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

  1. David J Williamson
  2. Cecilia Zaza
  3. Irene Carlon Andres
  4. Alessia Gentili
  5. James I Daly
  6. Harry Holmes
  7. Joseph Thrush
  8. Tobias Starling
  9. Stuart Neil
  10. Ray Owens
  11. Michael M Malim
  12. Christopher Tynan
  13. Sabrina Simoncelli
  14. Sergio Padilla-Parra
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Abstract

We present Alpha-BET, a structure-guided strategy leveraging AlphaFold to identify optimal ALFAtag insertion sites for minimally disruptive labeling of viral glycoproteins with high-affinity nanobodies. Applied to HIV-1 Env, SARS-CoV-2 S, and Nipah 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 virological applications.

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