Although all these Envs were susceptible to temsavir-mediated viral inhibition, as measured in a single-round pseudoviral neutralization assay, their susceptibility to temsavir varied (Table S1). Open in a separate window Figure 4 Temsavir affects the recognition of HIV-1-infected primary CD4+ T cells by bNAbs. reported that temsavir affects glycosylation, proteolytic processing, and overall conformation of Env. Here, we extend these results to a panel of primary Envs and infectious molecular clones (IMCs), where we observe a heterogeneous impact on Env cleavage and conformation. Our results suggest that the effect of temsavir on Env conformation is associated with its capacity to decrease Env processing. Indeed, we found that the effect of temsavir on Env processing affects the recognition of HIV-1-infected cells by broadly neutralizing antibodies and correlates with their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC). Keywords: HIV-1, Env glycoprotein, entry inhibitors, fostemsavir (BMS-663068), temsavir (BMS-626529), proteolytic cleavage, bNAbs, ADCC 1. Introduction The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is synthetized in the endoplasmic reticulum, where the gp160 precursor undergoes trimerization and the addition of glycans. During its transport to the Golgi, in addition to further modifications of complex sugars, each gp160 molecule is proteolytically cleaved by cellular proteases into the gp120 surface protein and a gp41 transmembrane subunit [1,2,3]. Env is then transported to the cell surface, where it is incorporated into budding viral particles. Env interaction with CD4 enables coreceptor binding and the subsequent gp41 conformational changes required for a fusion between the virus and the cell membrane [4]. The native unliganded Env is a metastable molecule sampling different conformations. Non-neutralizing antibodies (nnAbs) bind epitopes that are normally occluded in the unliganded trimer and, therefore, fail to recognize the native closed trimer, whereas broadly neutralizing antibodies (bNAbs) preferentially recognize this Env conformation [5,6,7,8,9,10]. Clinical trials are currently evaluating the potential of several bNAbs to control viral replication and decrease the size of the viral reservoir [11,12]. In addition to the recent progress made with the development of bNAbs used as prevention and treatment, new inhibitors of HIV-1 are being considered to treat infected individuals. Among them, temsavir (BMS-626529 and GSK2616713), a small molecule entry inhibitor administrated as a prodrug for fostemsavir (BMS-663068, GSK3684934, and RUKOBIA), was recently approved for the treatment of patients who have limited therapeutic options [13,14]. Temsavir is known to bind a conserved pocket in gp120 under its 20C21 loop, thereby preventing CD4 interaction [15,16]. This molecule also stabilizes the closed State 1 Env conformation, which is preferentially recognized by bNAbs [5,7]. We and others have reported that temsavir or its BMS-806 analog alter Env glycosylation and cleavage, thereby impacting its recognition by bNAbs [17,18]. Here, we used a panel of primary infectious molecular clones of HIV-1, expression plasmids for wild-type Env, or a cleavage-deficient mutant to evaluate the impact of temsavir on proteolytic Env processing and the recognition of bNAbs. Our findings suggest that the effect of temsavir on Env conformation is linked to its impact on Env cleavage. Albeit to different extents, this effect was observed with multiple Envs and R406 (Tamatinib) was generally associated with R406 (Tamatinib) a decreased recognition and antibody-dependent cellular cytotoxicity (ADCC) response mediated by bNAbs. 2. Materials and Methods 2.1. Ethics Statement Written informed consent was obtained from R406 (Tamatinib) all study participants, and research adhered to the ethical guidelines of the Centre de recherche du Centre hospitalier de lUniversit de Montral (CRCHUM), which was reviewed and approved by the CRCHUM institutional review board (ethics committee; approval number: CE 16.164CCA; approval date: 19 October MGC14452 2021). The research adhered to the standards indicated by the Declaration of Helsinki. 2.2. Cell Lines and Isolation of Primary Cells HEK 293T human embryonic kidney cells (obtained from ATCC) were maintained at 37 C under 5% CO2 in Dulbeccos modified Eagles medium (DMEM; Wisent, St. Bruno, QC, Canada) containing 5% fetal bovine serum (FBS; VWR, Radnor, PA, USA) and 100?g/mL penicillinCstreptomycin (Wisent). Primary human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells were isolated, activated, and cultured, as previously described [19]. Briefly, PBMCs were obtained by leukapheresis from six HIV-negative individuals (3 males and 3 females). CD4+ T cells were purified from rested PBMCs by negative selection (EasySep human CD4+ T cell enrichment R406 (Tamatinib) kit; STEMCELL Technologies, Vancouver, BC, Canada) and were activated with phytohemagglutinin-L (10 g/mL) for R406 (Tamatinib) 48 h and then maintained in an RPMI 1640 complete medium supplemented with rIL-2 (100 U/mL). 2.3. Plasmids and Proviral Constructs The vesicular stomatitis virus G (VSV-G)-encoding plasmid was previously described [20]. The sequence of full-length clade B HIV-1JRFL Env and clade A HIV-1BG505 (N332) were codon-optimized (GenScript).
Although all these Envs were susceptible to temsavir-mediated viral inhibition, as measured in a single-round pseudoviral neutralization assay, their susceptibility to temsavir varied (Table S1)
