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

David J. Williamson; Cecilia Zaza; Irene Carlón-Andrés; Alessia Gentili; Janet M. Daly; Harry Holmes; Joseph W. Thrush; Tobias Starling; Stuart J. D. Neil; Raymond J. Owens; Michael H. Malim; Christopher J. Tynan; Sabrina Simoncelli; Sergi Padilla‐Parra

doi:10.1101/2025.04.11.648405

BiophysicsbioRxivApr 11, 2025

Preprint

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

David J. Williamson iD, Cecilia Zaza iD, Irene Carlón-Andrés iD, Alessia Gentili iD, Janet M. Daly iD, Harry Holmes iD, Joseph W. Thrush iD, Tobias Starling, Stuart J. D. Neil iD, Raymond J. Owens iD, Michael H. Malim iD, Christopher J. Tynan iD, Sabrina Simoncelli iD, Sergi Padilla‐Parra iD

Abstract

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.

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