Structure-function studies of the membrane-interacting domains of HIV-1 Env spike

Project Summary
The first critical step for enveloped viruses, such as HIV-1, to enter host cells is viral membrane fusion.
Viral fusion proteins are fascinating protein folding machineries capable of adopting completely different
conformations during the fusion process; they are also important vaccine and therapeutic targets. Previous
studies have revealed both pre- and post-fusion conformations of the soluble fragments of many viral fusion
proteins, but less is known for structures of their fusion peptide, transmembrane and membrane-proximal
regions in the context of lipid bilayers. There is strong evidence for functional roles of the membrane-
interacting regions in fusion, and yet mechanistic studies on how they exert their functions remain scarce. We
hypothesize that membrane-interacting regions of other fusion proteins related to HIV-1 envelope
protein (Env) adopt defined oligomeric structures that are critical for the stability, function and
antigenicity of the full-length proteins in membrane. In the studies that we completed during the previous
funding period, we have determined the structures of the TM, membrane proximal external region, and
cytoplasmic tail of HIV-1 Env in bicelles that mimic lipid bilayers using the latest NMR technology. We find that
these regions all form well-ordered trimeric clusters and are conformationally coupled, and that disrupting them
can reduce fusion and alter the antigenic structure of the entire Env. In this application, we propose to apply
our NMR/bicelle technology to investigate the membrane regions of SIV Env and the recently emerged SARS-
CoV-2 spike (S), and to use cryo-electron microscopy to determine structures of the full-length proteins
reconstituted in lipid nanodiscs. We will define roles in membrane fusion of critical structural elements of these
regions by deep mutagenesis and functional assays. We will purse the following specific aims: 1) we will
investigate the membrane-interacting components of SIV Env; 2) we will investigate the membrane-interacting
components in the postfusion arrangement; 3) we will determine structures of the full-length SIV Env and
SARS-CoV-2 S in the context of membrane; 4) we will elucidate roles of the membrane-interacting domains of
HIV/SIV Env and SARS-CoV-2 S in their stability, function and antigenicity.

Grant Number: 5R01AI127193-10
NIH Institute/Center: NIH
Principal Investigator: Bing Chen