In a recent study posted to the bioRxiv* preprint server, researchers assessed the ability of spike (S) proteins from 24 animal or human coronaviruses (CoVs) to utilize angiotensin-converting enzyme 2 (ACE2) receptors across nine reservoirs of potential, intermediate, and human hosts.
Study: Determinants of species-specific utilization of ACE2 by human and animal coronaviruses. Image Credit: MartinJanca/Shutterstock.com
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Background
Spike glycoproteins in human CoVs (hCoVs), including severe acute respiratory syndrome CoV (SARS-CoV) and SARS-CoV-2, utilize the ACE2 receptor for invading human tissues. Therefore, the ability of CoVs to utilize human ACE2 (hACE2) and enter host cells is critical for effective transmission.
However, the ACE2-utilizing capability of human CoVs, bat CoVs and other CoVs of reservoirs, such as bats, humans, and intermediate-type hosts, are not well-characterized and require further investigation.
About the study
In the present study, researchers evaluated the capability of spike proteins (n=24) of various bat CoVs, civet CoVs, pangolin CoVs, and human CoVs to utilize the ACE2 receptors of reservoir bat species, civets, pangolins, raccoon dogs, camels, ferrets, pig hosts, and human beings.
SARS-CoV-2 S and ACE2 receptors obtained from several species were expressed, and the spike constructs from different bat CoVs, severe acute respiratory syndrome CoV (civets), SARS-CoV-2 (pangolins) and human CoVs were generated.
Vesicular stomatitis virus (VSV) particles were devoid of the glycoprotein gene, but codes for the green fluorescent protein (GFP) were pseudotyped with the spike proteins.
Subsequently, western blot analysis was performed. The team assessed species-based differences in ACE2 receptors' ability to regulate hCoV and animal CoV entry using expression constructs generated for the ACE2 orthologues.
These constructs were obtained from intermediate horseshoe bats (Rhinolophus affinis, Ra), common raccoon dogs (Nyctereutes procyonoides), greater horseshoe bats (Rhinolophus ferrumequinum, Rf), masked palm civets (Paguma larvata), Sunda pangolins (Manis javanica), camels (Camelus dromedaries), ferret (Mustela putorius furo), humans, and pigs (Sus scrofa domesticus).
Species-specific ACE2 usage by SARS-CoV-2 variants was analyzed. To assess SARS-CoV-2 infection kinetics, VSV-infected cells were quantified over time.
Further, quantitative cell-to-cell fusion assays were performed to determine the fusogenicity of SARS-CoV-2 S proteins in hACE2- or bat ACE2-expressing cells. To identify the mutation responsible for the loss of Rf ACE2 use, mutations were introduced separately into Omicron BA.2 S.
To determine the species-specificity of ACE2 use by animal CoVs related to human SARS-CoVs, human-derived or animal-derived ACE2 was overexpressed in human embryonic kidney (HEK)293T cells and their susceptibility to VSV infection, regulated by spike proteins from the pangolin, bat, human, and civet CoVs, was assessed.
Molecular modeling of S-ACE2 interactions was performed. To investigate whether COVID-19 vaccines could protect against bat CoV transmission to humans, serum samples from 10 individuals were assessed, among whom five had been administered one dose of ChAdOx1 nCoV-19 and two doses of BNT162b2 vaccines, and the remaining five had been administered three BNT162b2 vaccinations.
Results
SARS-CoV-2 Omicron VOCs used ACE2 more efficiently, but the R493Q mutation in Omicron BA.5 S disrupted the usage of ACE2 from Rhinolophus ferrumequinum bats.
Spike proteins from most CoVs exhibited species-specific differences in ACE2 utilization, partly due to variations in ACE2 residues 31, 41, and 354.
The T403R mutation allowed the RaTG13 bat CoV S to utilize all the ACE2 orthologs analyzed for viral entry.
The serum samples from COVID-19 vaccinees neutralized S proteins of several bat sarbecoviruses and prevented infection regulated by BtKY73 spike, which has 72% sequence similarity to SARS-CoV-2 spike protein, via bat ACE2 receptors.
Single amino acid changes in hCoV, and bat CoV spike proteins could markedly change their ability to use ACE2 receptors from different species.
The spike proteins of SARS-CoV-2 strains such as Wuhan-Hu-1, SARS-CoV-2 Delta variant of concern (VOC) and BA.1 sub-VOC, BA.2 sub-VOC, BA.4 sub-VOC, and BA.5 sub-VOC mediated infection in the HEK293T cells. These cells expressed ACE2 derived from pangolin, civet, camel, raccoon dog, pig, Rhinolophus affinis bats, and ferret.
Wuhan-Hu-1 strain and BA.5 sub-VOC spike proteins could not use ACE2 proteins of Rhinolophus ferrumequinum, Rf bats, and Omicron BA.5 spike could not utilize Rf angiotensin-converting enzyme 2 (ACE2) for infection.
All SARS-CoV-2 spike proteins promoted syncytia formation in cells that expressed hACE2 or Ra angiotensin-converting enzyme 2 (S3).
However, Wuhan-Hu-1 S and Omicron BA.5 spike proteins showed poor activity in mediating the fusion of cell membranes in Rf ACE2-expressing cells. Residue R493 in S protein and residue D31/H41 in Rf angiotensin-converting enzyme 2 affected the entry of BA.5 in host cells.
R493Q change in the CoV-2 spike disrupted ACE2 usage from Rf bats, widespread in Asia, North Africa, and Europe.
A few bat CoVs showed potential to spread across Northern Africa, Asia, and Europe. The spike proteins of bat CoVs showed species-specific differences in their ACE2-utilization ability to cause infection, partially determined by mutations at position 403 in the viral spike protein and position 31 in ACE2 receptors.
G354 in ACE2 was essential for hCoV-NL63 infection, whereas G354H and G354R mutations weakened the ACE2-NL63 spike protein interactions. The hCoV-NL63 showed the potential to infect multiple animal species, and G354 determined ACE2 use by the circulating human CoV.
The R493Q and T403R mutations in the spike proteins of SARS-CoV-2 and RaTG13, respectively, and G354R/H, D31N, and H41Y in ACE2 receptors, were essential for species-specificity and efficient ACE2 use by animal CoVs and hCoVs.
The R493Q mutation facilitated discriminating Omicron BA.4/5 from Omicron BA.2 and enhanced the replicative fitness of Omicron BA.4/5 in human cells.
The findings showed expanded ACE2 tropism from the Wuhan-Hu-1 strain to Omicron BA.1 sub-VOC and Omicron BA.2 sub-VOC, which was subsequently lost in Omicron BA.4/5. K31N/D/E/T mutations enhanced the usage of ACE2 orthologs for BtKY72 S-mediated infections.
Conclusion
Overall, the study findings highlighted the determinants of using ACE2 receptors of various CoVs and indicated that SARS-CoV-2 vaccinations could probably protect against future zoonotic transmission of SARS-CoV-associated bat viruses.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.