A nanoscale look at coronavirus infection
Date:
March 9, 2022
Source:
Stanford University
Summary:
Using super-resolution microscopy, scientists uncovered new details
about the location of viral molecules in a cell after coronavirus
infection.
FULL STORY ==========================================================================
A human cell being infected by a coronavirus is a crowded place as
the virus turns its host into a virus-replicating machine. Now, for
the first time, Stanford scientists have used super-resolution light
microscopy to sift through the crowd and determine where in the cell
viral molecules lie.
==========================================================================
W.E. Moerner, professor of chemistry, and Stanley Qi, assistant professor
of bioengineering and Institute Scholar at Stanford ChEM-H, have used
the method, which gives scientists a nanoscale view into the cell, to
pinpoint exactly where in the cell certain pieces of the coronavirus --
like the spike protein and the genetic material -- are at different
points post-infection. They found that, unlike what lower-resolution
confocal microscopy has indicated, the virus-replicating machinery and
the RNA product of that process are physically separated in the cell,
which could indicate new details about the viral life cycle.
Moerner, the Harry S. Mosher Professor in the School of Humanities and
Sciences and professor, by courtesy, of applied physics, and Qi studied
a coronavirus called HCoV-229E that, like its cousin SARS-CoV-2, is made
up of a spike protein-studded envelope surrounding a strand of RNA, the
virus' genetic material. That single strand of genomic RNA, or gRNA,
contains the instructions for making all the proteins that the virus
needs, including those that make copies of the gRNA and those that
assemble into the packaging that wraps around the RNA to make a new,
intact virus.
"When infected, the cell turns itself into a zombie, completely mind
controlled into producing more virus," said Qi, who is also an assistant professor of chemical and systems biology.
Scientists know a lot about which molecules are involved in which steps of viral life cycle. But precisely where in the cell all the virus' molecules
are during those steps has remained largely unanswered. Understanding
these subtle details could give greater insight into precisely how the
virus infects cells and help researchers find vulnerabilities or develop
better treatments for infection.
In the study, which was published in Cell Reports Methods Feb. 28,
the team zeroed in on two different forms of RNA: double-stranded RNA,
or dsRNA, which is an intermediate along the way to making new copies of
the virus, and gRNA, one strand of which gets injected into the cell, replicated and then packaged into new viruses. Knowing exactly where
in the cell those pieces are could tell scientists not only where the virus-replicating steps (dsRNA) and virus- assembly steps (gRNA) are
taking place, but how those steps are coordinated spatially.
========================================================================== Cellular galaxy Confocal fluorescence microscopy is a common method
for seeing objects within a cell by recording light emitted from
fluorescent labels or tags, not that different from the molecules
that give rise to "day-glo" socks. But confocal microscopy can only
be precise with structures that are about 250 nanometers (nm) across
or larger. Coronavirus particles are much smaller, at about 120 nm
in diameter, and the proteins and RNA within them even smaller. (For
reference, a strand of hair is about 100,000 nm thick.) "There is no
getting around the fundamental blurriness of confocal microscopy," said Moerner. "Many important cellular objects are very small; some 50 nm, some
10 nm, and some even smaller." Super-resolution fluorescence microscopy
uses carefully controlled single- molecule imaging to bring these
cellular objects into sharper focus, allowing scientists to see objects
as small as 10 nm across. Scientists can only look at a single cell at
a time using these techniques, and the experiments require lot of time
and specialized resources. Despite the challenges, the unrivaled detail
with which scientists can view the cell makes the process invaluable. And
that jump in clarity revealed something unexpected to Moerner and Qi.
The research team used two differently colored tags to look at their two molecules, magenta for gRNA and green for dsRNA. In addition to spots
of green and magenta, confocal images showed blurry white clouds that
suggested that dsRNA and gRNA could be in the same spot throughout the
cell, possibly enveloped together in some kind of particle. But by using super-resolution techniques, the team saw something very different.
========================================================================== "When I saw those images for the first time, it was like looking at
some amazing galaxy," said Moerner, who received the Nobel Prize in
chemistry in 2014 for developing the microscopy techniques that give
scientists these detailed views into the cell. The super-resolution
images showed a dark sky of bright magenta clusters and green stars --
and none of them ever overlapped.
Contrary to what confocal images had hinted at, dsRNA and gRNA are never
in the same place at the same time.
Separate experiments, in which they also looked at proteins from the
virus and the host cell, confirmed that the virus-replicating dsRNA and
the RNA product of that replication are never found floating through the
cell together. Their results confirmed that viral replication occurs
in a part of the cell known as the endoplasmic reticulum, or ER, as
was already known. The gRNA formed then buds off into the cell to get
packaged into a fully formed virus. Unlike what previous studies have
shown, however, Moerner and Qi now saw that in addition to being found
inside the ER, the virus-replicating dsRNA is also found in large (up
to 450 nm) spheres that do not contain any gRNA throughout the cell.
They suspect that these bubbles of dsRNA, which are not actively
replicating, might be a sort of temporary dsRNA storage while new viruses
are being packaged and shipped out.
Exploring antiviral treatments Viral infection is a complex process, and
while the team does not know exactly what drives the virus to produce
these temporary stores of dsRNA, they hope that super resolution can
also answer those questions and others in the future.
By learning more about when and where certain viral infection steps take
place, scientists may also be able to develop and evaluate treatments.
In this study, the researchers in the Moerner and Qi labs also joined
forces to look at what happens after treatment with the antiviral
remdesivir. They saw that the while the levels of gRNA and dsRNA overall decreased in the cell, the size of the dsRNA bubbles remained the same,
which supports their temporary storage theory. The team hopes that further studies with the super-resolution toolkit could help determine if other antivirals might target those spheres.
"When people don't have tools, they have no way of making new findings,"
said Qi.
"This is a great example of how you can't predict what you will find
until you go looking," said Moerner. "Much can be learned about the
biology of these complex systems with modern nanoscale optical tools."
Other Stanford coauthors include former graduate student Jiarui Wang, postdoctoral scholars Mengting Han and Leiping Zeng, graduate student
Anish Roy and former postdoctoral scholars Haifeng Wang and Leonhard
Mo"ckl.
Moerner is a professor in the School of Humanities in Sciences, a
faculty fellow at Stanford ChEM-H and a member of Bio-X and of the Wu
Tsai Neurosciences Institute. Qi is a member of Bio-X, the Maternal &
Child Health Research Institute, Stanford Cancer Institute and the Wu
Tsai Neurosciences Institute.
The work was supported by the National Institute of General Medical
Sciences and the National Institutes of Health. Wang is a Mona M. Burgess Stanford Bio- X Fellow.
========================================================================== Story Source: Materials provided by Stanford_University. Original written
by Rebecca McClellan. Note: Content may be edited for style and length.
========================================================================== Journal Reference:
1. Jiarui Wang, Mengting Han, Anish R. Roy, Haifeng Wang, Leonhard
Mo"ckl,
Leiping Zeng, W.E. Moerner, Lei S. Qi. Multi-color super-resolution
imaging to study human coronavirus RNA during cellular
infection. Cell Reports Methods, 2022; 2 (2): 100170 DOI:
10.1016/j.crmeth.2022.100170 ==========================================================================
Link to news story:
https://www.sciencedaily.com/releases/2022/03/220309104513.htm
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