constructs with and without the LLP2 motif were expressed in activated Jurkat E6 T-cells. YFP-Gp41 constructs (Figure 7A and Table S1) were introduced into T-cells via electroporation, activated by CD3&CD28 antibodies and the expression of the early T-cell activation marker CD69 was followed by flow cytometry (Figure 7 and Figure S9A). The T-cell activation marker CD25 was not evaluated as its expression levels did not alter following antibody activation (Figure S9B). YFP-gp41 expression levels and membrane surface expression were similar between gp41 constructs (Figure S10 and S11 respectively). To focus on inhibition by the gp41 CT we replaced the gp41 TMD with a CD8 TMD (termed gp41-full) (Figure 7A) as the gp41 TMD has been shown to inhibit CD3&CD28 induced activation in T-cells (10,11). CD69 expression was observed in non-activated, YFP-only expressing cells (Figure 7). This is possibly due to the electroporation as previously shown (61). To minimize the sampling error due to nonspecific induction of CD69 expression we utilized three gating strategies: i) CD69 positive, considered CD69 expression over non stained cells (Figure 7D), ii) high CD69, considered CD69 expression over the non-activated sample (Figure 7D and S12A), iii) Low CD69, the amount of total CD69 expressing cells minus the amount of High CD69 expressing cells (Figure 7D, S12B and C). CD69 expression was normalized to activated T-cells expressing YFP only. Results are summarized in Figure 7 B-D and Figure S12. In short, we found that gp41-full inhibited CD69 expression. Mutation in the YSPL internalization motif (28,62-64) slightly increased the inhibition of high CD69 expression but did not significantly affect total CD69 expression. Strikingly, CT truncation up to LLP2 (gp41-LLP2) resulted in a decrease in CD69 expression. Further, truncation of LLP2 (gp41-ΔLLP2) resulted in an increase of up to 30% in CD69 expression compared to gp41-LLP2. When the entire CT was


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truncated, CD69 expression was similar to ΔLLP2. Taken together, these results reveal LLP2 as an inhibitor of T-cell activation in the context of the full gp41 protein.


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Biochemistry


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Figure 7: CD69 inhibition by Gp41 variants expressed in CD3/CD28 activated Jurkat E6 T-cells. A) Scheme representing gp41 constructs used in this study. YFP was added to the N-terminus of each construct. All gp41 TMD sequences were replaced with CD8 TMD and additional modifications and truncations are shown. B and C) representatives flow charts and histograms. B-D) Jurkat E6 T-cells expressing YFP tagged gp41, stained with a PE-Cy7 tagged antibody against CD69. CD69 positive is considered PE-Cy7 signal over non stained, High CD69 is considered as PE-Cy7 signal over non activated sample and Low CD69 is the amount of total CD69 expressing cells minus the amount of High CD69 expressing cells. B) Flow charts of CD69 expression in cells expressing YFP gp41 variants. Percentages pertain to percentage of YFP expressing cells. C) Deletion of LLP2 results in an increase of CD69 expression pertaining to a loss of inhibition. CD69 expression represented as a histogram of YFP positive cells with stated gp41 variants. Count is normalized. D) Summary of mean CD69 expression in gp41 expressing T-cells. Red and blue depict the amount of cells expressing high and low CD69 out of the total CD69 expressing cells, respectively. Specific percentages are noted. Error bars pertain to total CD69 only. n=3. *P

The HIV gp41 Fusion Protein Inhibits T-Cell Activation through the

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