Mapping the Functional Architecture of Influenza A Virus-Induced Phase Separation through m⁶A Proximity Labeling

The precise spatial organization of biomolecules on the nanoscale is fundamental to cellular function. However, the molecular mechanisms governing this intricate architecture remain largely unexplored, particularly the role of RNA chemical modifications such as N6-methyladenosine (m⁶A) in orchestrating nanoscale order and functional compartmentalization. Here, we developed a spatially resolved m⁶A proximity labeling (m⁶APL) technique to directly map the subcellular localization and protein interaction network of m⁶A-modified transcripts. This approach enables in situ mapping of m⁶A sites, deciphers their RNA sequences, and identifies proteins in their immediate nanoscale vicinity. Applying m⁶APL, we discovered that influenza A virus (IAV) infection triggers the assembly of cytoplasmic, phase-separated inclusion bodies (IBs) that function as specialized, m⁶A-enriched replication hubs. We found that the upregulation of m⁶A on ribosome-related mRNAs, coupled with the enrichment of UGGT1 and SNRNP70 within these condensates, repurposes IBs into efficient factories for viral protein synthesis. Strikingly, we uncovered that SNRNP70 undergoes a spatial reallocation from the nucleus to the endoplasmic reticulum, driven by its strong affinity for the 3' conserved sequence of IAV mRNA. This redistribution is a critical event in nucleating the infection-induced biomolecular condensates. Our work establishes m⁶A-mediated interactions as a key principle driving the formation and function of these virus-host compartments.