Amber suppression technology for mapping site-specific viral-host protein interactions in mammalian cells

• Main Authors: Isa, Nur Firdaus, Bensaude, Olivier, Murphy, Shona
• Format: Article
• Language: English; English
• Published: Bio-protocol LLC 2022
• Subjects: QR355 Virology; RB Pathology
• Online Access: http://irep.iium.edu.my/96649/1/96649_Amber%20suppression%20technology%20for%20mapping%20site-specific.pdf; http://irep.iium.edu.my/96649/7/96649_Amber%20suppression%20technology_Scopus.pdf; http://irep.iium.edu.my/96649/; https://bio-protocol.org/e4315

Probing the molecular interactions of viral-host protein complexes to understand pathogenicity is essential in modern virology to help the development of antiviral therapies. Common binding assays, such as co-immunoprecipitation or pull-downs, are helpful in investigating intricate viral-host proteins interactions. However, such assays may miss low-affinity and favour non-specific interactions. We have recently incorporated photoreactive amino acids at defined residues of a viral protein in vivo, by introducing amber stop codons (TAG) and using a suppressor tRNA. This is followed by UV-crosslinking, to identify interacting host proteins in live mammalian cells. The affinitypurified photo-crosslinked viral-host protein complexes are further characterized by mass spectrometry following extremely stringent washes. This combinatorial site-specific incorporation of a photoreactive amino acid and affinity purification-mass spectrometry strategy allows the definition of viral-host protein contacts at single residue resolution and greatly reduces non-specific interactors, to facilitate characterization of viral-host protein interactions