Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry

Abstract

The discovery of SARS-like coronavirus in bats suggests that bats could be the natural reservoir of SARS-CoV. However, previous studies indicated the angiotensin-converting enzyme 2 (ACE2) protein, a known SARS-CoV receptor, from a horseshoe bat was unable to act as a functional receptor for SARS-CoV. Here, we extended our previous study to ACE2 molecules from seven additional bat species and tested their interactions with human SARS-CoV spike protein using both HIV-based pseudotype and live SARS-CoV infection assays. The results show that ACE2s of Myotis daubentoni and Rhinolophus sinicus support viral entry mediated by the SARS-CoV S protein, albeit with different efficiency in comparison to that of the human ACE2. Further, the alteration of several key residues either decreased or enhanced bat ACE2 receptor efficiency, as predicted from a structural modeling study of the different bat ACE2 molecules. These data suggest that M. daubentoni and R. sinicus are likely to be susceptible to SARS-CoV and may be candidates as the natural host of the SARS-CoV progenitor viruses. Furthermore, our current study also demonstrates that the genetic diversity of ACE2 among bats is greater than that observed among known SARS-CoV susceptible mammals, highlighting the possibility that there are many more uncharacterized bat species that can act as a reservoir of SARS-CoV or its progenitor viruses. This calls for continuation and expansion of field surveillance studies among different bat populations to eventually identify the true natural reservoir of SARS-CoV.

Fig. 1

Sequence alignment of SARS-CoV binding regions of ACE2s from 9 bats, civet and human. The GenBank accession numbers of bat, civet and human ACE2 are as follows: human (NM021804), civet (AY881174), Rf-HB (GQ999931), Rm-HB (GQ999932), Rs-GX (GQ999933), Rp-GX (EF569964), Hp-HN (GQ999934), Rp-GZ (GQ999935), Rs-HB (GQ999936), Md-YN(GQ999937) and Rpu-HB (GQ999938). The alignment was generated using ClustalX v1.83. In black are single, fully conserved residues. In gray are strongly conserved residues. In light gray are weakly conserved residues. Asterisks indicate residues that interact directly with the receptor-binding domain of the SARS-CoV S protein

Fig. 2

Testing of the ability of bat ACE2 proteins to mediate pseudovirus HIV/BJ01-S and live SARS- CoV infection. a HeLa cells transfected with plasmids encoding bat and human ACE2s were infected with pseudovirus HIV/BJ01-S. Infectivity was determined by measuring the activity of reporter luciferase as described in “Materials and methods”. HeLa cells transfected with pcDNA3.1 and human ACE2 were used as the negative and positive controls, respectively. All tests were performed in triplicate, and the experiments were repeated three times. The error bar represents the calculated standard deviation. I27T, N31K, K35E, and H41Y are mutants of MdACE2 that were made using a QuikChange II Site-Directed Mutagenesis Kit. b SARS-CoV live virus infection using the ACE2s from bats as described in “Materials and methods”. HeLa cells transfected with pcDNA3.1 and human ACE2 were used as the negative and positive controls, respectively. c Expression of bat or human ACE2. Lysates from HeLa cells transfected with plasmid expressing human or bat ACE2 were analyzed by western blot. Rabbit anti-bat ACE2 polyclonal antibody (upper panel) or β-actin monoclonal antibody (lower panel) was used as the primary antibody. Lane 1 vector pcDNA3.1 control; lanes 2–10 bat ACE2 from samples Rf-HB, Rm-HB, Rpu-HB, Hp-HN, Rp-HB, Rp-GZ, Rs-GX, Rs-HB and Md-YN; lanes 11–14 Md-YN ACE2 mutant I27T, N31K, K35E and H41Y; lane 15 human ACE2. The abbreviations of bat species are given in the main text

Fig. 3

Homologous structural modeling of SARS-CoV and Md-YN ACE2 (MdACE2) interactions. a Critical salt bridge between hACE2 Lys31 and Glu35 and the hydrophobic residues surrounding it, based on the experimentally determined crystal structure of SARS-CoV RBD complexed with hACE2 (PDB 2AJF). b Homologous structural modeling of the hydrogen bond between MdACE2 Asn31 and Lys35. The modeling was done in the program O [3]. c Critical salt bridge between hACE2 Lys353 and Glu38 and the hydrophobic residues surrounding it, based on the structure of SARS-CoV RBD complexed with hACE2. d Homologous structural modeling of the salt bridge between MdaACE2 Lys353 and SARS-CoV Glu38 and the hydrophobic residues surrounding it. Structural illustrations were prepared using the program Povscript [2]