SARS-CoV-2 Orf3a protein interaction mapping using unnatural amino acid incorporation.

Mapping transient protein-protein interactions remain a major challenge in studying viral host-pathogen interfaces. While some virus-host interactions are stable and readily captured, the majority are highly dynamic, reflecting the need for viral proteins to engage distinct host factors at different stages of the life cycle. Here, we employ a protein engineering strategy based on the site-specific incorporation of the unnatural acid p-azido-L-phenylalanine (AzF) to enable photo-crosslinking proteomic analysis of the SARS-CoV-2 accessory protein Orf3a in live cells. Genetic installation of AzF at residue K198 of Orf3a permitted UV-induced covalent capture of proximal host interacting proteins, overcoming challenges associated with membrane localization and limited protein abundance. A total of 248 high-confidence Orf3a-interacting proteins were reproducibly identified and subjected to gene ontology analysis, revealing enrichment in innate immune signaling, antiviral defense, RNA processing, and viral replication-associated pathways. Orf3a is an accessory protein that functions as a viroporin and traffics across multiple cellular compartments, and was found to interact with host RNA helicases, RNA-binding proteins, immune regulators, and metabolic enzymes implicated in SARS-CoV-2 infection. Together, these results demonstrate that genetically encoded, site-specific photo-crosslinking enables selective capture of transient interactions that are often missed by nonspecific 254 nm UV crosslinking approaches and highlights Orf3a as a multifunctional protein that engages diverse host pathways. More broadly, this study establishes a generalizable framework for leveraging unnatural amino acid-based protein engineering approaches to interrogate dynamic host-pathogen interactions.