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

. CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 8 A striking difference between SARS-CoV-2 and animal SL-CoVs is the presence 162 of a polybasic furin cleavage site at the S1/S2 boundary of the S protein (Fig.1) . Here, 163 we generated a SARS-CoV-2 S gene mutant with the furin cleavage site deleted to (Fig.3B ). 172 We also tested the receptor usage of these 14 ACE2 by SARS-CoV (Fig.3D ). The To help understand the molecular basis of different ACE2 receptor activities, we 181 first examined the overall sequence variation between these ACE2s. For this purpose, 182 we constructed a phylogenetic tree based on the nucleotide sequences of ACE2s (Fig.4) . 183 Interestingly, the phylogenetic clustering of ACE2s is correlated with their abilities to 184 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 9 support SARS-CoV-2 entry. For example, ACE2s in subclade IIA (human, rhesus 185 monkey and rabbit) and IIB (rat and mouse) were the most efficient and poorest receptor, 186 respectively, while ACE2s in clade I (from the remaining animals) were intermediate 187 between subclades IIA and IIB. This correlation suggests that sequence variations that 188 define for speciation are responsible for observed differences in receptor activity. The copyright holder for this preprint (which was not peer-reviewed) is the .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 K417, Y83 contacts N487, and K353 appears to be at the center of a hydrogen bond 208 network spanning seven RBD residues (Y449, G496, Q498, T500, N501, G502, Y505) 209 and eight ACE2 residues (D38, Y41, Q42, N330, K353, G354, D355, R357). The D30N, 210 Y83F and K353H substitutions are all predicted to disrupt these interactions in rat and 211 mouse ACE2 ( Figure 6 ). This is consistent with previous reports which pinpoint K353 212 as an important hotspot for both SARS-CoV-2 16 and SARS-CoV 33 binding. It has been 213 experimentally demonstrated that introduction of K353H into hACE2 significantly 214 reduces binding to SARS-CoV S1; in contrast, introduction of H353K into rat ACE2 215 significantly increases binding to SARS-CoV S1 34 Table S1 ). 220 Both Bat ACE2s are also inefficient receptors for viral entry (Fig.3B ). Since the 221 profile of residues at the receptor/RBD interface is significantly different from rat and 222 mouse ACE2, we examined other bat-specific residue substitutions that may be 223 contributing to receptor dysfunction. There are 8 and 10 critical residue substitutions in 224 the Rs bat and Tb bat ACE2s, respectively (Fig.5 ). Among these, we examined the 225 substitutions at positions Y41, H34 and E329 as they are only seen in bat ACE2s. The 226 Y41H substitution in both bat ACE2s appears to be disrupting the same H-bond network 227 that was disrupted by K353H in rat and mouse ACE2. Although Y41 is not as centrally 228 located in the H-bond network as K353, it directly contacts N501 from the RBD, which is 229 the same residue that is stabilized by K353. A second interaction which appears to be 230 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for t