Research

Illustration of the technological pipeline for a whole-body imaging-driven multiscale investigative approach to study virus infection of murine leukemia virus (MLV), HIV-1 and SARS-CoV-2. (Top) Longitudinal NBLI reveals infection of NLuc-expressing replication competent viruses informs subsequent focused studies at infected tissues. (Second from top) Intravital microscopy monitoring infected cells within tissues. (Middle, and second from bottom) Confocal immunohistochemistry or light sheet fluorescence microscopy of infected cells within the architecture of the tissue. (Bottom) Electron tomography characterization of virus infected cells. See Review for details.
(Image credit: Pradeep Uchil)

We utilize noninvasive whole-body bioluminescence imaging (NBLI) to monitor virus dissemination and spread in living animals. Whole body imaging guides us to infected tissues that we study at the single cell level using multi-photon laser scanning microscopy and immunohistochemistry. Electron tomography permits insights at the ultrastructural level. This approach allows us to study viral dissemination, pathogenesis and immune responses over 8 orders of magnitude.

Cryo-electron tomography of HIV-1 envelope binding to CD4 in membranes reveals asymmetric intermediates https://www.nature.com/articles/s41586-023-06762-6 that were previously predicted by smFRET experiments and https://elifesciences.org/articles/34271.
(Image credit: Wenwei Li)

Progress in the structural understanding of the HIV-1 and SARS-CoV-2 viral spike proteins has produced static images of viral pre-fusion and post-fusion conformations. To provide insights into the dynamics of the native trimer, we have established single molecule technologies to measure the conformational changes of individual spike molecules on the surface of intact viruses (Insert links https://pubmed.ncbi.nlm.nih.gov/25298114/ https://pubmed.ncbi.nlm.nih.gov/33242391/. Introducing a pair of donor and acceptor fluorophores allowed the direct observation of conformational changes through time-dependent changes in the efficiency of Förster resonance energy transfer (FRET). Our studies revealed that the unliganded spike proteins of HIV-1 and SARS-CoV-2 are dynamic, and that activation by receptors CD4 or ACE2 stabilizes pre-existing conformational states through a necessary structural intermediate. Conformational assays for the spike proteins of both viruses allow us to determine the conformational preferences of neutralizing antibodies and as such inform immunogen design for vaccines.

As an orthogonal structural method, we have established cryo-electron tomography (cryoET) to monitor the structure of viral spike proteins on the surface of native virus particles. Parallel cryoET and smFRET will provide a comprehensive understanding of the structure and dynamics of the HIV-1 and SARS-CoV-2 spike proteins. We have also established an experimental system that allows us to study how viral glycoproteins interact with receptors in biological membranes and how they are activated to mediate fusion between viral and cellular membranes. First insights have revealed 1) how asymmetric HIV-1 Env trimers can engage one and two CD4 molecules before binding to three CD4 molecules, and 2) how interaction of SARS-CoV-2 spike with receptor leads to spike refolding, driving membranes together for membrane fusion. Identifying fusion intermediates can inform antiviral therapies that target these steps, thereby interfering with viral spread.

(1) Spike is composed of 2 subunits, S1 (brown) S2 (tan). S1 binds to host receptor ACE2 (blue). (2) S1 sheds off and exposes S2. (3) S2 extends and inserts into the host cell membrane. (4) S2 refolds and pulls virus and host membranes together. (5) Spike reaches its postfusion state as membranes fuse. Antibodies (green) bind S2 and inhibit refolding to prevent membrane fusion. (Image credit: Michael W. Grunst) https://www.science.org/stoken/author-tokens/ST-2051/full This is the author's version of the work. It is posted here by permission of the AAAS for personal use, not for redistribution. The definitive version was published in Science on Aug 15, 2024, DOI: 10.1126/science.adn565.