YSM Issue 94.1

Virology & Molecular Biology
FOCUS
previous study found that, in SARS-CoV,
Nsp1 is also necessary for viral replication,
making it a vital component of sickness
progression and a strong candidate for
target therapeutics against coronaviruses.
With the COVID-19 outbreak,
coronavirus research became
critical, and scientists applied
what was already known about
an earlier coronavirus, SARS-
CoV, to make hypotheses
about SARS-CoV-2. Yale
Molecular Biophysics
and Biochemistry
professor Yong Xiong,
whose research group
studies how viruses
suppress and escape a host's
immune system, hypothesized
that SARS-CoV-2 Nsp1 is likely
critical for disease progression
and poisoning host cells.
To test this hypothesis, his
collaborator, associate professor
Sidi Chen, investigated twentyseven
of the twenty-nine proteins
encoded by the SARS-CoV-2
genome. Chen
t r a n s f e c t e d
each protein
individually
into human
l u n g
epithelial
cells and
found that
out of the
t w e n t y - s e v e n
S A R S - C o V - 2
proteins tested, Nsp1
caused the most
severe decrease in
cell viability. To
confirm that Nsp1
is the linchpin of
this phenotype, a new
population of cells
was transfected with a
mutated, defunct copy of
Nsp1. This group of cells
remained healthy, leading
Xiong and Chen to conclude in a
recent paper published
in Molecular Cell that
SARS-CoV-2 Nsp1 is “one of
the most potent pathogenicity
protein factors of SARS-CoV-2
in human cells of lung origin.”
www.yalescientific.org
Shifting Gears
After Xiong and his collaborators
knew with greater certainty what leads to
pathogenicity, they began to investigate
how Nsp1 led to this cell sickness. Nsp1
infection causes a large-scale shift in
the host cell's transcriptome, with
the expression of 9,262 genes
being altered as a result of this
protein’s presence in the cell. By
sequencing cellular mRNAs and
quantifying the amount of each
mRNA transcript present using
mRNA-seq, the research team was able to
determine which host genes were affected
by Nsp1 expression. Nsp1 expression led
to the decreased expression of 5,394 genes,
the majority of which are related to protein
synthesis, cellular metabolism, and the
immune system. To express the proteins
encoded in their own genome, cells
need the protein-production machinery,
the ribosome, and energy to translate
their mRNA transcripts into proteins.
By suppressing genes involved in these
processes, Nsp1 shuts down cellular protein
synthesis—hijacking the host cell, rerouting
resources to build viral machinery,
and dampening the cell's immune response
to allow the infection to occur.
The connection between Nsp1 expression
and the genes it upregulates is less clear than
those it downregulates. Nsp1 upregulates
the expression of 3,868 genes that encode
transcription factors that regulate higherorder
chromatin structure, homeobox
genes that are most known for driving body
patterning, DEAD-box genes that regulate
RNA metabolism, and regulators that drive
cell fate determination. How upregulation of
these genes might affect the pathogenicity of
SARS-CoV-2 is not yet understood. "Logically,
Nsp1 programs the cellular transcriptome
in order to redirect cellular resources to the
virus, but there is nothing specific that jumps
out to us," Xiong said. It is also unclear how
Nsp1 alters gene expression on a molecular
level as Nsp1 has no nuclear activity, meaning
that it never enters the host cell nucleus where
all the cell's genetic information is stored.
The Two-Pronged Approach
In the case of SARS-CoV, Nsp1 has
been shown to bind to the 40S, the small
ribosomal subunit, to block translation
of mRNA into protein and promote
cleavage and degradation of cellular mRNA.
However, the molecular mechanisms of
these activities remained unexplained.
Recent advancements in cryogenic electron
microscopy (cryo-EM) and Xiong’s role in
bringing this technology to Yale has made it
possible to use these clues from SARS-CoV
to look at SARS-CoV-2 activities at the
atomic scale.
Xiong used cryo-EM to investigate
how Nsp1 inhibits protein synthesis.
By freezing proteins down to cryogenic
temperatures (approximately below
negative 150 degrees Celsius), Xiong was
able to capture proteins in their native form
and image these native structures at the
resolution of 2.7 angstroms, about the width
of a water molecule. His lab found that the
C-terminus, or back end, of the Nsp1 protein
tightly binds to the mRNA entry channel
on the 40S subunit, while the N-terminus
interacts more loosely with subunit’s head
domain. “Think of a body with a neck and
head. Around the neck is the mRNA path,
where it is loaded and translated,” Ivan
Lomakin, an associate research scientist in
the Bunick lab and expert in human protein
synthesis, explained. “Part of Nsp1 binds
to this path. The other portion binds to the
head, which is a moving part that would
otherwise enable mRNA to slide along
the channel.” While the C-terminus
of Nsp1 physically sits in the entry
channel at the neck and
binds to the
ribosomal
RNA and
r i b o s o m a l
proteins uS3 and
uS5, the rest of the
Nsp1 molecule interacts with the
head domain of the ribosome.
The exact effect of this is unknown since
the N-terminus does not bind tightly to the
ribosome, so the cryo-EM image could not
precisely determine how the N-terminus
makes contact with the 40S subunit. Nsp1
also competes with some initiation factors
critical for eukaryotic translation for binding
to the 40S subunit and locks the 40S subunit
in a “closed” conformation, which is the state
where the ribosome is unable to load mRNA.
In addition to preventing mRNA from
loading onto the ribosome, previous studies
focusing on SARS-CoV have shown that Nsp1
prompts the cutting of host cell mRNA. mRNA
stability is determined by many structural
features within the mRNA transcript, which
March 2021 Yale Scientific Magazine 15