Coronavirus protein interaction mapping in bat and human cells reveals network rewiring governing immune evasion and zoonotic potential.

Coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can cause severe disease in humans, whereas reservoir hosts such as horseshoe bats remain asymptomatic. To investigate how host-specific protein-protein interactions (PPIs) influence infection, we generated comparative PPI maps for SARS-CoV-2 and its bat progenitor RaTG13, using affinity purification mass spectrometry (AP-MS) in human and greater horseshoe bat cells. We identify both conserved and virus- and host-specific interactions that regulate infection dynamics. Notably, SARS-CoV-2 requires a nonsynonymous mutation in the nucleocapsid to replicate in bat cells expressing human ACE2 and TMPRSS2. Strikingly, a single amino acid difference in Orf9b between viruses acts as a molecular switch that reprograms mitochondrial targeting: in human cells, enhanced translocase of outer mitochondrial membrane 70 (Tom70) binding promotes immune evasion, whereas in bat cells, strengthened interaction with the bat-enriched restriction factor mitochondrial amidoxime reducing component 2 (MTARC2) limits infection. These findings establish a general principle by which minimal sequence variation can reshape virus-host interactions and contribute to immune antagonism, host adaptation, and species barriers.