Probing conformational dynamics of HIV-1 Env in real time and in situ during virus entry

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Research Abstract: The envelope (Env) glycoprotein is a virus-encoded protein exposed on the surface of HIV- 1 virions and is the primary target of neutralizing antibodies against HIV-1. Env is structurally flexible or conformationally dynamic, allowing HIV-1 to enter susceptible cells via membrane fusion and evade antibody recognition. Thus, the highly dynamic Env is a critical target for developing HIV-1 vaccines, inhibitors, and cure strategies. However, a lack of knowledge on the structural dynamics of Env entangles the attempts to comprehend the mechanisms by which Env enables virus entry and facilitates immune evasion, which is at the core of Env-directed interventions. Env is a trimer in which each protomer consists of two subunits, gp120 and gp41. The surface-exposed gp120 contains receptor/coreceptor binding sites, and the transmembrane gp41 harbors the fusion machinery. Upon binding to the receptor CD4, gp120 undergoes conformational changes that lead to the exposure of a coreceptor-binding site. Subsequent interactions with the coreceptors will then activate a cascade of fusion-promoting refolding events in gp41, progressing from a metastable pre-fusion state, through the putative pre-hairpin intermediates, to the stable post-fusion six-helix bundle that leads to the fusion of viral and cellular membranes. High-resolution structures of Env have significantly furthered our understanding of Env architectures and antigenicity during virus entry. However, real-time conformational dynamics of Env that delineate the sequence of events that underlies the structural transformations and the fusogenic transitions remain largely elusive. Single-molecule Förster resonance energy transfer (smFRET) was applied upon introducing FRET-paired fluorophores into gp120 to reveal the dynamic features of virus-associated full-length Env. We have demonstrated that native Env is dynamic and, upon interaction with CD4, transitions from the pre- triggered conformation (State 1) through a partially open intermediate (State 2) to the fully activated open conformation (State 3). Many broadly neutralizing antibodies (bNAbs) demonstrate a preference for State 1-Env. Unexpectedly, the designed soluble Env, the detergent-solubilized Env, and the aldrithiol-2 inactivated virus- associated Env all exhibit a propensity to occupy State 2, offering a different perspective on the current spectra of Env-based vaccine design. Our studies on gp120 using smFRET have set the stage for our next goal—to investigate the less-understood fusion protein gp41. Specifically, we propose to use the dynamic-and-static in- situ imaging platform of smFRET and cryo-electron tomography (cryoET) to directly visualize real-time conformational dynamics/events of virus-embedded gp41 that correlate with membrane fusion and characterize potential drug-targeting gp41 intermediates structurally. The ultimate goal is to generate a fusion movie serving as the framework for developing HIV-1 interventions that direct against virus entry. We will also use our platform to specify conformational recognition preferences for virus-associated Env of gp41-directed neutralizing antibodies to inform the development of antibody cocktails and the “antibody-to-vaccine” strategy.