Structural Basis of Bat and Raccoon Dog Angiotensin-Converting Enzyme 2 (ACE2) Receptor Recognition by Sarbecoviruses

Understanding the mechanisms that facilitate cross-species transmission into human populations is essential for elucidating the evolutionary origins and pandemic potential of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This dissertation investigates the molecular basis of receptor recognition in two species relevant to the emergence of COVID-19: the horseshoe bat (Rhinolophus sinicus) as a potential natural host and the raccoon dog (Nyctereutes procyonoides) as a suspected intermediate host. Coronaviruses evolve to optimize receptor recognition in their natural hosts. A central question regarding the emergence of SARS-CoV-2 is why its receptor-binding domain (RBD) binds human angiotensin-converting enzyme 2 (ACE2) with high affinity despite limited apparent time for adaptation, and why some bat-derived RBDs paradoxically bind human ACE2 more strongly than bat ACE2. To address this question, we compared the RBDs of SARS-CoV-2 and BANAL-52, a related bat coronavirus, and examined their inter- actions with ACE2 from Rhinolophus sinicus and humans. Structural and biochemical analyses revealed that the BANAL-52 RBD is well adapted for binding to R. sinicus ACE2, with His498 of the BANAL-52 RBD forming a favorable interaction with His41 of R. sinicus ACE2. In contrast, the SARS-CoV-2 RBD preferentially recognizes human ACE2, largely due to His34 and Met82 in human ACE2, residues that broadly enhance RBD binding. These findings demonstrate that receptor recognition by SARS-CoV-2 and related bat coronaviruses is consistent with established structural principles and provide molecular in- sights into the evolutionary origins of COVID-19. Since the COVID-19 outbreak, raccoon dogs have been proposed as potential intermediate hosts involved in SARS-CoV-2 transmission to humans. To evaluate their role in the pandemic and define the species barrier for SARS-CoV-2 transmission, we analyzed the interaction between raccoon dog ACE2 and the SARS-CoV-2 spike protein. Biochemical data showed that raccoon dog ACE2 functions as an effective receptor for the SARS-CoV-2 spike protein, although less efficiently than human ACE2. Structural comparisons identified key differences in virus-binding residues between raccoon dog and human ACE2, including L24Q, Y34H, E38D, T82M, and R353K, which help explain their distinct receptor activities. These residue variations contribute to the molecular species barrier between raccoon dogs and humans and may inform broader assessments of mammalian susceptibility to SARS- CoV-2. Together, these studies provide a structural framework for understanding how SARS- CoV-2 and related coronaviruses overcome species barriers. The findings support the potential role of raccoon dogs as susceptible hosts capable of carrying SARS-CoV-2, while also showing that the pandemic potential of SARS-CoV-2 was strongly influenced by a spike protein already highly compatible with human ACE2. Overall, this work helps bridge the evolutionary gap between bat coronavirus reservoirs, potential hosts, and human infection.

Author dc:creator

• Hsueh, Fu-Chun

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/11299/281067

OAI identifier oai:identifier

oai:conservancy.umn.edu:11299/281067