Molecular evolution of entry receptor usage underlying zoonotic human betacoronaviruses

PROJECT SUMMARY Most human viruses originate from recent zoonotic spillover, but the upstream evolutionary processes in animal reservoirs that drive zoonosis-promoting traits remain poorly understood. Our long-term goal is to elucidate the evolutionary forces enabling animal viruses to acquire traits facilitating human spillover and adaptation, with a focus on viral entry receptor usage as a critical determinant of cross-species transmission. Toward this end, this proposal investigates the evolutionary dynamics underlying changes in receptor-binding specificity in beta-coronaviruses (CoVs) linked to past and potential future zoonoses: SARS-CoV-2, MERS- CoV, and HKU1 alongside their bat, rodent, and other animal relatives. Our central model is that long-term evolutionary arms races between viruses and wildlife hosts drive evolvable mechanisms of receptor- engagement promoting subsequent human spillover and adaptation. This model will be examined through three specific aims: (1) Identify mechanisms driving human receptor binding in bat SARS-related CoVs; (2) Dissect the origins and consequences of receptor-switching in bat MERS-related CoVs; and (3) Identify evolutionary origins of and functional constraints imposed by a newly discovered HKU1 CoV receptor. In each aim, we combine phylogenetic surveys across diverse animal CoVs with high-throughput mutagenesis screens to map the evolutionary, genetic, and structural mechanisms driving receptor-use transitions and their downstream evolutionary consequences. These studies will illuminate how host-virus dynamics shape receptor-binding architectures to enable zoonotic potential and post-spillover antigenic evolution. The resulting large-scale genotype-phenotype maps will inform computational models for assessing viral zoonotic risk and guide the design of broad-spectrum antibody and vaccine therapeutics for pandemic preparedness. Taken together, this work advances understanding of mechanisms of viral evolution while providing actionable insights for proactive ecological, diagnostic, and therapeutic interventions.