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Variations in cell-surface ACE2 levels alter direct binding of SARS-CoV-2 Spike protein and viral infectivity: Implications for measuring Spike protein interactions with animal ACE2 orthologs.

ABSTRACT: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, the most severe pandemic in a century. The virus gains access to host cells when the viral Spike protein (S-protein) binds to the host cell-surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interaction with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often these cells only transiently express ACE2 proteins and levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bi-cistronic vector with an easy to quantify reporter protein to normalize ACE2 expression. We found that both binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus is proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of reporter protein, Thy1.1. We also compared different ACE2 orthologs which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein while human ACE2 was the highest level detected and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variant's ability to potentially infect different animals.

Importance

SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here we describe a method to generate cells stably expressing equivalent levels of different ACE2 orthologs, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both binding of the viral Spike protein receptor binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to ACE2 levels at the cell surface. Adaptation of this method will allow for the creation of a library of stable transfected cells expressing equivalent levels of different vertebrate ACE2 orthologs which can be repeatedly used for identifying vertebrate species which may be susceptible to infection with SARS-CoV-2 and its many variants.

SUBMITTER: Kazemi S

PROVIDER: S-EPMC8562541 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Spike-receptor interaction is a critical determinant for the host range of coronaviruses. Here, we investigated all the five World Health Organization-designated variants of concern (VOC), including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), for their Spike receptor-binding domain (RBD)'s interactions with ACE2 orthologs of 18 animal species. We found that, compared to the RBD of an early isolate WHU01, the Alpha RBD has markedly increased affinity to cattle and pig ACE2 proteins and decreased affinity to horse and donkey ACE2 proteins. The RBDs of Beta and Gamma variants have almost completely lost affinity to bat, horse, and donkey ACE2 orthologs. Mainly due to the Q493R and N501Y mutations, the Omicron RBD showed markedly enhanced affinity to mouse ACE2. Molecular dynamic simulations further suggest that Omicron RBDs are optimal for electrostatic interactions with mouse ACE2. Interestingly, the Omicron RBD also showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of horse, donkey, pig, dog, cat, pangolin, American pika, and bat. The K417N, G496S, and Y505H substitutions were identified as three major contributors that commonly have negative impact on RBD binding to these eight ACE2 orthologs. These findings show that Spike mutations have been continuously shaping SARS-CoV-2's binding affinities to animal ACE2 orthologs and suggest the importance of surveillance of animal infection by circulating SARS-CoV-2 variants.IMPORTANCESpike-receptor interaction is a critical determinant for the host range of coronaviruses. In this study, we investigated the SARS-CoV-2 WHU01 strain and five WHO-designated SARS-CoV-2 variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and the early Omicron variant, for their Spike interactions with ACE2 proteins of 18 animal species. First, the receptor-binding domains (RBDs) of Alpha, Beta, Gamma, and Omicron were found to display progressive gain of affinity to mouse ACE2. More interestingly, these RBDs were also found with progressive loss of affinities to multiple ACE2 orthologs. The Omicron RBD showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of some livestock animals (horse, donkey, and pig), pet animals (dog and cat), and wild animals (pangolin, American pika, and Rhinolophus sinicus bat). These findings shed light on potential host range shift of SARS-CoV-2 VOCs, especially that of the Omicron variant.

Project description:PurposeSpike protein on SARS-CoV-2 virus plays an integral part during infection as cell entry depends on binding of this protein to human ACE2 receptor. Understanding of infectivity by these variants necessitates a comparative structural analysis of complexes of spike protein-receptor binding domain (RBD) of these variants to receptor.MethodologyWild type SARS-CoV-2 spike protein sequence was retrieved from the UniProt database, and mutations of five variants at receptor binding domain were manually incorporated and aligned using Clustal Omega. Crystal structure complexes of human ACE2 receptor with spike protein RBD domain of SARS-CoV-2 variants of wild type, α, β, and δ were extracted from the RCSB database. Wild type SARS-CoV-2 complex with receptor was used as template to generate model complexes of receptor with spike protein RBD of γ and omicron variants through WinCoot program. These were energy minimized and validated and molecular dynamic simulation was performed using Desmond simulation program.ResultsMutations are distributed across the entire length of RBD, but the maximum number of mutations are seen at 11 positions within binding interface motifs of six variant sequences. Interface of spike protein RBDs with human ACE2-receptor shows different mix of hydrogen bonded and ionic interactions. Alpha and β variants have few interactions, while γ and δ variants have higher number of interactions compared to wild type variant. Omicron variant, with 10 polar interactions including two ionic bonds, has the highest binding energy.ConclusionDifferent mutations on RBD of spike protein results in varying quantity and quality of interactions, thereby affecting potency of each variant. Variations in binding are due to interactions of mutant residues and induced conformational changes on loops of RBDs. Variants α and β have a low potency, while, γ, δ, and omicron have a higher potency. These results correlate with viral infectivity and place clinical observations in the right perspective.

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Variations in cell-surface ACE2 levels alter direct binding of SARS-CoV-2 Spike protein and viral infectivity: Implications for measuring Spike protein interactions with animal ACE2 orthologs.

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