Scientists Compare ACE2 and SARS-CoV-2 Interactions Between Species

The COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic disease.Like any other pathogenic Coronavirus, SARS-CoV-2 crossed the species barrier and caused human disease.This fact motivated the current research published on the preprint server. bioRxiv* In October 2020, we will investigate how coronavirus is involved in host cell receptors in various animals.

The importance of this study lies in the close interaction between humans and various types of livestock, where respiratory spread is very likely. Not only is this a constant threat to humanity, but it also poses financial challenges to livestock farmers and pet owners.

Research on the pathology of the virus and measurement of candidate therapies also use animals infected with the virus. Therefore, this study assists public health professionals, economic strategists, and those working with the COVID-19 animal model.

Viral binding via spike protein

The virus binds with high affinity to angiotensin converting enzyme 2 (ACE2) on host cells in the lungs and other organs. As a result, the virus fuses with the host’s cell membrane, causing cell invasion and infection.

The SARS-CoV-2 spike-ACE2 complex has been studied by crystallography and electron microscopy. This showed how amino acids interact with the spike receptor binding domain (RBD) in human ACE2.

Measuring potential spike RBD binding can help predict which animals are susceptible to the virus.

Variability of ACE2 sequence

Researchers in the current study have examined the conserved diverse sequences of the ACE2 receptor protein in many related species, especially the receptor binding domain (RBD).

All vertebrates had an ACE2 sequence homologous to human ACE2. The sequences of cynomolgus monkeys and rhesus monkeys were 95% and 94% identical to those of humans. For other mammals, it was on the order of 80-87%.

In the horseshoe bat, suspected of being a viral reservoir, the identity was only 81% of the total ACE2 protein and 76% of the 25 interacting amino acids. In fact, in many cases, the degree of identity varied widely across sequences and interacting residues.

In response to this finding, they observed one ACE2 protein from cattle (Torus boss) Is different from the human mutant in the entire sequence (79% identity), but showed high affinity binding to SARS-CoV-2 spike RBD (84% identity). This suggests that there may be many species of mammals that allow infection with this virus.

On the other hand, the binding site residues are less identical than the entire dog and rabbit sequences. Future studies need to establish the hypothesis that residues that interact with RBD are much more associated with binding than those outside this site.

Variable residues in the ACE2-RBD interface of individual species. With reference to human ACE2360, the various mutant interaction residues are colored red and the conserved 361 residues are colored cyan. For the rest of the ACE2 protein, the conserved residues are white and the 362 variable residues are bright red. Certain residues, such as M82, Q24, D30, and H34, are often mutated compared to 363 humans. D30 has been conservatively converted to glutamic acid, but H34, for example, has a more non-conservative change.

Binding affinity with ACE2 sequence

Researchers have compared the importance of different regions of the protein and individual amino acids to its structure. This type of study has previously demonstrated the presence of complementarity determining regions (CDRs) in antibodies that interact with the corresponding antigen.

A powerful tool used for variability analysis is Shannon entropy. Such tests have shown that ACE2 is highly conserved throughout the sequence. Of the 25 RBD-binding or interacting residues, 21 were well conserved and none had high Shannon entropy.

There were 5 fully conserved residues, 4 near the binding site and only 1 contact residue. Their preservation demonstrates their importance for cell invasion or interspecific protein structure via protease-mediated degradation.

The most variable were at positions 24 and 34, the latter in the center of the ACE2-RBD interface, with much higher Shannon entropy. Both of these are contact residues. Some researchers suggest that these positions may help predict the infectivity of certain SARS-CoV-2 mutants.

Binding affinity with matching residues

Residues that did not match human ACE2 were evaluated individually in the next step. They found that in the Greater Horseshoe Bat, only 5 of the 8 residues in contact with RBD are identical to human ACE2, but near 19/25. This may mean that ACE2 has a low affinity for the virus.

Another putative intermediate animal host of the virus was the scale armor, where only one contact and three adjacent residues did not match that of human ACE2. More data is needed to confirm its role in SARS-CoV-2 infection. Similar close matches include dogs and cats.

In cattle, three interacting residues at the binding site change, one is the contact residue. Cattle are known to carry the far-flung SARS-CoV-2 associated bovine coronavirus, and two coronaviruses in this family have already jumped over species barriers to infect humans with colds. ..

However, they bind to sialoglicans instead of ACE2. Nevertheless, the ability of cattle to function as a storage host for SARS-CoV-2 needs to be evaluated. In fact, the interaction of bovine coronavirus with SARS-CoV-2 can lead to the emergence of recombinants with dangerous consequences.

Highly conserved bovine ACE2 at the binding site

Bovine ACE2 is approximately 78% identical to humans, but binding site residues show higher (84%) conservation. Researchers investigated whether this would allow high-affinity binding to the target virus RBD, despite the low homology of the rest of the ACE2 sequence.

In an ELISA study, they found that bovine ACE2 binding appeared to be one-tenth that of human ACE2. This is mainly due to the high rate of dissociation from the complex once formed. Surprisingly, this low affinity was comparable to that of human ACE2 for SARS-CoV-1 RBD and was in the low nanomolar range compared to the latter sub-nanomolar concentration.

Ideal for 2 site binding models

When simulating the viral interaction with RBD using a two-site model, both human and bovine ACE2 showed a much more consistent fit. This may mean that an RBD multimer is formed and binds tightly to the surface of ACE2. If so, the presence of a second site would explain a 10x higher affinity.

Implications

“Such interactions are important to investigate in order to understand the details of the interaction between coronavirus spike RBD and cell surface ACE2,” the researchers commented. Although bovine ACE2 is one-fifth to one-tenth that of human ACE2, it binds to SARS-CoV-2 RBD with high affinity, and it seems that the virus can infect cattle.

The study also demonstrates the feasibility of using the biochemical properties of the receptor to predict how different species will treat the virus, especially in fast screening.

Researchers said,Further studies of various species of viral receptors, including the recently discovered neuropyrin-2 co-receptor of SARS-CoV-2, make prediction algorithms based on viral receptor sequence and structural modeling much more accurate and rapid. Development should be possible... “

*Important Notices

bioRxiv publishes unpeer-reviewed preliminary scientific reports and should not be considered definitive, guide clinical / health-related behaviors, or be treated as established information.