Broad and differential animal ACE2 receptor usage by SARS-CoV-2

Xuesen Zhao; Danying Chen; Robert Szabla; Mei Zheng; Guoli Li; Pengcheng Du; Shuangli Zheng; Xinglin Li; Chuan Song; Rui Li; Ju-Tao Guo; Murray Junop; Hui Zeng; Hanxin Lin

human population remains mysterious, although bat and pangolin were proposed to be 27 the natural reservoirs. Strikingly, comparing to the SARS-CoV-2-like CoVs identified in 28 bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) 29 glycoprotein. SARS-CoV-2 uses human ACE2 as its receptor to infect cells. Receptor 30 recognition by the S protein is the major determinant of host range, tissue tropism, and 31 pathogenesis of coronaviruses. In an effort to search for the potential intermediate or 32 amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 33 14 mammal species and found that ACE2 from multiple species can support the 34 infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage 35 site deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse 36 exhibited the highest and lowest receptor activity, respectively. Among the remaining 37 species, ACE2 from rabbit and pangolin strongly bound to the S1 subunit of 38 SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These wild animal market in Wuhan city would otherwise be a unique place to trace any 73 potential animal source; however, soon after the disease outbreak, the market was 74 closed and all the wild animals were cleared, making this task very challenging or even 75 impossible. As an alternative, wide screening of wild animals becomes imperative. 76 Several recent studies identified multiple SARS-COV-2-like CoVs (SL-CoVs) from 77 smuggled Malayan pangolins in China. These pangolin CoVs (PCoV) form two 78 phylogenetic lineages, PCoV-GX and PCoV-GD 8-11 . In particular, lineage PCoV-GD 79 was found to carry a nearly identical receptor-binding motif (RBM) in the spike (S) 80 protein to that of SARS-CoV-2 (Fig.1) . However, the genome of these pangolin Receptor recognition by the viral S protein is the major determinant of host range, 89 cell, tissue tropism, and pathogenesis of coronaviruses 14 . The S protein of 90 SARS-CoV-2 is a type I membrane glycoprotein, which can be cleaved to S1 and S2 91 subunit during biogenesis at the polybasic furin cleavage site (RRAR) (Fig.1) multiple animal ACE2 could serve as receptors for SARS-CoV-2 and the SARS-CoV-2 116 mutant. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest 117 receptor activity, respectively, with the other 10 ACE2s exhibiting intermediate activity. 118 The implications of our findings were discussed in terms of the natural reservoir, 119 zoonotic transmission, human-to-animal transmission, animal health, and animal model. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.19.048710 doi: bioRxiv preprint 7 confirm that hACE2 is the bone fide entry receptor for SARS-CoV-2. 10 animal ACE2s were between human/monkey and rat/mouse. Among these, ACE2 of 154 canine, feline, rabbit and pangolin could support virus entry at levels >50% of hACE2. 155 To examine receptor binding ability, we performed immunoprecipitation (IP) 156 analysis by using both S1 and receptor binding domain (RBD) as probe. Among the 14 157 different ACE2s tested, ACE2 from human, monkey, feline, rabbit and pangolin 158 exhibited significant and consistent association with S1 and RBD (Fig.3C) . Importantly, 159 these ACE2s correspond to the group of ACE2s that supported the most efficient virus 160 entry (Fig.3B ).