DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions

HIV-1 Envelope (Env) mediates viral-host membrane fusion after binding host-receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion-bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand-free, CD4-bound, inhibitor-bound, and antibody-bound SOSIPs, we measured inter-subunit distances throughout spin-labeled SOSIPs using double electron-electron resonance (DEER) spectroscopy and compared results to soluble and virion-bound Env structures, and single-molecule fluorescence resonance energy transfer (smFRET)-derived dynamics of virion-bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody-bound structures and shielding of non-neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra-subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter-subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion-bound Envs and demonstrate DEER's relevance for immunogen design. HIV-1 Env, the only target of neutralizing antibodies, is highly dynamic, and only snapshots of static conformations are available. Stadtmueller et al. used DEER spectroscopy to map conformations of soluble Env and its complexes with antibodies or the CD4 receptor. Results reveal similarities to virion-bound Env and buried non-neutralizing antibody epitopes, advancing knowledge of Env function and vaccine design.