Characterization of Key Spike RBD Residues Influencing SARS-CoV-2 Variant Adaptation to Avian ACE2

Weitong Yao¹Yujun Li^2*Huize Sun¹Danting Ma³Xiaojuan Tang^4,5Aiping Zeng^6*FANG HUANG^1*

• ¹Hubei Jiangxia Laboratory, Wuhan, China
• ²Biosafety Level 3 Laboratory, Shenzhen University, Shenzhen, China
• ³Tianjin Key Laboratory of Metabolic Diseases, Tianjin Medical University, Tianjin, China
• ⁴School of Chemical Biology and Biotechnology, Shenzhen Graduate School, Peking University, Shenzhen, Guangdong Province, China
• ⁵Shenzhen Bay Laboratory, Shenzhen, Guangdong, China
• ⁶Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hebei Province, China

The beta-coronavirus SARS-CoV-2 has been defined to infect mammals and other species along with virus evolution. The host range of different SARS-CoV-2 strains relies on the affinity of receptor-binding domains (RBDs) of the spike proteins and the host receptor ACE2. Our previous findings revealed that chicken ACE2 did not bind WT SARS-CoV-2 encoded RBD. Here, the ACE2 of chicken and its avian ortholog mallard were found to support binding with RBDs of Alpha, Beta, and Gamma variants, and enable the viral entry. Whereas, the RBD of BA.1 failed to bind avian ACE2, a triple-residue reversal mutant (S446G, S496G, H505Y) restored the ACE2 binding and efficient viral entry. Furthermore, several key residues within RBD were characterized as the determinant to support binding to avian ACE2. Indeed, investigation of such cross-species transmission patterns would help to understand virological features and to speculate host preferences of newly emerged SARS-CoV-2 variants.