SARS-related coronaviruses
with theoretical spillover potential more widespread than previously thought
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.

evolutionary tree doesn’t add up

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?

binding is an ancient characteristic

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.

Human ACE2
binding is just a hop away

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.”

deeper understanding

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.


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