key: cord-0335173-v5m5r173
authors: Prévost, Jérémie; Medjahed, Halima; Vézina, Dani; Chen, Hung-Ching; Hahn, Beatrice H; Smith, Amos B.; Finzi, Andrés
title: HIV-1 envelope glycoproteins proteolytic cleavage protects infected cells from ADCC mediated by plasma from infected individuals
date: 2021-10-26
journal: bioRxiv
DOI: 10.1101/2021.10.26.465908
sha: 035b44db6eb8e27e2a2da30539791768882b6e59
doc_id: 335173
cord_uid: v5m5r173

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.

"closed" conformation (State 1) to downstream CD4-bound conformations (States 2/3), which 23 are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the 24 premature "opening" of Env which exposes highly conserved epitopes recognized by non-25 neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity 26 (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the 27 "closed" conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage 28 and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected 29 individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not 30 wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC 31 responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure 32 of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC 33 responses. 34 35 1. Introduction 36

Cell-surface staining of HIV-1-transfected and HIV-1-infected cells was executed as 192 previously described [35, 61] . For transfected cells, we used the standard calcium phosphate 193 method to transfect 7 μg of each IMC into 2 × 10 6 293T cells. Binding of cell-surface HIV-1 Env 194 by anti-Env mAbs (5 µg/mL) or HIV+ plasma (1:1000 dilution) was performed at 48h post-195 transfection. Similarly, cell-surface staining of infected cells was performed at 48h post-infection. AquaVivid viability dye and cell proliferation dye (eFluor670; eBioscience) and used as target 208 cells. Autologous PBMC effectors cells, stained with another cellular marker (cell proliferation 209 dye eFluor450; eBioscience), were added at an effector: target ratio of 10:1 in 96-well V-bottom 210 plates (Corning). A 1:1000 final dilution of HIV+ plasma was added to appropriate wells and 211 cells were incubated for 5 min at room temperature. The plates were subsequently centrifuged 212 for 1 min at 300 x g, and incubated at 37°C, 5% CO 2 for 5h before being fixed in a 2% PBS-213 formaldehyde solution. Samples were acquired on an LSRII cytometer (BD Biosciences) and 214 data analysis was performed using FlowJo v10.5.3 (Tree Star). The percentage of ADCC was 215 calculated with the following formula: (% of p24+ cells in Targets 

To study the role of the furin cleavage site on Env conformation, we performed 228 mutagenesis on the infectious molecular clones (IMCs) of clade B transmitted/founder (T/F) 229 viruses CH058 and CH077. Envs from both viruses were previously shown to preferentially 230 sample the "closed" State 1 conformation [61] . We introduced substitutions in the primary 231 cleavage site at position 508 and 511 ( Figure 1A ), to replace the highly conserved arginine 232 residues with serine residues (R508S/R511S; referred as Cl-mutant), a double mutant known to 233 efficiently abrogate furin-dependant Env processing [64, [80] [81] [82] . We used protein radioactive 234 labelling of 293T cells transfected with the different IMC constructs followed by Env 235 immunoprecipitation to confirm the effect of the mutations on Env cleavage (Figure 1B-E). As 236 expected, Env glycoproteins expressed from the wild-type (WT) construct were efficiently 237 cleaved while their cleavage-deficient (Cl-) counterpart yielded little to no detectable gp120 in 238 the 293T cell lysates ( Figure 1B,D) . Although we observed some soluble gp120 in the 239 supernatant of CH058-transfected cells, this was likely due to the presence of second upstream 240 cleavage site, which matched the furin consensus sequence (RAKR). Supernatant of CH077-241 transfected cells did not contain gp120 consistent with an altered upstream cleavage site 242 (KAKR) ( Figure 1A ). Of note, two bands of gp160 with distinct molecular weights were observed 243 in cells transfected with Cl-variants, a phenotype previously observed that was linked to a 244 difference in glycosylation [83] [84] [85] . 245 246 Subsequently, we evaluated the ability of a panel of bNAbs and nnAbs to recognize the 247 cleaved (WT) and uncleaved (Cl-) Env at the surface of 293T cells. We selected these cells 248 since they don't express CD4 and it has been well documented that the presence of CD4 affects 249

Env conformation [26, 35, 86] . Cell were transfected with the different IMC constructs and virus-250 expressing cells were identified using Gag p24 staining ( Figure 1F -G). Cell-surface Env 251 expression was normalized using the conformation-independent 2G12 antibody. Cells 252 expressing WT Env were preferentially recognized by the bNAbs preferring the State 1 253 conformation (PGT126, VRC03, PG9) and recognizing the fusion peptide (PGT151, VRC34) 254 compared to those expressing the respective cleavage site mutants ( Figure 1F -G). Conversely, 255 the binding of nnAbs targeting the downstream conformations States 2/3 (19b, F240, 17b) and 256

State 2A (A32, C11) was significantly enhanced on cells expressing uncleaved Env ( Figure 1F -257 G). To confirm this phenotype in a physiologically more relevant culture system, we infected 258 activated primary CD4+ T cells with the different primary IMCs. Of note, all viruses were 259 pseudotyped with the VSV G glycoprotein to normalize the level of infection and to compensate 260 the inability of uncleaved Env to mediate viral fusion. Consistent with the 293T results, 261 productively-infected cells (p24+ CD4low) were more efficiently recognized by bNAbs when 262 expressing cleaved Env, and by nnAbs when expressing uncleaved Env ( Figure 1H -I). Overall, 263 these results support and extend previous observations indicating that furin cleavage favors the 264 adoption of the native "closed" conformation at the cell surface [40, 65, 84] . precipitated proteins were loaded onto SDS-PAGE gels and analyzed by autoradiography and 278 densitometry to calculate their processing indexes. The processing index is a measure of the 279 conversion of the mutant gp160 Env precursor to mature gp120, relative to that of the wild-type staining obtained in at least 3 independent experiments. Error bars indicate mean ± SEM. 288

Statistical significance was tested using an unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, 289 **** p < 0.0001).

We next investigated the effect of furin cleavage on Env conformation at the surface of 292 viral particles, since the viral membrane is known to be enriched in cholesterol, a lipid known to 293 stabilize Env State 1 conformation by interacting with gp41 membrane proximal external region 294 (MPER) [87] [88] [89] . Since virions expressing the Env Cl-variants were unable to infect even highly 295 permissive cells, we used a recently developed virus capture assay [79] ( Figure 2A ). 296

Specifically, we generated luciferase reporter pseudovirions that contained both HIV-1 Env and 297 VSV G glycoproteins, thus allowing captured virions to infect 293T cells in an Env-independent 298 manner (i.e., 293T infection is driven by the incorporated VSV G glycoprotein, Figure 2B ). 299

Virions harboring WT Env were captured more efficiently by bNAbs, while virions harboring 300 uncleaved Env were primarily bound by nnAbs ( Figure 2C ). The recognition of pseudovirions 301 was also assessed using purified anti-HIV-1 immunoglobulins from HIV+ asymptomatic donors 302

(HIV-IG) [90] . Since the vast majority of naturally-elicited antibodies targets Env in its "open" 303 conformation, HIV-IG polyclonal antibodies captured viral particles displaying immature Env in a 304 larger proportion ( Figure 2D ). HIV-IG specific capture of uncleaved or cleaved Env could be 305 further increased using the small molecule CD4mc BNM-III-170, which stabilizes the CD4-306 bound conformation ( Figure 2D ). Alternatively, treatment with the conformational blocker 307

Temsavir decreased the capacity of HIV-IG to capture viral particles bearing Cl-Envs ( Figure  308 2D), in agreement with its capacity to stabilize the "closed" conformation [20, 22, 55] . These 309 results indicate that uncleaved Env can be forced into "open" or "closed" conformations using 2G12 mAb. Data shown are the mean ± SEM from at least three independent experiments. 324

Statistical significance was tested using an unpaired t test or a Mann-Whitney U test based on 325 statistical normality (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant). 326 327

Knowing that alterations in the furin cleavage site increase the exposure of downstream 329 conformations at the surface of infected cells and lentiviral particles, we sought to determine 330 whether the presence of uncleaved Env at the surface of infected cells could also affect ADCC 331 responses mediated by plasma from HIV-1-infected donors. Activated primary CD4+ T cells 332 were infected with WT or cleavage defective CH058 and CH077 and then examined for their 333 susceptibility to ADCC killing following incubation with plasma from 15 different chronically HIV-334 1-infected individuals. As expected, HIV+ plasma binding was significantly higher on infected 335 cells expressing cleavage-deficient Env compared to WT Env ( Figure 3A -B). Moreover, 336

inhibition of Env cleavage led to strong ADCC responses, while WT-infected cells were 337 protected from these responses mediated by HIV+ plasma ( Figure 3C -D). Treatment with BNM-338 III-170 was found to enhance the binding of HIV+ plasma on both WT and Cl-mutant infected 339 cells, consistent with its ability to expose CD4i epitopes. Accordingly, CD4mc addition induced a 340 potent ADCC response against WT-infected cells, but did not further enhance the ADCC 341 response against cells expressing cleavage-deficient Env, suggesting that CD4i epitope 342 exposure by uncleaved Env is sufficient to trigger the elimination of infected cells by ADCC. 343

Conversely, the addition of State 1-stabilizing molecule Temsavir protected Cl-expressing cells 344 from ADCC by decreasing the binding of HIV+ plasma to uncleaved Env ( Figure 3A -D). Of note, 345

Temsavir didn't impact HIV+ plasma mediated ADCC against WT infected cells since they are 346 known to already express the Env in the "closed" conformation [26, 35-37, 40, 60, 61, 86, 91, 347 92] . Altogether, our results demonstrate the importance for HIV-1 to limit the presence of Env 348 gp160 precursor at the surface of infected cells to evade nnAbs-mediated ADCC responses. 349 bars indicate means ± SEM. Statistical significance was tested using a repeated measures one-363

way ANOVA with a Holm-Sidak post-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, 364 P < 0.0001; ns, nonsignificant). 365 366

In this study, we show that uncleaved HIV-1 Env trimers display a conformational including the synthetic peptide Dec-RVKR-CMK and the serine protease inhibitor α1-PDX, are 393 also being investigated, but their in vivo efficacy and toxicity remain to be determined [12, 13, 394 101-105] . If these broad-spectrum inhibitors end up being well tolerated and exhibit good 395 pharmacokinetic properties, they may also be useful as therapeutics against other viral 396 infections, including Influenza A, Ebola, Respiratory syncytial virus (RSV) and SARS-CoV-2, 397

where the acquisition of a furin cleavage site in the respective fusion glycoproteins appears to 398 confer a higher level of infectivity [106] [107] [108] [109] [110] 

Small-molecule CD4 mimics interact with a 601 highly conserved pocket on HIV-1 gp120

CD4-mimetic small molecules 605 sensitize human immunodeficiency virus to vaccine-elicited antibodies

Activation and Inactivation of Primary 609 Human Immunodeficiency Virus Envelope Glycoprotein Trimers by CD4-Mimetic 610 Compounds

A Small-Molecule CD4-Mimetic Compound Protects Bone 613

Marrow-Liver-Thymus Humanized Mice From HIV-1 Infection

Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

Co-receptor Binding Site Antibodies Enable CD4-623 Mimetics to Expose Conserved Anti-cluster A ADCC Epitopes on HIV-1 Envelope 624 Glycoproteins

Two Families of Env Antibodies Efficiently Engage Fc-Gamma 628 Receptors and Eliminate HIV-1-Infected Cells

A New Family of Small-Molecule CD4-Mimetic 632

Compounds Contacts Highly Conserved Aspartic Acid 368 of HIV-1 gp120 and Mediates 633

Antibody-Dependent Cellular Cytotoxicity

The HIV-1 Env gp120 638 Inner Domain Shapes the Phe43 Cavity and the CD4 Binding Site

Stabilizing the HIV-1 641 envelope glycoprotein State 2A conformation

Modulating HIV-1 envelope glycoprotein 647 conformation to decrease the HIV-1 reservoir

Analogues Stabilize the State-1 Conformation of the Human Immunodeficiency 653 Virus (HIV-1) Envelope Glycoproteins

Shedding-Resistant HIV-1 Envelope Glycoproteins Adopt 656 Downstream Conformations That Remain Responsive to Conformation-Preferring 657

Mutagenic stabilization and/or disruption 660 of a CD4-bound state reveals distinct conformations of the human immunodeficiency 661 virus type 1 gp120 envelope glycoprotein

664 Release of gp120 Restraints Leads to an Entry-Competent Intermediate State of the 665 HIV-1 Envelope Glycoproteins

The highly conserved layer-3 component of the 668 HIV-1 gp120 inner domain is critical for CD4-required conformational transitions

Conformation of HIV-1 Envelope governs rhesus CD4 usage and simian-672 human immunodeficiency virus replication. bioRxiv 2021

Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses

Envelope glycoproteins sampling states 2/3 are susceptible to ADCC by 681 sera from HIV-1-infected individuals

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Asymmetric 687 structures and conformational plasticity of the uncleaved full-length human 688 immunodeficiency virus (HIV-1) envelope glycoprotein trimer

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein 692 trimers adopt a native-like conformation

HIV type 1 Env precursor cleavage state affects 696 recognition by both neutralizing and nonneutralizing gp41 antibodies

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Entry Inhibitor BNM-III-170 as the Bis-Trifluoroacetate Salt

Pseudotype formation of murine leukemia virus with 778 the G protein of vesicular stomatitis virus

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Phenotypic properties 797 of transmitted founder HIV-1

Relative resistance of HIV-1 founder 801 viruses to control by interferon-alpha

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Uncleaved Oligomers from Virions

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Nef Proteins from HIV-1 Elite Controllers 828

Are Inefficient at Preventing Antibody-Dependent Cellular Cytotoxicity

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The lipid membrane of HIV-1 stabilizes the viral envelope 838 glycoproteins and modulates their sensitivity to antibody neutralization

Preparation and characterization of an intravenous solution of IgG 842 from human immunodeficiency virus-seropositive donors

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Env Conformation and Antibody-Dependent Cellular Cytotoxicity Responses

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90K, an interferon-stimulated gene 858 product, reduces the infectivity of HIV-1

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