key: cord-0980185-36oddcei authors: Wang, Qiong; Ye, Sheng-Bao; Zhou, Zhi-Jian; Li, Jin-Yan; Lv, Ji-Zhou; Hu, Bodan; Yuan, Shuofeng; Qiu, Ye; Ge, Xing-Yi title: Key mutations on spike protein altering ACE2 receptor utilization and potentially expanding host range of emerging SARS-CoV-2 variants date: 2022-04-11 journal: bioRxiv DOI: 10.1101/2022.04.11.487828 sha: c18713220edcc216bfd94d05f30b5f30f9fb9143 doc_id: 980185 cord_uid: 36oddcei Increasing evidence supports inter-species transmission of SARS-CoV-2 variants from human to domestic or wild animals during the ongoing COVID-19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS-CoV-2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS-CoV-2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, in order to evaluate the impact of S mutations, we constructed 20 Hela cell lines stably expressing ACE2 orthologs from different animals, and prepared 27 pseudotyped SARS-CoV-2 carrying different spike mutants, among which 20 bear single mutation and the other 7 were cloned from emerging SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.135), Lambda (B.1.429) and Mu (B.1.525). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS-CoV-2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS-CoV-2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammals ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS-CoV-2, potentially contributing to spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS-CoV-2 variants with these two mutations. of SARS-CoV-2, warning a potential concern about more severe 134 prevalence of the virus in the future, though more validation based on 135 living virus is still required to draw a solid conclusion for this issue. Pseudovirus and ACE2 cells 138 The genomic sequences of human SARS-CoV-2 strains with high Table) . Then, these mutations result showed that all the 20 mutant pseudoviruses were successfully 153 prepared ( Fig 1B) . In the test of mammalian ACE2, we found that the T478I or A262S 218 significantly increased ability of pseudovirus to utilize human and 219 monkey ACE2 compared to D614G only control, indicating these two 220 mutants enhance the utilization of primate ACE2s (Fig 3A and 3B ). overexpressing cells (Fig 4A, B) reptilian ACE2s, as did the 20 mutants above (S1 and S2 Figs). in altering ACE2 utilization (S2 Table) . These results suggested that 478 th 327 and 501 th could be potentially key site for interspecies transmission of 328 SARS-CoV-2 and raised the concern about the neglecting of circulation 329 of SARS-CoV-2 in poultry and wild birds. promote the utilization of rabbit ACE2 (Fig 3) . Notably, these mutations We thank all study participants for their generous participation and 504 contribution to this work. Competing interests 524 The authors declare that there is no conflict of interests. 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Each 689 mutation in each SARS-CoV-2 variant is indicated relative to the 690 reference WT sequence. NTD N-terminal domain Primers design for mutagenesis Table The information of SARS-CoV-2 mutants