ABSTRACT
BACKGROUND: Rodents, such as mice, are vulnerable targets, and potential intermediate hosts, of SARS-CoV-2 variants of concern, including Alpha, Beta, Gamma, and Omicron. N501Y in the receptor-binding domain (RBD) of Spike protein is the key mutation dictating the mouse infectivity, on which the neighboring mutations within RBD have profound impacts. However, the impacts of mutations outside RBD on N501Y-mediated mouse infectivity remain to be explored. RESULTS: Herein, we report that two non-RBD mutations derived from mouse-adapted strain, Ins215KLRS in the N-terminal domain (NTD) and H655Y in the subdomain linking S1 to S2, enhance mouse infectivity in the presence of N501Y mutation, either alone or together. This is associated with increased interaction of Spike with mouse ACE2 and mutations-induced local conformation changes in Spike protein. Mechanistically, the H655Y mutation disrupts interaction with N657, resulting in a less tight loop that wraps the furin-cleavage finger; and the insertion of 215KLRS in NTD increases its intramolecular interaction with a peptide chain that interfaced with the RBD-proximal region of the neighboring protomer, leading to a more flexible RBD that facilitates receptor binding. Moreover, the Omicron Spike that contains Ins214EPE and H655Y mutations confer mouse infectivity > 50 times over the N501Y mutant, which could be effectively suppressed by mutating them back to wild type. CONCLUSIONS: Collectively, our study sheds light on the cooperation between distant Spike mutations in promoting virus infectivity, which may undermine the high infectiousness of Omicron variants towards mice.