Even as the coronavirus disease 2019 (COVID-19) pandemic was unfolding, there was already evidence suggesting that the disease was zoonotic in origin. One theory that gained particular media attention was the likelihood of transmission from wild bats, potentially sold in the wet market in Wuhan, China.
Since then, transmission to several other species has been seen, including mink and Syrian golden hamster, and many theorize the ability of the disease to spread to other non-human primates. Researchers from Oregon State University have developed a system to assess the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind to angiotensin-converting enzyme 2 (ACE2) orthologs.
The spike protein of SARS-CoV-2 is essential to the disease’s pathogenicity. It is formed of two subunits, S1 and S2. S1 contains a receptor-binding domain (RBD) that binds to several receptors, primarily ACE2, to permit viral cell entry. The N-terminal domain in S2 is responsible for membrane fusion.
The researchers transfected cells from a human rectal cancer cell line that were capable of supporting beta-coronavirus replication. These HRT-18G cells were transfected with vectors containing bicistronic mRNA that encoded for different ACE2 orthologs and mouse Thy1.1, which acted as a cell surface reporter protein. It is known that alternate ACE2 orthologs can allow species to escape SARS-COV-2 infection or prove susceptible to it. Several studies have modeled these effects, and some in vitro studies have even proven susceptibility or lack thereof. However, the researcher’s method should allow more rapid and trustworthy results and can easily be adapted to examine more orthologs.
As the mRNA in the DNA plasmid vectors were translated by the same mRNA as the ACE2, relative expression of Thy1.1 could infer ACE2 expression without creating new antibodies to detect each ACE2 ortholog or using hACE2 antibodies that may not bind to the orthologs with the same affinity. Using basic hACE2 in the plasmid showed 95% of cells expressing Thy1.1. A commercially available labeled RBD protein was incubated alongside the cells, and flow cytometry showed interaction in a concentration-dependent manner. SARS-CoV-2 pseudovirus expressing the spike protein and GFP proved to infect these cells with only five minutes of exposure.
Once the basic function of the method had been established, the scientists tested their hypothesis that Thy1.1 expression would increase with ACE2 expression and increased RBD binding and infectivity. To explore the interaction of transient hACE2 with the spike protein, both WT HRT-18G and transfected HRT-18G were stained with Thy1.1 and fluorescently labeled RBD – showing a strong positive correlation between Thy1.1 expression and RBD binding. They also discovered that when GFP labeled SARS-COV-2 pseudoviruses carrying the spike protein, the percentage of infected GFP-positive cells was highest in cells expressing high levels of ACE2, further confirming their hypothesis that cells expressing higher levels of ACE2 would be more susceptible to infection.
To test the different ACE2 orthologs, additional HRT18G cell lines were generated using the aforementioned system. These cell lines expressed either domestic feline or mouse ACE2 orthologs at equivalent levels. These cells were stained with antibodies for hACE2 – showing no positive results. SARS-CoV-2 RBD interactions were tested by incubating the cells with fluorescently labeled RBD and measuring binding with flow cytometry.
The mouse ACE2 showed no interaction between the ACE2 and spike protein – which is curious, as transmission to several rodent species has been observed in vivo. However, several other studies have also shown that Mus musculus ACE2 cannot interact with SARS-CoV-2. Human ACE2 showed the strongest affinity, followed by feline ACE2. Thy1.1. expression proved that all ACE2 orthologs were expressed equivalently. Confirmation using the previously mentioned SARS-CoV-2 pseudoviruses showed the same pattern, with hACE2 cells showing the most infection, followed by feline and then murine ACE2.
While the researchers have provided some insight into the ability of SARS-CoV-2 to spread to mice and cats, the greatest benefit of their research is the new method for examining the ability of SARS-CoV-2 to bind to ACE2 orthologs. Several studies have reported conflicting findings on the same ortholog, which could be due to the monoclonal antibody used to detect expression.
With this system, different orthologs can be compared without worrying about binding affinity or taking the time to find a monoclonal antibody that will bind effectively to the desired ortholog. The potential insights of this system could provide valuable information into the ability of SARS-CoV-2 to spread to other species and the likelihood of another zoonotic event. This could prove invaluable to public health policymakers and scientists attempting to model the future of the pandemic.
A preprint version of the group’s study is available on the bioRxiv server while the article undergoes peer review.
Surce: News Medical