ACE2 binding is an ancestral and evolvable trait of sarbecoviruses

Abstract

Two different sarbecoviruses have caused major human outbreaks in the past two decades ^1,2 . Both of these sarbecoviruses, SARS-CoV-1 and SARS-CoV-2, engage ACE2 through the spike receptor-binding domain ^2–6 . However, binding to ACE2 orthologues of humans, bats and other species has been observed only sporadically among the broader diversity of bat sarbecoviruses ^7–11 . Here we use high-throughput assays ¹² to trace the evolutionary history of ACE2 binding across a diverse range of sarbecoviruses and ACE2 orthologues. We find that ACE2 binding is an ancestral trait of sarbecovirus receptor-binding domains that has subsequently been lost in some clades. Furthermore, we reveal that bat sarbecoviruses from outside Asia can bind to ACE2. Moreover, ACE2 binding is highly evolvable—for many sarbecovirus receptor-binding domains, there are single amino-acid mutations that enable binding to new ACE2 orthologues. However, the effects of individual mutations can differ considerably between viruses, as shown by the N501Y mutation, which enhances the human ACE2-binding affinity of several SARS-CoV-2 variants of concern ¹² but substantially decreases it for SARS-CoV-1. Our results point to the deep ancestral origin and evolutionary plasticity of ACE2 binding, broadening the range of sarbecoviruses that should be considered to have spillover potential.

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Table 2: Resources

Antibodies
Sentences	Resources
For each library, 45 OD of induced culture was washed twice with PBS-BSA (0.2 mg/mL), and RBD surface expression was labeled via a C-terminal c-Myc tag with 1:100 diluted FITC-conjugated chicken anti-c-Myc antibody (Immunology Consultants Lab, CMYC-45F) in 3mL PBS-BSA.	c-Myc tag suggested: None anti-c-Myc suggested: None
One day post-transfection, cells were infected with VSV (G*ΔG-luciferase), and after 2 hr, infected cells were washed 5x with DMEM before adding medium supplemented with anti-VSV G antibody (I1-mouse hybridoma supernatant diluted 1:40, from ATCC CRL-2700)	*GΔG-luciferase) , suggested: None ant…**

SciScore for 10.1101/2021.07.17.452804: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Antibodies
Sentences	Resources
For each library, 45 OD of induced culture was washed twice with PBS-BSA (0.2 mg/mL), and RBD surface expression was labeled via a C-terminal c-Myc tag with 1:100 diluted FITC-conjugated chicken anti-c-Myc antibody (Immunology Consultants Lab, CMYC-45F) in 3mL PBS-BSA.	c-Myc tag suggested: None anti-c-Myc suggested: None
One day post-transfection, cells were infected with VSV (G*ΔG-luciferase), and after 2 hr, infected cells were washed 5x with DMEM before adding medium supplemented with anti-VSV G antibody (I1-mouse hybridoma supernatant diluted 1:40, from ATCC CRL-2700)	*GΔG-luciferase) , suggested: None anti-VSV G** suggested: (Fitzgerald Industries International Cat# 10R-2673, RRID:AB_11192005)
Detection of S was done with mouse monoclonal ANTI-FLAG M2 antibody (Sigma F3165) and Alexa Fluor 680 AffiniPure	ANTI-FLAG suggested: (Sigma-Aldrich Cat# F3165, RRID:AB_259529)
Experimental Models: Cell Lines
Sentences	Resources
293T cells were transfected using Lipofectamine 2000 (Life Technologies) with a S encoding-plasmid in Opti-MEM transfection medium and incubated for 5 hr at 37°C with 8% CO2 supplemented with DMEM containing 10% FBS.	293T suggested: None
VSV pseudovirus entry assays: HEK293T (293T) cells (ATCC CRL-11268) and 293T cells with stable transfection of human ACE242 were cultured in 10% FBS, 1% PenStrep DMEM at 37°C in a humidified 8% CO2 incubator.	HEK293T suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
Recombinant DNA
Sentences	Resources
pcDNA3.4 expression plasmids were transfected into HD 293F cells for protein expression.	pcDNA3.4 suggested: RRID:Addgene_131198)
Transient expression of R. affinis ACE2-Fc: The R. affinis 787 (GenBank: QMQ39222.1) and R. affinis 9479 (GenBank: QMQ39227.1) ACE2 ectodomains constructs were synthesized by GenScript and placed into a pCMV plasmid.	pCMV suggested: RRID:Addgene_20783)
Software and Algorithms
Sentences	Resources
Marginal likelihood ancestral sequence reconstruction was performed with FastML (version 3.11)49 using the amino acid sequence alignment, the maximum likelihood nucleotide tree topology from RAxML, the LG+G substitution matrix, re-optimization of branch lengths, and FastML’s likelihood-based indel reconstruction model.	FastML suggested: (Fastml, RRID:SCR_016092) RAxML suggested: (RAxML, RRID:SCR_006086)
The resulting CCSs are available on the NCBI Sequence Read Archive, BioSample SAMN18316101.	NCBI Sequence Read Archive suggested: (NCBI Sequence Read Archive (SRA, RRID:SCR_004891)
Yeast library sorting experiments were conducted on a BD FACSAria II with FACSDiva software (version 8.0.2).	FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
ACE2 labeling of RBD+ cells was measured on a BD LSRFortessa X50 flow cytometer and data was processed via FlowJo (version 10)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
Normalized cell entry levels of pseudovirus generated on different days (biological replicates) were plotted in Graph Prism as individual points, and average cell entry across biological replicates was calculated as the geometric mean.	Graph Prism suggested: None

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Results from JetFighter: We did not find any issues relating to colormaps.

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ACE2 binding is an ancestral and evolvable trait of sarbecoviruses

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