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

> **NIH NIH R37** · YALE UNIVERSITY · 2023 · $829,461

## Abstract

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.

## Key facts

- **NIH application ID:** 10761955
- **Project number:** 2R37AI150560-05
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** WALTHER H MOTHES
- **Activity code:** R37 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $829,461
- **Award type:** 2
- **Project period:** 2019-07-18 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10761955

## Citation

> US National Institutes of Health, RePORTER application 10761955, HIV-1 Env protein structure and function assessed by parallel smFRET and cryoET (2R37AI150560-05). Retrieved via AI Analytics 2026-05-29 from https://api.ai-analytics.org/grant/nih/10761955. Licensed CC0.

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