An Asymmetric Opening of HIV-1 Envelope Mediates Antibody-Dependent Cellular Cytotoxicity.

Contact for Reagent and Resource Sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, James B. Munro ( james.munro@tufts.edu ).

Experimental Model and Subject Details Ethics statement

Written informed consent was obtained from all study participants [the Montreal Primary HIV Infection Cohort ( Fontaine et al., 2011 ; Fontaine et al., 2009 ) and the Canadian Cohort of HIV Infected Slow Progressors ( International et al., 2010 ; Kamya et al., 2011 ; Peretz et al., 2007 )], and research adhered to the ethical guidelines of CRCHUM and was reviewed and approved by the CRCHUM institutional review board (ethics committee, approval number CE 16.164 - CA). Research adhered to the standards indicated by the Declaration of Helsinki. All participants were adult and provided informed written consent prior to enrolment in accordance with Institutional Review Board approval.

Cell lines and isolation of primary cells

HEK293T human embryonic kidney cells (obtained from ATCC) were grown as previously described ( Richard et al., 2015 ; Veillette et al., 2014b ). HEK293T cells were derived from female human embryonic kidney cells, into which the simian virus 40 T-antigen was inserted. Primary human PBMCs, and CD4+ T cells were isolated, activated and cultured as previously described ( Richard et al., 2015 ; Veillette et al., 2014b ). Briefly, PBMCs were obtained by leukapheresis from 7 HIV-uninfected healthy adult (6 males, 1 female) donors. CD4+ T lymphocytes were purified from resting PBMCs by a negative selection using immunomagnetic beads per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC). CD4+ T lymphocytes were activated with phytohemagglutinin-L (10 μg/ mL) for 48 hours and then maintained in RPMI 1640 complete medium supplemented with rIL-2 (100 U/mL). Primary CD4 T cells were isolated from PBMCs obtained from 7 viremic untreated HIV-1-infected individuals (5 males, 2 females). Purified CD4+ T cells were activated with PHA-L at 10 μg /ml for 36 hours and then cultured for 6 days in RPMI-1640 complete medium supplemented with rIL-2 (100U/ml). Sera Sera from nine deidentified untreated HIV-infected (8 males, 1 female) and three HIV-uninfected healthy adult male donors were heat-inactivated and conserved as previously described ( Richard et al., 2015 ; Veillette et al., 2014b ).

Method Details Plasmids and site-directed mutagenesis

The sequence of full-length clade B HIV-1 JRFL Env ( Wu et al., 2006 ) was codon-optimized (GenScript) and cloned into the expression plasmid pcDNA3.1 or PCGGS. Site-directed mutagenesis was performed using the QuikChange II XL site-directed mutagenesis protocol (Stratagene), and a stop codon was introduced to replace the codon for Gly 711, truncating the cytoplasmic tail (ΔCT) and enhancing cell-surface expression of selected HIV-1 JRFL Env. Point mutations were introduced at positions R508S and R511S to allow changes on the gp120-gp41 cleavage site (Bosch et al., 1990). Mutations were either added individually or in combination into pcDNA3.1-JRFL Env or the PCGGS-JRFL Env. The presence of the desired mutations was determined by automated DNA sequencing. The numbering of the HIV-1 Env amino acid residues is based on that of the prototypic HXBc2 strain of HIV-1, where position 1 is the initial methionine ( Korber, 1998 ). Viral production, infections, ex vivo amplification and detection of infected cells HIV-1 viruses were produced and titrated as previously described ( Veillette et al., 2015 ). Viruses were then used to infect activated primary CD4 T cells by spin infection at 800 × g for 1 h in 96-well plates at 25 °C. In order to expand endogenously-i nfected CD4 T cells, primary CD4 T cells were isolated from PBMCs from HIV-1-infected individuals. Purified CD4+ T cells were activated with PHA-L at 10 μg/ml for 36 hours and then cultured for 6 to 8 days in RPMI-1640 complete medium supplemented with rIL-2 (100 U/ml) ( Richard et al., 2015 ).

Antibodies Anti-HIV-1 cluster

A monoclonal antibodies A32, and C11 were either conjugated with Alexa-Flour 647 probe (Invitrogen) or not and used for cell-surface staining of HIV-1 Env expressing cells. The following Abs were used alone or in combination with anti-cluster A mAbs for cell-surface staining: A32, C11, 17b, 19b, GE2JG8, N12-i2, (1μg/ml) and the Fab fragments, Fab’2 fragments or full 17b (5μg/ml) mAb.. The monoclonal anti-CD4 OKT4 (1μg/ml) (14–0048-82; eBiosciences) was used to measure cell surface levels of CD4, as previously shown ( Richard et al., 2015 ). The secondary goat anti-mouse and anti-human antibodies coupled to Alexa Fluor 647 (Invitrogen) were used as secondary Abs. smFRET imaging HIV-1 JR-FL virions with a single peptide-tagged gp120 domain were generated, purified, and fluorescently labeled as described ( Munro et al., 2014 ). Briefly, HEK293T cells were transfected with pNL4–3 ΔEnv ΔRT plasmid, and a 20:1 ratio of wild-type HIV-1 JR-FL gp160 plasmid to gp160 with the V1-Q3 and V4-A1 peptide insertions. Virus was collected 24 hours post-transfection and pelleted in PBS over a 5% sucrose cushion at 20,000× g for 2 hours. The virus was resuspended in labeling buffer (50 mM HEPES pH7, 10 mM CaCl 2 , 10 mM MgCl 2 ), and incubated overnight at room temperature with 0.5 μM each of LD550-cadaverine and LD650-coenzyme A (Lumidyne Technologies), 0.65 μM transglutaminase (Sigma), and 5 μM acyl carrier protein synthase (AcpS), which labels the Q3 and A1 peptides, respectively. The virus was then incubated with 0.02 mg/ml DSPE-PEG 2000 -biotin (Avanti Polar Lipids) for 30 minutes at room temperature with gentle mixing. Virus was purified away from unbound fluorophore and lipid by ultracentrifugation over a 5–20% Optiprep (Sigma) gradient in an SW41 rotor (Beckman Coulter) for 1 hour at 35,000 rpms. The gradients were fractionated and the fractions containing labeled HIV-1 JR-FL virions were identified by p24 Western blot, pooled, and stored at −80°C until use in imaging experiments.

Virus preparations with HIV-1 JR-FL Env ΔCT or Env

CL-, or with HIV-1 BG505 Env were generated in exactly the same manner. The purified labeled virions were immobilized on passivated, streptavidin-coated quartz microscope slides. smFRET imaging was performed on a custom-built prism-based TIRF microscope controlled by Micro-Manager software (Open Imaging) as described ( Das et al., 2018 ). All ligands were introduced to the labeled virus and incubated for 1 hour at room temperature prior to surface immobilization.

Flow cytometry analysis of cell-surface staining

To assess the Env conformation of wildtype codon-optimized HIV-1 JR-FL Env by flow cytometry analysis, 3 × 10 5 2 93T cells were transfected by the calcium phosphate method with the Env-expressing plasmids along with a pIRES-GFP vector, at a ratio of 2 μg of pcDNA3.1 or JRFL ΔCT Env variants to 0.5 μg of green fluorescence protein (GFP). Sixteen hours post-transfection, cells were washed with fresh medium and epitope exposure was evaluated 24 h later. The recombinant Alexa-Fluor-conjugated C34-Ig protein ( Si et al., 2004 ) was used to detect HR1 exposure in the presence or absence of sCD4 (10μg/ml), as previously shown ( Pacheco et al., 2017 ). Cell-surface staining was performed as previously described ( Richard et al., 2015 ; Veillette et al., 2015 ). Binding of HIV-1-Env expressing cells by sera (1:1000 dilution), anti-Env mAbs or anti-CD4 mAbs was performed with or without BNM-III-170 (50μM) or its equivalent volume of vehicle (DMSO) at 48h post-infection/transfection. Cells infected with HIV-1 primary isolates were stained intracellularly for HIV-1 p24, using the Cytofix/Cytoperm Fixation/ Permeabilization Kit (BD Biosciences, Mississauga, ON, Canada) and the fluorescent anti-p24 mAb (PE-conjugated anti-p24, clone KC57; Beckman Coulter/Immunotech). The percentage of infected or transfected cells (p24 + cells or GFP + , respectively) was determined by gating the living cell population on the basis of the AquaVivid viability dye staining. Samples were analyzed on an LSRII cytometer (BD Biosciences), and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA).

ADCC FACS-based assay

Measurement of ADCC using the FACS-based assay was performed at 48h post-infection as previously described ( Richard et al., 2014 ; Richard et al., 2015 ; Richard J., 2018 ; Veillette et al., 2014b ). Briefly, infected primary CD4+ T cells were stained with viability (AquaVivid; Thermo Fisher Scientific) and cellular (cell proliferation dye eFluor670; eBioscience) markers and used as target cells. Autologous PBMC effectors cells, stained with another cellular marker (cell proliferation dye eFluor450; eBioscience), were added at an effector: target ratio of 10:1 in 96-well V-bottom plates (Corning, Corning, NY). Briefly, infected primary CD4+ T cells were incubated with autologous PBMC (Effector: Target ratio of 10:1) in presence of A32 (0.3125, 0,625, 1,25, 2,5 or 5 μg/ml,) and 17b (5 μg/ml) or17b Fab fragments alone or in combination, or with HIV+ sera (1:1000), in presence of 50 μM of BNM-III-170 or with equivalent volume of vehicle (DMSO). The plates were subsequently centrifuged for 1 min at 300 g, and incubated at 37C, 5% CO 2 for 5 to 6 h before being fixed in a 2% PBS-formaldehyde solution. Samples were analyzed on an LSRII cytometer (BD Biosciences). Data analysis was performed using FlowJo vX.0.7 (Tree Star). The percentage of ADCC was calculated with the following formula: (% of p24+ cells in Targets plus Effectors) - (% of p24+ cells in Targets plus Effectors plus sera) / (% of p24+ cells in Targets) by gating on infected lived target cells. ELISA The gp120 inner domain stabilized ID2 ( Tolbert et al., 2016 ) (0.1μg/ml) was diluted in PBS. Bovine serum albumin (BSA) (0.1μg/ml) was used a negative control. Proteins were adsorbed to plates (MaxiSorp; Nunc) overnight at 4°C. Coated wells were subsequently blocked with blocking buffer (tris-buffered saline (TBS) containing 0.1% Tween-20 and 2% (w/v) BSA for 1:30 hour at room temperature. HIV+ sera (1:1000 dilution) were diluted in blocking buffer and added to the coated wells for 1:30 hour at room temperature. Wells were washed four times with washing buffer (TBS containing 0.1% Tween-20). Horseradish peroxidase-conjugated (HRP) antibody specific of human IgG (Pierce) was added for 1 hour at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with the LB 941 TriStar luminometer (Berthold Technologies).

Cryo-EM grid preparation

Freshly thawed Aldrithiol-2 (AT-2)-inactivated HIV-1 particles (Lot#P4311, final concentration 335μg/mL in TNE buffer based on capsid protein concentration) were mixed with the CD4mc BNM-III-170 (5μM) and two anti-HIV-1 antibodies, 17b (5μg/mL) and A32 (5μg/mL). The mixture was pre-incubated at 37°C for 30 min prior to EM grid preparation. Two μL of this mixture was dispensed on glow-discharged Quantifoil R2/2 grids before plunge freezing in liquid ethane cooled by liquid nitrogen using Vitrobot (FEI-MarkIV). A total of 1033 movies was collected using a K2 camera mounted on a Talos Arctica transmission electron microscope placed in the Melbourne Advanced Microscopy Facility (MAMF) at the Bio21 Institute. In-situ single particle reconstruction was performed using the Scipion suite is described in Figure S4 ( de la Rosa-Trevin et al., 2016 ). Structural model building Model building in the final cryo-EM density was performed by manually docking the crystal structure of the 17b antibody-bound HIV-1 (gp41/gp120) 3 hetero-trimer (PDB ID: 5VN3) in UCSF Chimera ( Pettersen et al., 2004 ). The initial placement of the model was followed by independent rigid docking of each gp41/gp120 hetero-dimer into the segmented Cryo-EM density using the SeggeR tool ( Pintilie et al., 2010 ) in UCSF Chimera. The position of each gp41-gp120 heterodimer was fine tuned in light of the segmented cryo-EM map produced by SeggeR and prior knowledge of epitope binding positions of the 17b and A32 antibodies obtained from crystal structures, 5VN3 (for 17b epitope) and 4YBL (for A32 epitope), respectively.

Quantification and Statistical Analysis Analysis of flow cytometry data

Statistics were analyzed using GraphPad Prism version 6.01 (GraphPad). Every data set was tested for statistical normality and this information was used to apply the appropriate (parametric or nonparametric) statistical test. P values