HIV-1 Env protein structure and function assessed by parallel smFRET and cryoET

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Summary The HIV-1 envelope protein (Env) mediates virus entry into cells while protecting functional centers from antibodies. As the only virus protein on the surface of virus particles, HIV-1 Env is a major target for vaccine development. We initiated studies on HIV-1 Env by establishing single-molecule Förster Resonance Energy Transfer (smFRET) to monitor the sampling of a single protomer in the context of a Env trimer on the surface of a virus particle. smFRET data indicated that HIV-1 Env is conformationally dynamic and samples at least three conformational states. Associating the observed FRET states with existing HIV-1 Env structural information revealed a conformational state on the surface of viruses that cannot be explained by current high-resolution structures. The controversy surrounding smFRET data prompted us to establish cryo-electron tomography (cryoET) as an orthogonal structural method. The advantage of cryoET and smFRET is that both methods allow for characterization of the structure and dynamics of Env on the surface of viruses. In addition, we identified experimental conditions that arrest a high number of Env trimers at various stages of the entry process to allow a structural characterization by cryoET and subsequent subtomogram averaging. We succeeded with a system where we incubate native HIV-1 viruses with virus-like particles (VLP) carrying receptor CD4 and coreceptor molecules. We observed Env-CD4 opposing CD4 membranes asymmetric conformational CD4 of availability on the surface of viruses that cannot be explained by current high-resolution structures. refined states. A detailed understanding of the structure and dynamics of the HIV-1 Env protein is important for the complexes to cluster at membrane-membrane interfaces thereby bringing membranes closer together. Subtomogram averaging and classification revealed that Env bound one molecule when membranes were further apart, then engaged two, and finally three CD4 molecules as moved closer together. HIV-1 Env trimers bound to one and two CD4 molecules adopted conformational states. These cryoET studies recall earlier smFRET work in our laboratory that a intermediate in the opening of the Env trimer corresponds to an asymmetric trimer with a single bound. I n this proposal, we will apply our cryoET methodology to next structurally characterize the transition Env from CD4 to coreceptor followed by activation into the pre-hairpin intermediate. Reenergized by the of cryoET and advances on smFRET, we will pursue our efforts to characterize a Finally, we are proposing smFRET methods to meet the increasingly complex structural insights into distinct Env conformational conformational state development of immunogens for vaccines and small molecule inhibitors against Env.