HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound “closed” conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature “opening” of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the “closed” conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.


Introduction
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is a class I viral membrane fusion protein which mediates viral entry using the CD4 cellular receptor. The envelope gp160 precursor is synthesized in the endoplasmic reticulum (ER) and oligomerizes as a trimer [1,2]. Subsequently, the trimeric Env traffics through the trans-Golgi network (TGN) to reach the plasma membrane and to be incorporated into nascent HIV-1 virions [3][4][5]. During its transit through the secretory pathway, Env undergoes important post-translational modifications, including N-linked and O-linked glycosylation as well as proteolytic cleavage [6][7][8][9][10]. The addition of high-mannose oligosaccharides takes place in the ER and these glycans are further processed to acquire complex modifications in the TGN [11]. Concomitantly, proprotein convertases present in the TGN, including furin and furin-like proteases, catalyze the cleavage of the immature gp160 polyprotein [12][13][14][15] into two functional non-covalently linked subunits: the exterior gp120 subunit, which is responsible for viral attachment and the transmembrane gp41 subunit, which mediates

Ethics Statement
Written informed consent was obtained from all study participants (the Montreal Primary HIV Infection Cohort [66,67] and the Canadian Cohort of HIV Infected Slow Progressors [68][69][70]), 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). The research adhered to the standards indicated by the Declaration of Helsinki. All participants were adults and provided informed written consent prior to enrolment in accordance with Institutional Review Board approval.

Cell Lines and Primary Cells
293T human embryonic kidney cells (obtained from ATCC) and TZM-bl cells (NIH AIDS Reagent Program) were maintained at 37 • C under 5% CO 2 in Dulbecco's Modified Eagle Medium (DMEM) (Wisent, St. Bruno, QC, Canada), supplemented with 5% fetal bovine serum (FBS) (VWR, Radnor, PA, USA) and 100 U/mL penicillin/streptomycin (Wisent). 293T cells were derived from 293 cells, into which the simian virus 40 T-antigen was inserted. TZM-bl were derived from HeLa cells and were engineered to stably express high levels of human CD4 and CCR5 and to contain the firefly luciferase reporter gene under the control of the HIV-1 promoter [71]. Primary human PBMCs and CD4+ T cells were isolated, activated, and cultured as previously described [26]. Briefly, PBMCs were obtained by leukapheresis from six HIV-negative individuals (all males), and primary CD4+ T lymphocytes were purified from resting PBMCs by negative selection using immunomagnetic beads per the manufacturer's instructions (StemCell Technologies, Vancouver, BC, Canada) and were activated with phytohemagglutinin-L (10 µg/mL) for 48 h and then maintained in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) complete medium supplemented with rIL-2 (100 U/mL).

Antibodies and Sera
The following Abs were used to assess Env conformation at the cell surface: conformationindependent anti-gp120 outer-domain 2G12 (NIH AIDS Reagent Program), broadly-neutralizing antibodies anti-CD4 binding site VRC03 (NIH AIDS Reagent Program), anti-V2 apex PG9 (Polymun Scientific, Klosterneuburg, Austria), anti-V3 glycan PGT126, anti-gp41-gp120 interface PGT151 (IAVI), anti-V3 glycan 10-1074 (kindly provided by Michel Nussenzweig) and anti-fusion peptide VRC34 (kindly provided by John Mascola) as well as non-neutralizing antibodies anti-gp41 C-C loop F240, anti-V3 crown 19b, anti-coreceptor binding site 17b, anti-cluster A A32 and C11 (NIH AIDS Reagent Program). The HIV-IG polyclonal antibody consists of anti-HIV immunoglobulins purified from a pool of plasma from HIV+ asymptomatic donors (NIH AIDS Reagent Program). Goat anti-human and anti-mouse antibodies pre-coupled to Alexa Fluor 647 (Invitrogen, Rockford, IL, USA) were used as secondary antibodies in flow cytometry experiments. Plasma from HIV-infected individuals was collected, heat-inactivated and conserved at −80 • C until use. In most experiments, the 2G12 monoclonal Ab (mAb) was used to normalize Env expression because of its conformation independence. Both viruses used in the study (CH058 and CH077) are well recognized by 2G12 despite polymorphism found at position 295 in CH077 Env.

Small Molecules
The small-molecule CD4-mimetic compound BNM-III-170 was synthesized as described previously [72]. The HIV-1 attachment inhibitor Temsavir (BMS-626529) was purchased from APExBIO (Houston, TX, USA). The compounds were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and diluted to 50 µM in phosphatebuffered saline (PBS) for cell-surface staining and virus capture assay or in RPMI-1640 complete medium for ADCC assays.

Radioactive Labeling and Immunoprecipitation of Envelope Glycoproteins
293T cells (3 × 10 5 ) were transfected by the calcium phosphate method with the different IMCs. One day after transfection, cells were metabolically labeled for 16 h with 100 µCi/mL of [ 35 S]methionine-cysteine ([ 35 S] Protein Labeling Mix; PerkinElmer, Waltham, MA, USA) in DMEM lacking methionine and cysteine and supplemented with 5% dialyzed fetal bovine serum. Cells were subsequently lysed in RIPA buffer (140 mM NaCl, 8 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 , 1% NP40, 0.05% sodium dodecyl sulfate (SDS), 1.2 mM sodium deoxycholate). Precipitation of radiolabeled envelope glycoproteins from the whole-cell lysates or found in the supernatant was performed with a pool of sera from HIV-1-infected individuals in the presence of 50 µL of 10% Protein A-Sepharose (Cytiva, Marlborough, MA) at 4 • C. The precipitated proteins were loaded onto SDS-PAGE gels and analyzed by autoradiography and densitometry to calculate their processing indexes. The processing index is a measure of the conversion of the mutant gp160 Env precursor to mature gp120, relative to wild-type Env trimers. The processing index is calculated with the following formula: processing index = ([total gp120]mutant × [gp160]WT)/([gp160]mutant × [total gp120]WT).

Viral Production and Infections
VSV-G-pseudotyped HIV-1 viruses were produced and concentrated as previously described [60]. Viruses were then used to infect activated primary CD4+ T cells from healthy HIV-1 negative donors by spin infection at 800× g for 1 h in 96-well plates at 25 • C. Viral preparations were titrated directly on primary CD4+ T cells to achieve similar levels of infection among the different IMCs tested (around 10% of p24+ cells). To assess viral infectivity, TZM-bl reporter cells were seeded at a density of 2 × 10 4 cells/well in 96-well luminometer-compatible tissue culture plates (PerkinElmer) 24 h before infection. Normalized amounts of viruses (according to reverse transcriptase activity [78]) in a final volume of 100 µL were then added to the target cells and incubated for 48 h at 37 • C. The medium was then removed from each well, and the cells were lysed by the addition of 30 µL of passive lysis buffer (Promega, Madison, WI, USA) and one freeze-thaw cycle. An

Virus Capture Assay
The HIV-1 virus capture assay was previously reported [79]. Pseudoviral particles were produced by transfecting 2 × 10 6 293T cells with pNL4.3 R-E-Luc (NIH AIDS Reagent Program) (3.5 µg), HIV-1 CH058 (3.5 µg), and VSV-G (1 µg) using the standard calcium phosphate method. Forty-eight hours later, virus-containing supernatant was collected, and cell debris were removed by centrifugation (1500 rpm for 10 min). Anti-Env antibodies was immobilized on white MaxiSorp ELISA plates (Thermo Fisher Scientific) at a concentration of 5 µg/mL in 100 µL of PBS overnight at 4 • C. Unbound antibodies were removed by washing twice the plates twice with PBS. Plates were subsequently blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. After washing plates twice with PBS, 200 µL of virus-containing supernatants were added to the wells. After 4 to 6 h incubation, virions were removed, and the wells were washed three times with PBS. Virus capture by any given antibody was visualized by adding 1 × 10 4 293T cells per well in complete DMEM. To measure recombinant virus infectivity, 1 × 10 4 293T cells were directly mixed with 100 µL of virus-containing supernatants per well. Forty-eight hours post-infection, cells were lysed by the addition of 30 µL of passive lysis buffer (Promega) and one freeze-thaw cycle. An LB 941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 µL of luciferin buffer (15 mM MgSO 4 , 15 mM KH 2 PO 4 [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 µL of 1 mM D-luciferin potassium salt (Prolume).

Flow Cytometry Analysis of Cell-Surface and Intracellular Staining
Cell-surface staining of HIV-1-transfected and HIV-1-infected cells was executed as previously described [35,61]. For transfected cells, we used the standard calcium phosphate method to transfect 7 µg of each IMC into 2 × 10 6 293T cells. Binding of cell-surface HIV-1 Env by anti-Env mAbs (5 µg/mL) or HIV+ plasma (1:1000 dilution) was performed at 48 h post-transfection. Similarly, cell-surface staining of infected cells was performed at 48 h post-infection. After cell-surface staining, transfected cells and infected cells were permeabilized using the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Mississauga, ON, Canada) and stained intracellularly using PE-conjugated mouse anti-p24 mAb (clone KC57; Beckman Coulter, Brea, CA, USA; 1:100 dilution). The percentage of transfected or infected cells (p24+) was determined by gating on the living cell population according to a viability dye staining (Aqua Vivid, Thermo Fisher Scientific). Samples were acquired on an LSRII cytometer (BD Biosciences), and data analysis was performed using FlowJo v10.5.3 (Tree Star, Ashland, OR, USA).

FACS-Based ADCC Assay
Measurement of ADCC using the FACS-based assay was performed at 48 h postinfection as previously described [26,36]. Briefly, HIV-1-infected primary CD4+ T cells were stained with AquaVivid viability dye and cell proliferation dye eFluor670 (Thermo Fisher Scientific) and used as target cells. Autologous PBMC effectors cells, stained with the cell proliferation dye eFluor450 (Thermo Fisher Scientific), were added at an effector: target ratio of 10:1 in 96-well V-bottom plates (Corning, Glendale, AZ, USA). A 1:1000 final dilution of HIV+ plasma was added to appropriate wells and cells were incubated for 5 min at room temperature. The plates were subsequently centrifuged for 1 min at 300× g and incubated at 37 • C, 5% CO 2 for 5 h before being fixed in a 2% PBS-formaldehyde solution. Samples were acquired on an LSRII cytometer (BD Biosciences) and data analysis was performed using FlowJo v10.5.3 (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.

Statistical Analysis
Statistics were analyzed using GraphPad Prism version 9.1.0 (GraphPad, San Diego, CA, USA). Every data set was tested for statistical normality and this information was used to apply the appropriate (parametric or nonparametric) statistical test. p values < 0.05 were considered significant; significance values are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Conformation of HIV-1 Uncleaved Env at the Surface of Infected Cells and Viral Particles
To study the role of the furin cleavage site on Env conformation, we performed mutagenesis on the infectious molecular clones (IMCs) of clade B transmitted/founder (T/F) viruses CH058 and CH077. Envs from both viruses were previously shown to preferentially sample the "closed" State 1 conformation [61]. We introduced substitutions in the primary cleavage site at position 508 and 511 ( Figure 1A), to replace the highly conserved arginine residues with serine residues (R508S/R511S; referred as Cl− mutant), a double mutant known to efficiently abrogate furin-dependant Env processing [64,[80][81][82]. We used protein radioactive labelling of 293T cells transfected with the different IMC constructs followed by Env immunoprecipitation to confirm the effect of the mutations on Env cleavage (Figure 1B-E). As expected, Env glycoproteins expressed from the wild-type (WT) construct were efficiently cleaved while their cleavage-deficient (Cl−) counterpart yielded little to no detectable gp120 in the 293T whole-cell lysates ( Figure 1B,D). Although we observed some soluble gp120 in the supernatant of CH058-transfected cells, this was likely due to the presence of a second upstream cleavage site, which matched the furin consensus sequence (RAKR). The supernatant of CH077-transfected cells did not contain gp120 consistent with an altered upstream cleavage site (KAKR) ( Figure 1A). Of note, two bands of gp160 with distinct molecular weights were observed in cells transfected with Cl− variants, a phenotype previously observed that was linked to a difference in Env trafficking and localization [83][84][85].  Error bars indicate the mean ± SEM. Statistical significance was tested using an unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
Subsequently, we evaluated the ability of a panel of bNAbs and nnAbs to recognize the cleaved (WT) and uncleaved (Cl−) Env at the surface of 293T cells. We selected these cells since they do not express CD4 and it has been well documented that the presence of CD4 affects Env conformation [26,35,86]. Cells were transfected with the different IMC constructs and virus-expressing cells were identified using Gag p24 staining ( Figure 1F,G). Cell-surface Env expression was normalized using the conformation-independent 2G12 antibody. Cells expressing WT Env were preferentially recognized by the bNAbs preferring the State 1 conformation (PGT126, VRC03, PG9) and recognizing the fusion peptide (PGT151, VRC34) compared to those expressing the respective cleavage site mutants ( Figure 1F,G). Conversely, the binding of nnAbs targeting the downstream conformations States 2/3 (19b, F240, 17b) and State 2A (A32, C11) was significantly enhanced on cells expressing uncleaved Env ( Figure 1F,G). To confirm this phenotype in a physiologically more relevant culture system, we infected activated primary CD4+ T cells with the different primary IMCs. Of note, all viruses were pseudotyped with the VSV G glycoprotein to normalize the level of infection and to compensate for the inability of uncleaved Env to mediate viral fusion. Consistent with the 293T results, productively-infected cells (p24+ CD4 low ) were more efficiently recognized by bNAbs when expressing cleaved Env, and by nnAbs when expressing uncleaved Env ( Figure 1H,I). Overall, these results support and extend previous observations indicating that furin cleavage favors the adoption of the native "closed" conformation at the cell surface [40,65,84].
We next investigated the effect of furin cleavage on Env conformation at the surface of viral particles, since the viral membrane is known to be enriched in cholesterol, a lipid known to stabilize Env State 1 conformation by interacting with the gp41 membraneproximal external region (MPER) [87][88][89]. Since virions expressing the Env Cl−variants were unable to infect even highly permissive cells, we used a recently developed virus capture assay [79] (Figure 2A). Specifically, we generated luciferase reporter pseudovirions that contained both HIV-1 Env and VSV G glycoproteins, thus allowing captured virions to infect 293T cells in an Env-independent manner (i.e., 293T infection is driven by the incorporated VSV G glycoprotein, Figure 2B). Virions harboring WT Env were captured more efficiently by bNAbs, while virions harboring uncleaved Env were primarily bound by nnAbs ( Figure 2C). The recognition of pseudovirions was also assessed using purified anti-HIV-1 immunoglobulins from HIV+ asymptomatic donors (HIV-IG) [90]. Since the vast majority of naturally-elicited antibodies targets Env in its "open" conformation [26,27,34], HIV-IG polyclonal antibodies captured viral particles displaying immature Env in a larger proportion ( Figure 2D). HIV-IG specific capture of uncleaved or cleaved Env could be further increased using the small molecule CD4mc BNM-III-170, which stabilizes the CD4-bound conformation ( Figure 2D). Alternatively, treatment with the conformational blocker Temsavir decreased the capacity of HIV-IG to capture viral particles bearing Cl− Envs ( Figure 2D), in agreement with its capacity to stabilize the "closed" conformation [20,22,55]. These results indicate that uncleaved Env can be forced into "open" or "closed" conformations using small molecule Env antagonists.  (19b, F240, 17b, A32, C11) or (D) HIV-IG. RLU values obtained using the different antibodies were normalized to the signal obtained with the conformation-independent 2G12 mAb. Data shown are the mean ± SEM from at least three independent experiments. Statistical significance was tested using an unpaired t-test or a Mann-Whitney Utest based on statistical normality (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, nonsignificant).

Impact of HIV-1 Env Proteolytic Cleavage on ADCC Responses Mediated by HIV+ Plasma
Knowing that alterations in the furin cleavage site increase the exposure of downstream conformations at the surface of infected cells and lentiviral particles, we sought to determine whether the presence of uncleaved Env at the surface of infected cells could also affect ADCC responses mediated by plasma from HIV-1-infected donors. Activated primary CD4+ T cells were infected with WT or cleavage defective CH058 and CH077 and then examined for their susceptibility to ADCC killing following incubation with plasma from 15 different chronically HIV-1-infected individuals. As expected, HIV+ plasma binding was significantly higher on infected cells expressing cleavage-deficient Env compared to WT Env ( Figure 3A,B). Moreover, inhibition of Env cleavage led to strong ADCC responses, while WT-infected cells were protected from these responses mediated by HIV+ plasma (Figure 3C,D). Treatment with BNM-III-170 was found to enhance the binding of  (19b, F240, 17b, A32, C11) or (D) HIV-IG. RLU values obtained using the different antibodies were normalized to the signal obtained with the conformation-independent 2G12 mAb. Data shown are the mean ± SEM from at least three independent experiments. Statistical significance was tested using an unpaired t-test or a Mann-Whitney U-test based on statistical normality (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, nonsignificant).

Impact of HIV-1 Env Proteolytic Cleavage on ADCC Responses Mediated by HIV+ Plasma
Knowing that alterations in the furin cleavage site increase the exposure of downstream conformations at the surface of infected cells and lentiviral particles, we sought to determine whether the presence of uncleaved Env at the surface of infected cells could also affect ADCC responses mediated by plasma from HIV-1-infected donors. Activated primary CD4+ T cells were infected with WT or cleavage defective CH058 and CH077 and then examined for their susceptibility to ADCC killing following incubation with plasma from 15 different chronically HIV-1-infected individuals. As expected, HIV+ plasma binding was significantly higher on infected cells expressing cleavage-deficient Env compared to WT Env ( Figure 3A,B). Moreover, inhibition of Env cleavage led to strong ADCC responses, while WT-infected cells were protected from these responses mediated by HIV+ plasma (Figure 3C,D). Treatment with BNM-III-170 was found to enhance the binding of HIV+ plasma on both WT and Cl− mutant infected cells, consistent with its ability to expose CD4i epitopes. Accordingly, CD4mc addition induced a potent ADCC response against WT-infected cells, but marginally enhanced the ADCC response against cells expressing cleavage-deficient Env, suggesting that CD4i epitope exposure by uncleaved Env is sufficient to trigger the elimination of infected cells by ADCC. Conversely, the addition of State 1-stabilizing molecule Temsavir protected Cl− expressing cells from ADCC by decreasing the binding of HIV+ plasma to uncleaved Env ( Figure 3A-D). Of note, Temsavir did not impact HIV+ plasma mediated ADCC against WT infected cells since they are known to already express the Env in the "closed" conformation [26,[35][36][37]40,60,61,86,91,92]. Altogether, our results demonstrate the importance for HIV-1 to limit the presence of Env gp160 precursor at the surface of infected cells to evade nnAbs-mediated ADCC responses.
Viruses 2021, 13, x FOR PEER REVIEW 10 of 17 HIV+ plasma on both WT and Cl-mutant infected cells, consistent with its ability to expose CD4i epitopes. Accordingly, CD4mc addition induced a potent ADCC response against WT-infected cells, but marginally enhanced the ADCC response against cells expressing cleavage-deficient Env, suggesting that CD4i epitope exposure by uncleaved Env is sufficient to trigger the elimination of infected cells by ADCC. Conversely, the addition of State 1-stabilizing molecule Temsavir protected Cl-expressing cells from ADCC by decreasing the binding of HIV+ plasma to uncleaved Env ( Figure 3A-D). Of note, Temsavir did not impact HIV+ plasma mediated ADCC against WT infected cells since they are known to already express the Env in the "closed" conformation [26,[35][36][37]40,60,61,86,91,92]. Altogether, our results demonstrate the importance for HIV-1 to limit the presence of Env gp160 precursor at the surface of infected cells to evade nnAbsmediated ADCC responses.

Discussion
In this study, we show that uncleaved HIV-1 Env trimers display conformational flexibility which favors the sampling of downstream "more open" conformations at the surface of infected cells and pseudoviral particles. Cell-surface expression of uncleaved gp160 leads to an efficient recognition of infected cells by non-neutralizing CD4i antibodies naturally present in plasma from HIV-1-infected individuals and as a consequence, leads to a significantly higher susceptibility to ADCC responses. Conversely, efficient cleavage by endogenous furin allows Env trimers to sample a metastable "closed" conformation (State 1), thus protecting HIV-1-infected cells from ADCC responses mediated by HIV+ plasma. Beyond the well-established role of furin cleavage on viral infectivity, efficient proteolytic cleavage of Env trimers thus appears to allow HIV-1 to evade humoral immune responses. These results are important in the context of recent findings showing that several interferon-inducible cellular antiviral factors affect Env gp160 precursor processing [93][94][95][96][97]. Among them, IFITM proteins impair Env cleavage through direct interaction with Env, while GBP2 and GBP5 restrict furin protease activity [93,96]. The antiviral activity of both families of proteins can be overcome by HIV-1 through Env substitutions or by increasing Env expression through the deletion of the accessory Vpu protein, respectively [97][98][99][100].
According to the Los Alamos National Laboratory HIV sequence database, very few mutations are naturally found in the furin cleavage site, especially for the basic residues found at positions 508, 510, and 511 which are more than 99.7% conserved. Given the importance of an effective Env cleavage to generate infectious viral particles, therapeutic interventions designed to specifically inhibit this proteolytic cleavage could result in a loss in infectivity with a concomitant increase in ADCC responses against infected cells. A recent study has shown that conformational blockers, such as Temsavir, can interfere with proper Env cleavage by reducing its conformational flexibility [63]. Additional drugs inhibiting directly the furin protease activity, including the synthetic peptide Dec-RVKR-CMK and the serine protease inhibitor α 1 -PDX, are also being investigated, but their in vivo efficacy and toxicity remain to be determined [12,13,[101][102][103][104][105]. If these broad-spectrum inhibitors end up being well-tolerated and exhibit good pharmacokinetic properties, they may also be useful as therapeutics against other viral infections, including Influenza A, Ebola, Respiratory syncytial virus (RSV), and SARS-CoV-2, where the acquisition of a furin cleavage site in the respective fusion glycoproteins appears to confer a higher level of infectivity [106][107][108][109][110].
Author Contributions: J.P. and A.F. conceived the study; J.P. and A.F. designed experimental approaches; J.P., H.M., D.V. and A.F. performed, analyzed, and interpreted the experiments; H.-C.C., B.H.H. and A.B.S.III supplied novel/unique reagents; J.P. and A.F. wrote the paper. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by grants from the National Institutes of Health to A.F. (R01 AI148379, R01 AI129769, and R01 AI150322) and BHH (R01 AI162646 and UM1 AI164570). Support for this work was also provided by P01 GM56550/AI150471 to A.B.S. and A.F. This work was partially supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases, by a CIHR Foundation grant #352417, a CIHR Team Grant #422148 and a Canada Foundation for Innovation grant #41027 to A.F. A.F. is the recipient of a Canada Research Chair on Retroviral Entry #RCHS0235 950-232424. J.P. is the recipient of a CIHR doctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the CRCHUM Institutional Review Board (ethics committee, approval number CE 16.164-CA).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: All data are contained within the article.