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Computational biophysical characterization of the SARS-CoV-2 spike protein binding with the ACE2 receptor and implications for infectivity.

ABSTRACT: SARS-CoV-2 is a novel highly virulent pathogen which gains entry to human cells by binding with the cell surface receptor - angiotensin converting enzyme (ACE2). We computationally contrasted the binding interactions between human ACE2 and coronavirus spike protein receptor binding domain (RBD) of the 2002 epidemic-causing SARS-CoV-1, SARS-CoV-2, and bat coronavirus RaTG13 using the Rosetta energy function. We find that the RBD of the spike protein of SARS-CoV-2 is highly optimized to achieve very strong binding with human ACE2 (hACE2) which is consistent with its enhanced infectivity. SARS-CoV-2 forms the most stable complex with hACE2 compared to SARS-CoV-1 (23% less stable) or RaTG13 (11% less stable). Notably, we calculate that the SARS-CoV-2 RBD lowers the binding strength of angiotensin 2 receptor type I (ATR1) which is the native binding partner of ACE2 by 44.2%. Strong binding is mediated through strong electrostatic attachments with every fourth residue on the N-terminus alpha-helix (starting from Ser19 to Asn53) as the turn of the helix makes these residues solvent accessible. By contrasting the spike protein SARS-CoV-2 Rosetta binding energy with ACE2 of different livestock and pet species we find strongest binding with bat ACE2 followed by human, feline, equine, canine and finally chicken. This is consistent with the hypothesis that bats are the viral origin and reservoir species. These results offer a computational explanation for the increased infection susceptibility by SARS-CoV-2 and allude to therapeutic modalities by identifying and rank-ordering the ACE2 residues involved in binding with the virus.

SUBMITTER: Chowdhury R

PROVIDER: S-EPMC7500280 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.ImportanceSARS-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.

Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (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 interactions 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 the 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 bicistronic vector with an easy-to-quantify reporter protein, Thy1.1. We found that both the binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus are proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of 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 had 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 variants' abilities 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 different orthologs of ACE2, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both the binding of the viral spike protein receptor-binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to the ACE2 levels at the cell surface. This method will allow the creation of a library of stably transfected cells expressing similar levels of different vertebrate ACE2 orthologs, which can be used repeatedly for identifying vertebrate species that may be susceptible to infection with SARS-CoV-2 and its many variants.

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Computational biophysical characterization of the SARS-CoV-2 spike protein binding with the ACE2 receptor and implications for infectivity.

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