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10.1128/JVI.02652-15. antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers 1-Methylpyrrolidine during natural infection recognize more open Env conformations (states 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and cross-linking decreased the exposure of pNAb epitopes on cell surface gp160; however, after detergent solubilization, cross-linked and BMS-806-treated gp160 sampled non-state-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume state-2-like conformations. Asymmetry of 1-Methylpyrrolidine the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a state-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage for a persistent virus like HIV-1. KEYWORDS: Env, cleavage, furin, processing, conformation, cryo-electron microscopy, structure, antibody, asymmetry INTRODUCTION Human immunodeficiency virus (HIV-1), the etiologic agent of AIDS, utilizes a metastable envelope glycoprotein (Env) trimer to engage host receptors and enter target cells (1). The functional Env trimer consists of three gp120 exterior subunits and three gp41 transmembrane subunits (1,C3). During virus entry, gp120 engages the receptors, CD4 and CCR5/CXCR4, and gp41 fuses the viral and cell membranes (4,C16). Env is the only virus-specific protein on the viral surface and is targeted by host antibodies (17,C20). In infected cells, the HIV-1 Env trimer is synthesized in the rough endoplasmic reticulum (ER), where signal peptide cleavage, folding, trimerization, and the addition of high-mannose glycans take place (21,C24). The resulting gp160 Env precursor is transported to the Golgi apparatus, where some of the glycans are modified to complex types and proteolytic cleavage by host furin-like proteases produces the gp120 and gp41 subunits (25,C41). The proteolytically processed, mature Env trimers are transported to Elf3 the cell surface and incorporated into virions. On the membrane of primary HIV-1, Env exists in a pretriggered, closed conformation (state 1) that resists the binding of commonly elicited antibodies (42,C47). Binding to the receptor, CD4, on the target cell releases the restraints that maintain Env in state 1, allowing transitions through a default intermediate conformation (state 2) to the 1-Methylpyrrolidine prehairpin intermediate (state 3) (42, 48, 49). In the more open state-3 Env conformation, a trimeric coiled coil composed of the gp41 heptad 1-Methylpyrrolidine repeat (HR1) region is formed and exposed, as is the gp120 binding site for the second receptor, either CCR5 or CXCR4 (6, 7, 50,C56). Binding to these chemokine receptors is thought to promote the insertion of the hydrophobic gp41 fusion peptide into the target cell membrane 1-Methylpyrrolidine and the formation of a highly stable six-helix bundle, which mediates virus-cell membrane fusion (14,C16, 57,C60). The ability of HIV-1 to establish persistent infections in humans requires an Env trimer that minimally elicits neutralizing antibodies and resists the binding of antibodies generated during the course of natural.