Results obtained by direct biochemical methods and X-ray crystallographic studies showed that α HCoV-NL63 [84,232], β2 SARS-CoV [233] and β2 SARS-CoV-2 [195] use their S1-CTD RBD to bind to a host receptor, angiotensin-converting enzyme 2 (ACE2, a zinc peptidase), that is essential for virus entry into cells. As shown in Figure 4b, ACE2 is a homodimeric type I transmembrane protein having an orientation with the N-terminus outside and the C-terminus inside the cytoplasm [195]. The virus-binding site (VBS) of these three CoVs is not the peptidase active site but the outer surface of the ACE2 N-terminal lobe. Analyses of cocrystal structures between RBDs of HCoV-NL63 (pdb: 3kbh [232]), SARS-CoV (pdb: 2ajf [234]) or SARS-CoV-2 (pdb: 6m0j [235]) and the human ACE2 (hACE2) receptor indicated aa residues covering the CoV–ACE2 interfaces, divided into a common region of hACE2 recognized by all three ACE2-recognizing CoVs (a hotspot region) and unique regions bound by HCoV-NL63, SARS-CoV or SARS-CoV-2 (Figure 4b, middle row). Evidence indicating that HCoV-NL63 and SARS-CoV bind to the same hotspot region on hACE2 and that their binding is important for infection was obtained from infection inhibition studies showing that the SARS-CoV RBD can inhibit lentivirus infections mediated by the S protein of either SARS-CoV or HCoV-NL63 into hACE2-expressing HEK293T cells [236]. Likewise, the use of the HCoV-NL63 RBD as a competitive inhibitor can inhibit infections of murine leukemia viruses (MLVs) mediated by SARS-CoV S protein into hACE2-expressing HEK293T cells [237]. In addition, aa changes in the hotspot region in hACE2, either L353A or D38A substitution, resulted in a significant reduction of binding interactions between the SARS-CoV or HCoV-NL63 RBD and hACE2 and reduction of MLV infections mediated by SARS-CoV or HCoV-NL63 S protein [237]. The results of these studies suggested that the hotspot region on the hACE2 VBS is a potential target for development of drugs against ACE2-binding CoVs. It should be noted that MLN-4760, an ACE2 inhibitor that binds to the ACE2 catalytic center and induces hACE2 conformational changes, did not affect interactions of SARS-CoV S1 with the hACE2 surface and did not affect SARS-CoV S protein-mediated infection. Likewise, binding of SARS-CoV S1 to hACE2 did not affect hACE2 catalytic activity [238].