Asymmetric HIV-1 envelope trimers bound to one and two CD4 molecules are intermediates during membrane binding

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD4. Although high resolution structures of Env complexed with soluble domains of CD4 have been determined, the binding process is less understood on native membranes. Here, we apply cryo-electron tomography (cryo-ET) to monitor Env-CD4 interactions at membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing opposing membranes closer together. Additionally, Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound one, two, and finally three CD4 molecules, upon which Env adopted a partially open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which likely has consequences for antibody-mediated immune responses and vaccine immunogen design.