Molecular mechanism by which SARS-CoV-2 Orf9b suppresses the Tom70-Hsp90 interaction to evade innate immunity.

The Tom70-Hsp90 interaction is critical for MAVS-mediated interferon (IFN) production. Upon RNA virus infection, cytosolic Hsp90 recruits key innate immune signaling proteins to MAVS on mitochondria through its interaction with Tom70. To evade this innate immune response, SARS-CoV-2 Orf9b binds to Tom70, thereby disrupting the Tom70-Hsp90 interaction and suppressing IFN production. Despite its importance, the molecular mechanism underlying the Orf9b-mediated IFN antagonism has remained unclear. Here, using an integrative approach including cryo-electron microscopy and 19F NMR spectroscopy, we show that Orf9b inhibits the Tom70-Hsp90 interaction through a bipartite mechanism. Through comprehensive structural, thermodynamic, and kinetic analyses, we reveal a previously unrecognized interplay between the rigid and dynamically disordered regions of Orf9b that blocks Hsp90 access to Tom70. Collectively, our results provide a high-resolution mechanistic framework for understanding Orf9b-mediated suppression of the host innate immune response.