Carlon Andres, I. | Search bioRxiv

bioRxiv2025

Alpha-BET: Functional labeling of envelope glycoproteins with single domain antibodies for in-virus single molecule imaging

We present Alpha-BET, a structure-guided strategy leveraging AlphaFold to identify optimal ALFA-tag insertion sites for minimally disruptive labeling of viral glycoproteins with high-affinity nanobodies. Applied to HIV-1 Env, SARS-CoV-2 S, and NiV G, Alpha-BET preserves structural integrity and function. For HIV-1 Env, we demonstrate super-resolution DNA-PAINT MINFLUX 3D imaging enabled by tag insertion, showcasing its power for visualizing native trimers in single virions and potential for broader applications in virus research.

biophysics↗

bioRxiv2020

Structure and Dynamics of HIV-1 Env Trimers on Native Virions Engaged in Living T Cells

The HIV-1 envelope glycoprotein (Env) mediates viral entry into the host cell. Although the highly dynamic nature of Env intramolecular conformations has been shown with single molecule spectroscopy in vitro, the bona fide Env intra- and intermolecular mechanics when engaged in live T cells remains unknown. We used both, two photon fast fluorescence lifetime imaging detection of single-molecule Forster Resonance Energy Transfer and single molecule photobleaching to reveal transitions between intramolecular and intermolecular conformations that mediate Env clustering. Furthermore, we show that three broad neutralizing anti-Env antibodies directed to different epitopes destabilise Env intramolecular dynamics and their clusters when engaged to living T cells. Importantly, our results show that Env clustering is a common conformation across different HIV-1 Env strains, which depends on efficient virus maturation, and that is disrupted upon binding of Env to CD4 or to neutralizing antibodies. Thus, this study illuminates how different intramolecular conformations and clusters of Env mediate HIV-1 Env-T cell interactions in real time and therefore might control immune evasion.

immunology↗