Computational optimization of the SARS-CoV-2 receptor-binding-motif affinity for human ACE2

The coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the coronavirus disease 2019 pandemic, and the closely related SARS-CoV coronavirus enter cells by binding at the human angiotensin converting enzyme 2 (hACE2). The stronger hACE2 affinity of SARS-CoV-2 has been connected with its higher infectivity. In this work, we study hACE2 complexes with the receptor-binding domains (RBDs) of the human SARS-CoV-2 and human SARS-CoV viruses, using all-atom molecular dynamics simulations and computational protein design with a physics-based energy function. The molecular dynamics simulations identify charge-modifying substitutions between the CoV-2 and CoV RBDs, which either increase or decrease the hACE2 affinity of the SARS-CoV-2 RBD. The combined effect of these mutations is small, and the relative affinity is mainly determined by substitutions at residues in contact with hACE2. Many of these findings are in line and interpret recent experiments. Our computational protein design calculations redesign positions 455, 493, 494, and 501 of the SARS-CoV-2 receptor binding motif, which contact hACE2 in the complex and are important for ACE2 recognition. Sampling is enhanced by an adaptive importance sampling Monte Carlo method. Sequences with increased affinity replace CoV-2 glutamine by a negative residue at position 493; serine by a nonpolar or aromatic residue or an asparagine at position 494; and asparagine by valine or threonine at position 501. Substitutions at positions 455 and 501 have a smaller effect on affinity. Substitutions suggested by our design are seen in viral sequences encountered in other species, including bat and pangolin. Our results might be used to identify potential virus strains with higher human infectivity and assist in the design of peptide-based or peptidomimetic compounds with the potential to inhibit SARS-CoV-2 binding at hACE2.     

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002–2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.     

Making sense of spike D614G in SARS-CoV-2 transmission

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), the etiologic agent of the current coronavirus disease 2019(COVID-19) pandemic, has evolved to adapt to human host and transmission over the past 12 months. One prominent adaptive mutation is the asparagine-to-glycine substitution at amino acid position 614 in the viral spike protein(D614G), which has become dominant in the currently circulating virus strains. Since spike protein determines host ranges, tissue tropism, and pathogenesis through binding to the cellular receptor of angiotensin converting enzyme 2(ACE2), the D614G mutation is hypothesized to enhance viral fitness in human host, leading to increased transmission during the global pandemic. Here we summarize the recent progress on the role of the D614G mutation in viral replication, pathogenesis, transmission, and vaccine and therapeutic antibody development. These findings underscore the importance in closely monitoring viral evolution and defining their functions to ensure countermeasure efficacy against newly emerging variants.     

SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.     

ACE2, B0AT1, and SARS-CoV-2 spike protein: Structural and functional implications

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a public health crisis and led to tremendous economic devastation. The spike protein (S) of SARS-CoV-2 hijacks the angiotensin converting enzyme 2 (ACE2) as a receptor for virus entry, representing the initial step of viral infection. S is one of the major targets for development of the antiviral drugs, antibodies, and vaccines. ACE2 is a peptidase that plays a physiologically important role in the renin–angiotensin system. Concurrently, it also forms dimer of heterodimer with the neutral amino acid transporter B⁰AT1 to regulate intestinal amino acid metabolism. The symptoms of COVID-19 are closely correlated with the physiological functions of ACE2. In this review, we summarize the functional and structural studies on ACE2, B⁰AT1, and their complex with S of SARS-CoV-2, providing insights into the various symptoms caused by viral infection and the development of therapeutic strategies.     

Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells

The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor-human angiotensin converting enzyme 2 (ACE2). Here, by using lentivirus based pseudotypes bearing spike protein, we demonstrated that entry of SARS-CoV-2 into host cells was dependent on clathrin-mediated endocytosis, and phosphoinositides played essential roles during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 were not required for efficient virus entry. Finally, we showed that the current predominant Delta variant, although with high infectivity and high syncytium formation, also entered cells through clathrin-mediated endocytosis. These results provide new insights into SARS-CoV-2 cellular entry and present proof of principle that targeting viral entry could be an effective way to treat different variant infections.     

Comparative genomic analysis reveals varying levels of mammalian adaptation to coronavirus infections

Severe acute respiratory coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is of zoonotic origin. Evolutionary analyses assessing whether coronaviruses similar to SARS-CoV-2 infected ancestral species of modern-day animal hosts could be useful in identifying additional reservoirs of potentially dangerous coronaviruses. We reasoned that if a clade of species has been repeatedly exposed to a virus, then their proteins relevant for viral entry may exhibit adaptations that affect host susceptibility or response. We perform comparative analyses across the mammalian phylogeny of angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2, in order to uncover evidence for selection acting at its binding interface with the SARS-CoV-2 spike protein. We uncover that in rodents there is evidence for adaptive amino acid substitutions at positions comprising the ACE2-spike interaction interface, whereas the variation within ACE2 proteins in primates and some other mammalian clades is not consistent with evolutionary adaptations. We also analyze aminopeptidase N (APN), the receptor for the human coronavirus 229E, a virus that causes the common cold, and find evidence for adaptation in primates. Altogether, our results suggest that the rodent and primate lineages may have had ancient exposures to viruses similar to SARS-CoV-2 and HCoV-229E, respectively.     

Aromatic amino acids in the juxtamembrane domain of severe acute respiratory syndrome coronavirus spike glycoprotein are important for receptor-dependent virus entry and cell-cell fusion

The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Delta19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Delta19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Delta19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.     

Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.     

Facing the wrath of enigmatic mutations: a review on the emergence of severe acute respiratory syndrome coronavirus 2 variants amid coronavirus disease-19 pandemic

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging respiratory virus responsible for the ongoing coronavirus disease 19 (COVID-19) pandemic. More than a year into this pandemic, the COVID-19 fatigue is still escalating and takes hold of the entire world population. Driven by the ongoing geographical expansion and upcoming mutations, the COVID-19 pandemic has taken a new shape in the form of emerging SARS-CoV-2 variants. These mutations in the viral spike (S) protein enhance the virulence of SARS-CoV-2 variants by improving viral infectivity, transmissibility and immune evasion abilities. Such variants have resulted in cluster outbreaks and fresh infection waves in various parts of the world with increased disease severity and poor clinical outcomes. Hence, the variants of SARS-CoV-2 pose a threat to human health and public safety. This review enlists the most recent updates regarding the presently characterized variants of SARS-CoV-2 recognized by the global regulatory health authorities (WHO, CDC). Based on the slender literature on SARS-CoV-2 variants, we collate information on the biological implications of these mutations on virus pathology. We also shed light on the efficacy of therapeutics and COVID-19 vaccines against the emerging SARS-CoV-2 variants.     

Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2

SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2–S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.     

Single-cell RNA analysis on ACE2 expression provides insights into SARS-CoV-2 potential entry into the bloodstream and heart injury

Coronavirus disease-2019 (COVID-19) is a global pandemic with high infectivity and pathogenicity, accounting for tens of thousands of deaths worldwide. Recent studies have found that the pathogen of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), shares the same cell receptor angiotensin converting enzyme II (ACE2) as SARS-CoV. The pathological investigation of COVID-19 deaths showed that the lungs had characteristics of pulmonary fibrosis. However, how SARS-CoV-2 spreads from the lungs to other organs has not yet been determined. Here, we performed an unbiased evaluation of cell-type-specific expression of ACE2 in healthy and fibrotic lungs, as well as in normal and failed adult human hearts, using published single-cell RNA-seq data. We found that ACE2 expression in fibrotic lungs mainly locates in arterial vascular cells, which might provide a route for bloodstream spreading of SARS-CoV-2. Failed human hearts have a higher percentage of ACE2-expressing cardiomyocytes, and SARS-CoV-2 might attack cardiomyocytes through the bloodstream in patients with heart failure. Moreover, ACE2 was highly expressed in cells infected by respiratory syncytial virus or Middle East respiratory syndrome coronavirus and in mice treated by lipopolysaccharide. Our findings indicate that patients with pulmonary fibrosis, heart failure, and virus infection have a higher risk and are more susceptible to SARS-CoV-2 infection. The SARS-CoV-2 might attack other organs by getting into the bloodstream. This study provides new insights into SARS-CoV-2 blood entry and heart injury and might propose a therapeutic strategy to prevent patients from developing severe complications.     

Experimental Evidence for Enhanced Receptor Binding by Rapidly Spreading SARS-CoV-2 Variants

Rapidly spreading new variants of SARS-CoV-2 carry multiple mutations in the viral spike protein which attaches to the angiotensin converting enzyme 2 (ACE2) receptor on host cells. Among these mutations are amino acid changes N501Y (lineage B.1.1.7, first identified in the UK), and the combination N501Y, E484K, K417N (B.1.351, first identified in South Africa), all located at the interface on the receptor binding domain (RBD). We experimentally establish that RBD containing the N501Y mutation results in 7-fold stronger binding to the hACE2 receptor than wild type RBD. The E484K mutation only slightly enhances the affinity for the receptor, while K417N attenuates affinity. As a result, RBD from B.1.351 containing all three mutations binds 3-fold stronger to hACE2 than wild type RBD but 2-fold weaker than N501Y. However, the recently emerging double mutant E484K/N501Y binds even stronger than N501Y. The independent evolution of lineages containing mutations with different effects on receptor binding affinity, viral transmission and immune evasion underscores the importance of global viral genome surveillance and functional characterization.     

Differential binding of SARS-CoV-2 Spike protein variants to its cognate receptor hACE2 using molecular modeling based binding analysis

The current emergence of novel coronavirus, SARS-CoV-2 and its ceaseless expansion worldwide has posed a global health emergency that has adversely affected the humans. With the entire world striving to understand the newly emerged virus, differences in morbidity and infection rate of SARS-CoV-2 have been observed across varied geographic areas, which have been ascribed to viral mutation and evolution over time. The homotrimeric Spike (S) glycoprotein on the viral envelope surface is responsible for binding, priming, and initiating infection in the host. Our phylogeny analysis of 1947 sequences of S proteins indicated there is a change in amino acid (aa) from aspartate (Group-A) to glycine (Group-B) at position 614, near the receptor- binding domain (RBD; aa positions 331-524). The two variants are reported to be in circulation, disproportionately across the world, with Group-A dominant in Asia and Group-B in North America. The trimeric, monomeric, and RBD of S protein of both the variant groups (A & B) were modeled using the Swiss-Model server and were docked with the human receptor angiotensin-converting enzyme 2 (hACE2) employing the PatchDock server and visualized in PyMol. Group-A S protein''s RBD bound imperceptibly to the two binding clefts of the hACE2 protein, on the other hand, Group-B S protein''s RBD perfectly interacted inside the binding clefts of hACE2, with higher number of hydrogen and hydrophobic interactions. This implies that the S protein''s amino acid at position 614 near the core RBD influences its interaction with the cognate hACE2 receptor, which may induce its infectivity that should be explored further with molecular and biochemical studies.     

Evolutionary pathways to SARS-CoV-2 resistance are opened and closed by epistasis acting on ACE2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infects a broader range of mammalian species than previously predicted, binding a diversity of angiotensin converting enzyme 2 (ACE2) orthologs despite extensive sequence divergence. Within this sequence degeneracy, we identify a rare sequence combination capable of conferring SARS-CoV-2 resistance. We demonstrate that this sequence was likely unattainable during human evolution due to deleterious effects on ACE2 carboxypeptidase activity, which has vasodilatory and cardioprotective functions in vivo. Across the 25 ACE2 sites implicated in viral binding, we identify 6 amino acid substitutions unique to mouse—one of the only known mammalian species resistant to SARS-CoV-2. Substituting human variants at these positions is sufficient to confer binding of the SARS-CoV-2 S protein to mouse ACE2, facilitating cellular infection. Conversely, substituting mouse variants into either human or dog ACE2 abolishes viral binding, diminishing cellular infection. However, these same substitutions decrease human ACE2 activity by 50% and are predicted as pathogenic, consistent with the extreme rarity of human polymorphisms at these sites. This trade-off can be avoided, however, depending on genetic background; if substituted simultaneously, these same mutations have no deleterious effect on dog ACE2 nor that of the rodent ancestor estimated to exist 70 million years ago. This genetic contingency (epistasis) may have therefore opened the road to resistance for some species, while making humans susceptible to viruses that use these ACE2 surfaces for binding, as does SARS-CoV-2.

This study suggests that ancient events in mammalian cardiovascular evolution determined the host range of SARS-CoV-2 millions of years before the current pandemic. These physiological constraints are so inflexible that escape from SARS-CoV-2 susceptibility would likely have required significant alterations to the human cardiovascular system.     

Infection of SARS-CoV-2 causes severe pathological changes in mouse testis

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected more than 600 million people worldwide. Several organs including lung, intestine, and brain are infected by SARS-CoV-2. It has been reported that SARS-CoV-2 receptor angiotensin-converting enzyme-2 (ACE2) is expressed in human testis. However, whether testis is also affected by SARS-CoV-2 is still unclear. In this study, we generate a human ACE2 (hACE2) transgenic mouse model in which the expression of hACE2 gene is regulated by hACE2 promoter. Sertoli and Leydig cells from hACE2 transgenic mice can be infected by SARS-CoV-2 pseudovirus in vitro, and severe pathological changes are observed after injecting the SARS-CoV-2 pseudovirus into the seminiferous tubules. Further studies reveal that Sertoli and Leydig cells from hACE2 transgenic mice are also infected by authentic SARS-CoV-2 virus in vitro. After testis interstitium injection, authentic SARS-CoV-2 viruses are first disseminated to the interstitial cells, and then detected inside the seminiferous tubules which in turn cause germ cell loss and disruption of seminiferous tubules. Our study demonstrates that testis is most likely a target of SARS-CoV-2 virus. Attention should be paid to the reproductive function in SARS-CoV-2 patients.     

Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2

SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia pandemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2. As a result, we predicted that SARS-CoV-2 tends to utilize ACE2s of various mammals, except murines, and some birds, such as pigeon. This prediction may help to screen the intermediate hosts of SARS-CoV-2.     

Insights on cross-species transmission of SARS-CoV-2 from structural modeling

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global pandemic that has infected more than 31 million people in more than 180 countries worldwide. Like other coronaviruses, SARS-CoV-2 is thought to have been transmitted to humans from wild animals. Given the scale and widespread geographical distribution of the current pandemic and confirmed cases of cross-species transmission, the question of the extent to which this transmission is possible emerges, as well as what molecular features distinguish susceptible from non-susceptible animal species. Here, we investigated the structural properties of several ACE2 orthologs bound to the SARS-CoV-2 spike protein. We found that species known not to be susceptible to SARS-CoV-2 infection have non-conservative mutations in several ACE2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. Our models also allow us to predict affinity-enhancing mutations that could be used to design ACE2 variants for therapeutic purposes. Finally, our study provides a blueprint for modeling viral-host protein interactions and highlights several important considerations when designing these computational studies and analyzing their results.     

Generation of enzymatically competent SARS-CoV-2 decoy receptor ACE2-Fc in glycoengineered Nicotiana benthamiana

Human angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding and cell entry. Administration of high concentrations of soluble ACE2 can be utilized as a decoy to block the interaction of the virus with cellular ACE2 receptors and potentially be used as a strategy for treatment or prevention of coronavirus disease 2019. Human ACE2 is heavily glycosylated and its glycans impact on binding to the SARS-CoV-2 spike protein and virus infectivity. Here, we describe the production of a recombinant soluble ACE2-fragment crystallizable (Fc) variant in glycoengineered Nicotiana benthamiana. Our data reveal that the produced dimeric ACE2-Fc variant is glycosylated with mainly complex human-type N-glycans and functional with regard to enzyme activity, affinity to the SARS-CoV-2 receptor-binding domain, and wild-type virus neutralization.     

Mechanism and evolution of human ACE2 binding by SARS-CoV-2 spike

Spike glycoprotein of SARS-CoV-2 mediates viral entry into host cells by facilitating virus attachment and membrane fusion. ACE2 is the main receptor of SARS-CoV-2 and its interaction with spike has shaped the virus’ emergence from an animal reservoir and subsequent evolution in the human host. Many structural studies on the spike:ACE2 interaction have provided insights into mechanisms driving viral evolution during the on-going pandemic. This review describes the molecular basis of spike binding to ACE2, outlines mechanisms that have optimised this interaction during viral evolution, and suggests directions for future research.