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

In this study, we examined the receptor activity of 14 ACE2 orthologues. The 244 results suggested that wild type and mutant SARS-CoV-2 lacking the furin cleavage site 245 in S protein could use ACE2 from a broad range of animal species to enter host cells. 246 Below we discuss the implication of our findings in terms of natural reservoir, zoonotic 247 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 The copyright holder for this preprint (which was not peer-reviewed) is the .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 12 SARS-CoV-2 entry (Fig.3B ). Although these data were obtained by using HIV1-based 253 pseudotyped virus, for ACE2 of Rs bat, civet, and mouse, the data is consistent with in 254 vitro infection data using infectious virus 3 . Receptor usage by coronaviruses has been 255 well known to be a major determinant of host range, tissue tropism, and pathogenesis 256 14,35,36 . It is therefore reasonable to assume that SARS-CoV-2 would be able to infect all 257 these animals. As a matter of fact, several in vivo infection and seroconversion studies 258 have confirmed that SARS-CoV-2 can infect rhesus monkey 37 , feline, ferret, and canine As described above, it seems that dogs are not as susceptible as cats to 315 SARS-CoV-2 38,45 . Interestingly, this is in agreement with results from IP analysis that 316 showed cat ACE2 could bind to S1 or RBD more efficiently than dog ACE2 (Fig.3C) . 317 Structural models further suggest that, at those critical RBD-binding residues, dog and 318 cat ACE2 share 4 substitutions (Q24L, D30E, D38E, and M82T), while dog ACE2 has 319 an additional substitution, H34Y (Fig.5) . Based on structural modeling, both Q24L and 320 M82T are predicted to be disruptive, while both D30E and D38E are tolerable (Table 321 . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 15 S1). H34Y in dog ACE2 is predicted to disrupt the hydrogen bond with Y453 of RBD 322 (Table S1 ). These atomic interactions explain why dog ACE2 binds to S1 or RBD less 323 efficiently compared to cat ACE2, and both are less efficient than human ACE2. 324 In addition to cat and dog, rabbits are also often raised as household pets. Our 325 results indicate that rabbit ACE2 is an efficient receptor ( Fig. 3B and 3C) , suggesting 326 that rabbit may be more susceptible to SARS-CoV-2 infection than cat. 327 Currently, there is no evidence that infected pets can transmit the virus back to 328 human; however, this may be possible and should be investigated. Out of an abundance 329 of caution it would be best when one is infected to have both human and pets 330 quarantined, and the pets tested as well. for SARS-CoV-2 as mouse ACE2 still supports some entry for SARS-CoV (Fig.3D) , 349 but not SARS-CoV-2. An alternative way to make a mouse-adapted SARS-CoV-2 strain 350 could be achieved by rational design of the S gene. Based on the structural model, we 351 know that receptor dysfunction of mouse ACE2 is due to disruptive D30N, L79T, M82S, 352 Y83F, E329A and K353H substitutions (Fig.5, Fig.6 and Table S1 ). Therefore, by 353 specifically introducing mutations into the RBM of S gene it may be possible to restore 354 or at least partly restore interactions with these ACE2 substitutions. Consequently, the 355 engineered virus may