Determination of the factors responsible for the tropism of SARS-CoV-2-related bat coronaviruses to Rhinolophus bat ACE2

Abstract

The efficiency of infection receptor use is the first step in determining the species tropism of viruses. After the coronavirus disease 2019 pandemic, a number of SARS-CoV-2-related coronaviruses (SC2r-CoVs) were identified in Rhinolophus bats, and some of them can use human angiotensin converting enzyme 2 (ACE2) for the infection receptor without acquiring additional mutations. This means that the potential of certain SC2r-CoVs to cause spillover from bats to humans is "off-the-shelf." However, both SC2r-CoVs and Rhinolophus bat species are highly diversified, and the host tropism of SC2r-CoVs remains unclear. Here, we focus on two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and determine how the tropism of SC2r-CoVs to Rhinolophus bat ACE2 is determined at the amino acid resolution level.

Keywords: ACE2; Rhinolophus bat; SARS-CoV-2; coronavirus; spike.

FIG 1

Different ACE2 tropism of the two Laotian SC2r-CoVs. (A) Maximum likelihood tree of SC2r-CoVs and SARS-CoV-2 (strain Wuhan-Hu-1) based on their nucleotide sequences corresponding to RBD in S. SARS-CoV-1 (strain Tor2) and two SC1r-CoVs (WIV1 and LYRa11) are included as an outgroup. *, >0.8 bootstrap value; **, >0.9 bootstrap value. The scale bar indicates genetic distance. The usability of human ACE2 for SC2r-CoV infection is indicated with ○ (yes) or × (no), respectively, and the clade of SC2r-CoVs that can use human ACE2 is shaded in brown. (B) Geological distributions of Rhinolophus bat species. The habitat information originates from the IUCN Red List of Threatened Species website (https://www.iucnredlist.org/). Note that habitat information for R. cornutus is not available. Also, the habitat information for R. marshalli (B236 was isolated) and R. malayanus (B52 was isolated) is included. (C) Pseudovirus assay. HIV-1-based reporter viruses pseudotyped with the S proteins of B52 or B236 were prepared. The pseudoviruses were inoculated into a series of HOS-TMPRSS2 cells stably expressing Rhinolophus bat ACE2 cells at 1 ng HIV-1 p24 antigen. The infectivity (relative light unit) in each target cell is shown. The host species in which ACE2 is preferred by B52 or B236 are indicated in green and orange, respectively. Data are expressed as the mean with SD. Assays were performed in quadruplicate. Statistically significant differences (*P < 0.05) between B52 and B236 were determined by a two-sided Student’s t-test. (D and E) Phylogenetic relationship of Rhinolophus bat species. (D) Time-calibrated species tree for Rhinolophus bat species generated by TimeTree5 (16). MYA, million years ago. (E) Maximum likelihood tree of Rhinolophus bat ACE2 sequences. The scale bar indicates genetic distance.

FIG 2

Interaction between Rhinolophus bat ACE2 and the S proteins of two Laotian SC2r-CoVs. (A) Inverse correlation of the ACE2 susceptibility to B236 and B52 infection. The boxed region is zoomed in on the right panel. (B) Association between B52 and B236 infectivity and ACE2 polymorphism among animal species. The association between the relative infectivity [log₁₀(B236 infectivity/B52 infectivity)] for each ACE2 protein and each polymorphic amino acid site was evaluated by one-way ANOVA. Dashed line, P = 0.1. Outlier species (human and R. pearsonii; gray dots in Fig. 2A) were excluded from the analysis. (C) Amino acid sites associated with the B236 and B52 infection tropisms. Heatmaps of the Z scores of B236 infectivity, B52 infectivity, and relative infectivity are shown on the left. (D–F) Structural insights into the binding of S RBD and ACE2 proteins. (D) The scheme of interaction between the SARS-CoV-2 S receptor binding motif (top) and human ACE2 (bottom). The salt bridge or hydrogen bond is indicated in black, and the van der Waals interaction is indicated in brown. Q493 of SARS-CoV-2 S and K31 and E35 of human ACE2 are indicated in black. This information is referred to in a previous report (17). RBM, receptor binding motif. (E) Amino acid alignment of the RBDs of B52 and B236. Residues with nonsynonymous substitutions between B52 and B236 are shaded in gray. The alignment was plotted by Multiple Align Show (https://www.bioinformatics.org/sms/index.html). (F) The structural model of the complex of B236 S RBD (blue) and the homology model ACE2 of R. cornutus (leftmost, green), R. pusillus (the second from the left, green), R. macrotis (the second from the right, orange), or R. sinicus (rightmost, orange), respectively. The model was reconstructed by using the co-structure of B236 S RBD and human ACE2 (PDB: 7PKI, https://www.rcsb.org/structure/7PKI) (6) as templates and homology models. The residue 493 of B236 S RBD and the residues 31 and 35 of ACE2s are indicated as stick models. Dashed lines indicate salt bridges. (G) The structural model of the complex of B52 S RBD (red) and the homology model ACE2 of R. cornutus (leftmost, green), R. pusillus (the second from the left, green), R. macrotis (the second from the right, orange), or R. sinicus (rightmost, orange), respectively. The model was reconstructed by using the co-structure of B236 S RBD and human ACE2 (PDB: 7PKI, https://www.rcsb.org/structure/7PKI) (6) as templates and homology models. The residue 493 of B52 S RBD and the residues 31 and 35 of ACE2s are indicated as stick models. Dashed lines indicate hydrogen bonds.

FIG 3

ACE2 tropism is determined by the interaction between residues 31/35 of ACE2 and residue 493 of S. (A) Western blotting. A representative blot of pseudovirus is shown. HIV-1 p24 is an internal control for the pseudovirus. kDa, kilodalton. (B and D) Pseudovirus assay. HIV-1-based reporter viruses pseudotyped with the S proteins of B236, B52, or their derivatives were prepared. The pseudoviruses were inoculated into a series of HEK293 cells transiently expressing Rhinolophus bat ACE2 cells at 2 ng HIV-1 p24 antigen, and the percentages of infectivity compared to that of the virus pseudotyped with B52 are shown. The numbers in the panel indicate the fold change of the B236 value to the B52 value in each target cell. (C) Western blotting. A representative blot of the cells transiently expressing flag-tagged Rhinolophus bat ACE2 cells is shown. TUBA is an internal control for the cells. kDa, kilodalton. In (B) and (D),the data are expressed as the mean with SD. Assays were performed in quadruplicate. The numbers in the panel indicate the fold change versus parental S. Statistically significant differences (*P < 0.05) between B236 and B52 were determined by a two-sided Student’s t-test.