Abstract 2061 The mechanistic basis for immune evasion by Orf9b of SARS-CoV-2

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is considered the worst pandemic of the 21st century. Despite the successes in vaccine production and treatment, there remains an urgent need to understand how SARS-CoV-2 evades the innate immune system to prevent future outbreaks. An accessory protein of SARS-CoV-2, open reading frame 9b (Orf9b), is a crucial antagonist of the antiviral response. Orf9b binds to the translocase of mitochondrial outer membrane 70 (Tom70), preventing the interaction of Tom70 with heat shock protein 90 (Hsp90). This inhibition subsequently blocks the interferon response against SARS-CoV-2. However, despite its importance, the molecular mechanism of how Orf9b inhibits the Tom70-Hsp90 interaction remains unclear. Whereas Orf9b binds to the hydrophobic pocket in the C-terminal domain of Tom70, Hsp90 is thought to interact with the N-terminal domain of Tom70 through its EEVD motif. To elucidate the structural basis of this mechanism, we employ single particle cryo-electron microscopy (cryo-EM) and fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR). In combination with other biophysical approaches, such as isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we reveal the structural basis by which Orf9b inhibits the binding of Tom70 to Hsp90. Our results demonstrate the mechanistic basis for the competitive binding of Orf9b and Hsp90 to Tom70. We expect that our results will provide mechanistic insights into how SARS-CoV-2 inhibits the host innate immune response through Tom70 antagonization. This project was funded by the Welch Foundation (Grant No. A-2028-20230405).