Bastiray, A. | Search bioRxiv

bioRxiv2025

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

The Tom70-Hsp90 interaction is critical for activating 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, the SARS-CoV-2 protein Orf9b binds to Tom70, thereby disrupting the Tom70-Hsp90 interaction and suppressing IFN production. Despite its importance, the molecular mechanism underlying Orf9b-mediated inhibition of IFN signaling remains unclear. Here, using an integrative approach combining cryo-electron microscopy, 19F NMR spectroscopy, and isothermal titration calorimetry (ITC), we show that Orf9b inhibits Hsp90 binding to Tom70 through a bipartite mechanism. The helix and intrinsically disordered tail of Orf9b sterically block the access of two distinct structural units of Hsp90 to Tom70. We also find that Orf9b-mediated allosteric conformational changes in Tom70 do not contribute to the inhibition of the Hsp90 binding. Comprehensive structural, thermodynamic, and kinetic analyses further reveal that Orf9b primarily slows the association kinetics between Hsp90 and Tom70. Collectively, our results provide a high-resolution mechanistic framework for understanding Orf9b-mediated suppression of the host innate immune response.

biophysics↗

bioRxiv2022

The energy landscape reshaped by strain-specific mutations underlies the long-range epistasis in NS1 evolution of influenza A virus

The mechanisms underlying how individual mutations affect the protein energy landscape are crucial for understanding how proteins evolve. However, predicting mutational effects remains challenging because of epistasis--the nonadditive interactions between mutations. Here, we investigate the biophysical mechanism of strain-specific epistasis in the nonstructural protein 1 (NS1) of the influenza A virus (IAV). To understand the molecular basis of epistasis, we conducted comprehensive analyses of four NS1s of IAV strains that emerged between 1918 and 2004. We find that strain-specific mutations of NS1s are near-neutral with respect to the association with the p85{beta} subunit of PI3K. However, the individual residues on the p85{beta}-binding interface show long-range epistatic interactions with strain-specific mutations. We reveal that strain-specific mutations reshaped the energy landscape of NS1, leading to long-range epistasis. Our findings offer a high-resolution mechanism of how near-neutral mutations silently alter the biophysical energy landscapes, resulting in diverse background effects during molecular evolution.

biophysics↗