To infect our cells, SARS-CoV-2’s spike protein turns the key on the molecular doorway formed by our ACE2 protein. Mutations that adapted its spike protein to human ACE2 helped the virus make its way out of bats, where SARS-related coronaviruses have circulated for thousands of years, and eventually trigger the COVID-19 pandemic. After the smaller SARS epidemic in the early 2000s, COVID-19 is the second viral outbreak driven by a coronavirus that acquired the key to our cells.
New work, published Thursday in the journal Nature from scientists at Fred Hutchinson Cancer Research Center and the University of Washington, suggests that to safeguard against other coronaviruses that could gain the ability to sneak into our cells, we need to think globally. The researchers found that the ability to bind ACE2 — a crucial trait in species-jumping coronaviruses — could be a more widespread possibility than previously thought. Instead of being a late evolutionary development, the ability to bind ACE2 is an ancient property of bat SARS-related coronaviruses — and found in coronaviruses outside of Asia.
The researchers found that many of the coronaviruses that already bind one species’ ACE2 can jump to another species’ ACE2 (including humans’) by switching a single protein building block, or amino acid. Binding to human ACE2 is not the only requirement for spillover into humans, but it’s an important hurdle a coronavirus must clear to start a pandemic, said Dr. Tyler Starr, the postdoctoral fellow in Hutch evolutionary biologist Dr. Jesse Bloom’s lab who led the work.
Researchers who are working on broad coronavirus vaccines and treatments in preparation for future viral spillovers must take these more distantly related viruses into account, he said.
“People who are working on surveillance and therapeutic antibody development need to expand their breadth,” Starr said. “If you don’t test viruses [from around the world] you’re missing the plot. We don’t know where the next pandemic might come from.”
The findings also highlight the care that should be taken in handling and sampling coronaviruses with unknown ACE2 binding potential, he said.
Starr, who joined Bloom’s lab to study HIV and quickly pivoted to SARS-CoV-2 when the COVID-19 pandemic struck, was inspired to study the origins of ACE2 binding in SARS-related coronaviruses (known as sarbecoviruses) after researching the hunt for the origins of SARS-CoV-1, which caused an epidemic in Asia in 2003.
“It was a 10-year process, and there was a debate the whole time,” he said.
Although many bat viruses with genetic similarity to SARS-CoV-1 were found across China during this time, they were genetically distinct, and none of these viruses were found to use ACE2 proteins as their entry receptor.
It wasn’t until 2013 that researchers announced they’d found a related bat coronavirus whose key fit the lock of the human ACE2, marking these bat viruses as a proximal source of the SARS-CoV-1 epidemic. Scientists worried about future spillovers intensified their searches for more ACE2-binding bat coronaviruses, which continued to be found in just a single province in Southern China, even though sarbecoviruses are found throughout the world.
Many bat sarbecoviruses in Asia, Europe, and Africa did not appear to use ACE2 as their entry point, leading researchers to conclude that it was a trait that had appeared late in coronavirus evolution.
But when Starr studied the tree of sarbecovirus evolution, he wasn’t convinced.
“SARS-CoV-2 sprung from a branch where we weren’t looking [for a new spillover event],” Starr said.
He suspected that the hyper-focus on ACE2-binding bat coronaviruses in one region of the world had blinded scientists to the origins of ACE2 binding in coronaviruses.
“It seemed like there was potential that using ACE2 is a trait writ large,” he said. “Using ACE2 in general could be a more widespread trait than was previously appreciated.”
What if the coronaviruses that didn’t use ACE2 to infect were the exception, instead of the rule?
The region of the coronavirus spike protein that interacts with ACE2 is called the receptor-binding domain, or RBD. To assess whether ACE2 binding is a new or old characteristic, Starr amassed 45 RBD genes from across the four known, closely related subgroups of RBDs across the SARS-related coronavirus family. These subgroups, also known as clades, describe related RBDs that can be traced to the same branch point off the sarbecovirus evolutionary tree. SARS-Cov-2 and SARS-CoV-1 RBDs fall into different clades with closely related bat viruses, both in Asia. Starr’s survey included two RBDs from a clade of sarbecoviruses circulating in bats in Europe and Africa, which diverged from Asian sarbecoviruses hundreds or thousands of years ago.
Then, using a high-throughput system in which he enlists yeast to display just the RBD segment of the spike protein, Starr screened each RBD’s ability to bind ACE2 receptors from different host species, including human, civet, pangolin, mouse and two species of bat found in China.
As expected, nearly all RBDs in SARS-CoV-2 and SARS-CoV-1’s clades bound each species' ACE2 to a greater or lesser degree. But RBDs from the third clade of Asian sarbecoviruses didn’t bind any ACE2 from any species Starr tested, as had previously been suspected for this clade. And, in a first for any sarbecovirus found outside Asia, Starr saw that a bat virus from Kenya bound to two bat ACE2s.
That virus is from “a distinct clade on the evolutionary tree, and obviously, geographically, that's a much larger range that a single province in China, or a larger region in Southeast Asia, where ACE2 binding as a general property was thought to emerge,” Starr said.
Collaborating with University of Washington biochemist Dr. David Veesler, Starr confirmed that the Kenyan virus does use bat ACE2 to enter cells.
“This suggests that in Africa and Europe, viruses are probably also using ACE2, and it's this one clade in Southeast Asia that lost ACE2 binding that's so heavily sampled that is actually the outlier,” Starr said.
But because of how distantly related the Kenyan virus is to the Asian sarbecoviruses that scientists have focused on, Starr expects that many related but yet-to-be tested sarbecoviruses also use ACE2 as their entry point.
Though human ACE2 binding isn’t the only requirement for a sarbecovirus to trigger a pandemic, Starr’s findings suggests that scientists should change the geographic breadth of viral surveillance and perform wider, more careful sampling to monitor sarbecovirus spillover potential, he said.
Starr said that his study also can inform work on therapeutics and vaccines. To guard against a future spillover, researchers are working to design pan-sarbecovirus vaccines and therapeutic antibodies. But concentrating too much on SARS-CoV-1 and -2 and ignoring distantly related sarbecoviruses could leave us vulnerable to spillover events that may happen elsewhere on the sarbecovirus family tree, he said.
Starr used computational methods to reconstruct the ancestral RBD that existed before Asian and non-Asian sarbecovirus lineages diverged, and found that the ancestor also bound one of the bat ACE2 variants. According to Starr’s reconstruction, broader ACE2 binding arose in the ancestors of Asian sarbecoviruses as they diverged from the European and African viruses, then was lost in one clade as its ancestors diverged again from those that would give rise to SARS-Cov-1 and -2.
A lot of the mutations in SARS-CoV-2 variants, like delta and omicron, cluster in their RBD. This means SARS-CoV-2’s RBD is evolutionarily flexible — it can take a lot of mutations but still do its job, Starr said. That helped this virus acquire the ability to bind human ACE2 and jump into us. How easy would it be, Starr wondered, for other sarbecoviruses to make the same leap?
Pretty easy, it turns out.
Starr used deep mutational scanning, an approach Bloom’s team often uses to assess the effect of mutations on a protein’s function, to test how mutations affected a given RBD’s ability to bind ACE2.
He tested 14 RBDs and found that single amino acid switches in an RBD’s protein sequence could dramatically improve its binding to its ACE2 target. Such single mutations could also help an RBD acquire the ability to bind a new ACE2 species target. Starr found two individual amino acid changes that conferred human ACE2 binding on the Kenyan sarbecovirus that already targeted a bat ACE2.
“So human ACE2 binding is easily accessible in that region, where we don’t have much sampling of viruses to begin with,” he said. And more generally, "sarbecoviruses can cross species boundaries pretty easily, probably due in part to the evolvability of the RBD interface [that contacts ACE2]” that these deep mutational scanning experiments reveal.
He also showed that whether an RBD mutation improves ACE2 binding depends on which virus it’s found in. For example, N501Y, an amino acid change that enhances human ACE2 binding by SARS-CoV-2 variants of concern, reduces ACE2 binding in SARS-CoV-1.
This means that scientists can’t extrapolate from one virus to another, Starr said.
“After 2003, a lot of people were working on SARS-CoV-1, under the premise that we should be prepared for the next pandemic,” he said. “If we were monitoring viruses in nature and we saw that N501Y, we would’ve said, ‘It’s nothing to be concerned about.’ But we would have been misled.”
Starr is working to better understand nuances of human ACE2 in the sarbecovirus branch that gave rise to SARS-CoV-1 and -2’s subgroups, as well as why these viruses’ RBDs are better at binding a range of species’ ACE2s. He also wants to understand why these changes occurred.
He thinks it may be related to fact that bats which harbor coronaviruses often have several ACE2 variants and live in caves containing many bat species infected with many sarbecoviruses. Viruses whose RBDs were flexible and able to use differing ACE2s would be the most likely to stick around and keep infecting more bats. Bats, in turn, continue to evolve new flavors of ACE2 to evade the coronaviruses, setting up an evolutionary arms race that could have driven human ACE2-binding as a side effect, Starr said.
Figuring out what it was about bats in Southeast Asia that drove human ACE2 binding could also help scientists better understand how likely it is to emerge in bat coronaviruses in Europe and Asia. Are there similarities or differences in bat ACE2 variants or bat ecology that make this property more or less likely to emerge elsewhere?
Moreover, said Starr, he’s hoping to shed light on general principles at play in viral pandemics. The forces that helped SARS-CoV-2 spill over into humans may be at play in other unrelated viruses. This could help scientists trying to develop antibody therapeutics that work against a range of potential pandemic pathogens.
“There are a hundred other viruses people think could be the source of the next pandemic,” Starr said.
This research was funded by the National Institute of Allergy and Infectious Diseases, the National Institute of General Medical Sciences, a Pew Biomedical Scholars Award, the Burroughs Wellcome Fund, Fast Grants, the Bill & Melinda Gates Foundation, the Russian Foundation for Basic Research, the Damon Runyon Cancer Foundation and the Howard Hughes Medical Institute.
Sabrina Richards, a staff writer at Fred Hutchinson Cancer Center, has written about scientific research and the environment for The Scientist and OnEarth Magazine. She has a PhD in immunology from the University of Washington, an MA in journalism and an advanced certificate from the Science, Health and Environmental Reporting Program at New York University. Reach her at srichar2@fredhutch.org.
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