The 442th amino acid residue of the spike protein is critical for the adaptation to bat hosts for SARS-related coronaviruses

Abstract

Bats carry diverse severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). The suspected interspecies transmission of SARSr-CoVs from bats to humans has caused two severe CoV pandemics, the SARS pandemic in 2003 and the recent COVID-19 pandemic. The receptor utilization of SARSr-CoV plays the key role in determining the host range and the interspecies transmission ability of the virus. Both SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as their receptor. Previous studies showed that WIV1 strain, the first living coronavirus isolated from bat using ACE2 as its receptor, is the prototype of SARS-CoV. The receptor-binding domain (RBD) in the spike protein (S) of SARS-CoV and WIV1 is responsible for ACE2 binding and medicates the viral entry. Comparing to SARS-CoV, WIV1 has three distinct amino acid residues (442, 472, and 487) in its RBD. This study aimed at exploring whether these three residues could alter the receptor utilization of SARSr-CoVs. We replaced the three residues in SARS-CoV (BJ01 strain) S with their counterparts in WIV1 S, and then evaluated the change of their utilization of bat, civet, and human ACE2s using a lentivirus-based pseudovirus infection system. To further validate the S-ACE2 interactions, the binding affinity between the RBDs of these S proteins and the three ACE2s were verified by flow cytometry. The results showed that the single amino acid substitution Y442S in the RBD of BJ01 S enhanced its utilization of bat ACE2 and its binding affinity to bat ACE2. On the contrary, the reverse substitution in WIV1 S (S442Y) significantly attenuated the pseudovirus utilization of bat, civet and human ACE2s for cell entry, and reduced its binding affinity with the three ACE2s. These results suggest that the S442 is critical for WIV1 adapting to bats as its natural hosts. These findings will enhance our understanding of host adaptations and cross-species infections of coronaviruses, contributing to the prediction and prevention of coronavirus epidemics.

Keywords: ACE2; Receptor binding domain; Receptor utilization; SARS-CoV; SARSr-CoV; Spike.

Fig. 1

Generation of cells stably expressing civet, bat and human ACE2. (A) Detection of ACE2 expression in HeLa cells by western-blot using anti-HA tag antibodies. Lanes 1 to 3: transiently transfectedcells expressing civet (c), bat (b) and human (h) ACE2; Lane 4: HeLa cell acts as a negative control; Lanes 5 to 7: cell lines stably expressing civet, bat and human ACE2 cell. (B) HeLa cells stably expressing civet, bat and human ACE2 were verified by immunofluorescence. ACE2 expression was detected with mouse anti-HA antibody followed by Alexa 555-conjugated goat anti-mouse IgG. Nuclei were stained with DAPI (6-diamidino-2-phenylindole). The top row shows HeLa cells as a negative control. Rows 2 to 4 show HeLa cells stably expressing hACE2, bACE2, and cACE2. The columns (from left to right) show cells of brightfield, staining of cell nuclei (blue), staining of expressed ACE2-HA (red fluorescence of Alexa 555), and the merged double-stained image.

Fig. 2

Construction and functional analysis of pseudoviruses. (A) Sequence homology of the RBD of the spike gene of human SARS-CoV BJ01 (H-SARS-CoV-BJ01), bat SARSr-CoV WIV1 (B-SL-CoV-WIV1) and bat SARSr-CoV Rp3 (B-SL-CoV-RP3). The * represents different amino acid sites, and the▲represents the same sites among the key residues between H-SARS-CoV-BJ01 and B-SL-CoV-WIV1. (B) Detection of pseudoviruses bearing different S proteins by western-blot. Pseudoviruses were probed with C-oV S protein polyclonal pntibody (top) and with P24-specific mAbs (bottom) as a control. (C) Measurement of pseudovirus infectivity by determining luciferase activity. HIV-1-luciferase pseudotyped with S protein of the H-SARS-CoV-BJ01, B-SL-CoV-WIV1 and their variants, was incubated with HeLa-bACE2, HeLa-cACE2, HeLa-hACE2 and Vero-E6 cells. After 48 h infection, cell lysates were measured as luciferase activity. The results were presented as the means and standard deviations of data from three independent experiments. The error bars indicate standard deviations.

Fig. 3

Detection of expression and purification of RBD protein. (A) Detection of RBD protein in supernatant and lysate by western-blot using anti-S tag mAbs respectively. (B) Detection of the purified protein by western-blot using anti-S tag mAbs and coomassie blue staining.

Fig. 4

Solution RBD binding assays of bat, civet, and human ACE2. HeLa cells stably expressing bat (A), civet (B), and human ACE2 (C) were resuspended with BJ01-RBD, WIV1-RBD and Rp3-RBD proteins and their mutants (50 μg/mL) and then performed flow cytometry. The gray shaded area represents the control of HeLa cells without ACE2 expression.