Project Summary: The envelope (Env) glycoprotein exposed on the surface of HIV-1 virions is essential for
virus entry into susceptible cells and is the primary target of neutralizing antibodies, small molecules, and fusion
inhibitors. The structural dynamics of Env promote HIV-1 entering susceptible cells via membrane fusion and
facilitate antibody evasion. However, a lack of knowledge about the time-resolved structural dynamics and
allostery of Env impedes the attempts to comprehend the mechanisms by which Env enables virus entry and
facilitates immune evasion. Env is trimeric, in which each protomer is composed of two subunits: exterior gp120
for binding to cellular receptors/coreceptors and membrane-anchored gp41 for driving fusion. Upon interacting
with cellular receptors/coreceptors, gp120 undergoes large-scale conformational changes, further activating a
series of fusion-promoting structural refolding in gp41. Refolding in gp41 progresses from a prefusion state
through intermediates to the post-fusion that is generally believed to drive membrane fusion. Structures of Env
have provided atomic details of Env conformations at fusion endpoints. However, substantial knowledge gaps in
the Env-mediated fusion mechanism cannot be bridged by conventional structural tools. These gaps include
time, sequence, reversibility, transitional paths, and transient intermediate steps of structural changes of Env.
The proposed research is based on the hypothesis that Env is a highly cooperative dynamic machine in which
conformational changes reflected in gp120 and gp41 during fusion occur in well-ordered spatial and temporal
equilibrium. Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) studies with fluorescent
labels placed on gp120 demonstrated that native Env intrinsically interchanges between multiple conformations
and has responsive conformational sampling to CD4 activation, neutralizing antibodies, and inhibitors. This
project will elucidate spatiotemporal information of Env in its native state, with focused investigations on the less-
studied gp41 and its association with gp120, and ultimately provide a time-resolved stepwise spatial framework
of the fusion process. Multiple synergic methods, including smFRET in the context of intact virions, an integrated
enhanced sampling molecular dynamics (MD)-smFRET, AI-guided multiscale simulations/modeling for mapping
Env allostery, cell-based virological assays, and photophysical analysis will be used. The research is built upon
recently established minimally invasive smFRET imaging of Env using genetic code expansion combined with
click chemistry. It will be further extended to multi-perspective for monitoring gp120-gp41 associated
cooperativity and capturing structural changes of gp41. The conformational dynamics of Env will be monitored
from three different structural perspectives. Neutralizing antibodies and fusion inhibitors will be used to facilitate
revealing conformational cooperativities of Env and spatiotemporally resolving individual fusion intermediates.
The expected results will provide new insights about the time-resolved structural dynamics and allostery of Env
that can inform the design of Env-centric interventions such as vaccines, antibody therapy, and anti-viral drugs.
