HIV-1 Fusion on Native Membranes Captured by Cryo-Electron Tomography

HIV-1 Fusion on Native Membranes Captured by Cryo-Electron Tomography Summary Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding to host cells through interactions of the viral envelope glycoprotein (Env) with the cellular receptor CD4 and co-receptors CCR5 or CXCR4. While structures of Env in complex with soluble domains of these receptors have been determined in vitro, the understanding of how these proteins interact on membranes to mediate fusion remains incomplete. Specifically, it is poorly understood how Env, CD4, CCR5/CXCR4 and membranes interact in a higher-order manner at membrane-membrane interfaces, and how the conformational changes of Env, triggered by the engagement of receptors, lead to membrane fusion. These molecular events need to be investigated in their native environment on membranes, where virus fusion occurs. Using a virus-like particle (VLP) system and cryo-electron tomography (cryoET), we have recently reconstituted prefusion membrane-membrane interfaces and revealed unprecedented intermediates of HIV-1 Env bound to one, two, and three CD4 molecules. At these interfaces, Env–CD4 complexes organized into clusters and rings, bringing the opposing membranes closer. Although adopted to an open conformation, the Env trimer bound to three CD4 molecules did not engage the membrane-embedded co-receptor, likely due to steric constraints imposed by membrane-associated CD4 molecules. Consequently, key structures such as HIV-1 Env bound to co-receptors and Env intermediates during refolding that drive membrane fusion are still missing. Here, we propose to extend our research into the dynamics of HIV-1 fusion using cryoET and integrate molecular dynamics (MD) simulations to understand how these components orchestrate the fusion process, to determine the structural intermediates during HIV-1 Env refolding that drive fusion, and to investigate the mechanisms by which fusion inhibitors block HIV-1 infection. Specifically, we will 1) determine if HIV-1 Env-CD4 receptor clustering is necessary or advantageous for HIV-1 Env fusion, 2) determine how Env binds to coreceptor followed by the refolding of gp41 that drives membrane fusion, and 3) investigate the timeline of action for various HIV-1 fusion inhibitors and their effects on the conformational transitions of Env during fusion.