JANET IWASA, UNIV. UTAH

FeatureA computer simulation of the structure of the coronavirus SARS-CoV-2.

HOW THE CORONAVIRUS
INFECTS OUR CELLS

Scientists are unpicking SARS-CoV-2’s life cycle. By Megan Scudellari
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SOURCE: STRUCTURAL IMAGE FROM LORENZO CASALINO, UNIV.
CALIFORNIA, SAN DIEGO (REF. 1); GRAPHIC: NIK SPENCER/NATURE

The coronavirus sports a luxurious
sugar coat. “It’s striking,” thought
Rommie Amaro, staring at her computer simulation of one of the trademark spike proteins of SARS-CoV-2,
which stick out from the virus’s
surface. It was swathed in sugar
molecules, known as glycans.
“When you see it with all the glycans, it’salmost unrecognizable,” says Amaro, a computational biophysical chemist at the Universityof California, San Diego.
Many viruses have glycans covering theirouter proteins, camouflaging them from thehuman immune system like a wolf in sheep’s
clothing. But last year, Amaro’s laboratorygroup and collaborators created the mostdetailed visualization yet of this coat, basedon structural and genetic data and rendered
atom-by-atom by a supercomputer. On
22 March 2020, she posted the simulation to
Twitter. Within an hour, one researcher asked
in a comment: what was the naked, uncoatedloop sticking out of the top of the protein?
Amaro had no idea. But ten minutes later,
structural biologist Jason McLellan at the
University of Texas at Austin chimed in: theuncoated loop was a receptor binding domain(RBD), one of three sections of the spike thatbind to receptors on human cells (see ‘Ahidden spike’).
In Amaro’s simulation, when the RBD
lifted up above the glycan cloud, two glycans
swooped in to lock it into place, like a kickstandon a bicycle. When Amaro mutated the glycans
in the computer model, the RBD collapsed.
McLellan’s team built a way to try the same
experiment in the lab, and by June 2020, thecollaborators had reported that mutating thetwo glycans reduced the ability of the spike
protein to bind to a human cell receptor1 — arole that no one has previously recognized incoronaviruses, McLellan says. It’s possible thatsnipping out those two sugars could reducethe virus’s infectivity, says Amaro, although
researchers don’t yet have a way to do this.
Since the start of the COVID-19 pandemic,
scientists have been developing a detailed
understanding of how SARS-CoV-2 infects
cells. By picking apart the infection process,they hope to find better ways to interrupt it
through improved treatments and vaccines,
and learn why the latest strains, such as the
Delta variant, are more transmissible.
What has emerged from 19 months of work,
backed by decades of coronavirus research, isa blow-by-blow account of how SARS-CoV-2
invades human cells (see ‘Life cycle of the pandemic coronavirus’). Scientists have discovered key adaptations that help the virus to grab
on to human cells with surprising strength andthen hide itself once inside. Later, as it leaves
cells, SARS-CoV-2 executes a crucial processing step to prepare its particles for infecting
even more human cells. These are some of thetools that have enabled the virus to spread soquickly and claim millions of lives. “That’s why
it’s so difficult to control,” says Wendy Barclay,
a virologist at Imperial College London.

Barbed and readyIt starts with the spikes. Each SARS-CoV-2
virion (virus particle) has an outer surface
peppered with 24–40 haphazardly arranged
spike proteins that are its key to fusing withhuman cells2. For other types of virus, suchas influenza, external fusion proteins are
relatively rigid. SARS-CoV-2 spikes, however,are wildly flexible and hinge at three points,
according to work published in August 2020by biochemist Martin Beck at the Max Planck
Institute of Biophysics in Frankfurt, Germany,and his colleagues3.
That allows the spikes to flop around, sway
and rotate, which could make it easier for themto scan the cell surface and for multiple spikes
to bind to a human cell. There are no similar
experimental data for other coronaviruses,
but because spike-protein sequences arehighly evolutionarily conserved, it is fair toassume the trait is shared, says Beck.
Early in the pandemic, researchers confirmed that the RBDs of SARS-CoV-2 spike
proteins attach to a familiar protein called theACE2 receptor, which adorns the outside ofmost human throat and lung cells. This receptor is also the docking point for SARS-CoV, thevirus that causes severe acute respiratory
syndrome (SARS). But compared with SARSCoV, SARS-CoV-2 binds to ACE2 an estimated

A HIDDEN SPIKE

The spike protein of SARS-CoV-2 is coated insugar molecules, or glycans, which disguise it
from the immune system. It can hinge at threepoints on the stalk, giving it flexibility.
Receptor-binding
domainGlycanS1
subunit

S2
subunit

Hip
Knee
Stalk
Ankle

2–4 times more strongly4, because several
changes in the RBD stabilize its virus-binding
hotspots5.
Worrying variants of SARS-CoV-2 tend tohave mutations in the S1 subunit of the spike
protein, which hosts the RBDs and is responsible for binding to the ACE2 receptor. (A second
spike subunit, S2, prompts viral fusion with thehost cell’s membrane.)
The Alpha variant, for example, includes
ten changes in the spike-protein sequence,
which result in RBDs being more likely to stayin the ‘up’ position6. “It is helping the virus
along by making it easier to enter into cells,”
says Priyamvada Acharya, a structural biologist at the Duke Human Vaccine Institute in
Durham, North Carolina, who is studying the
spike mutations.
The Delta variant, which is now spreading
around the world, hosts multiple mutationsin the S1 subunit, including three in the RBD
that seem to improve the RBD’s ability to bindto ACE2 and evade the immune system7.

Restricted entry
Once the viral spikes bind to ACE2, otherproteins on the host cell’s surface initiate aprocess that leads to the merging of viral andcell membranes.
The virus that causes SARS, SARS-CoV, uses
either of two host protease enzymes to breakin: TMPRSS2 (pronounced ‘tempress two’) or
cathepsin L. TMPRSS2 is the faster route in,but SARS-CoV often enters instead throughan endosome — a lipid-surrounded bubble —which relies on cathepsin L. When virions enter
cells by this route, however, antiviral proteins
can trap them.
SARS-CoV-2 differs from SARS-CoV because
it efficiently uses TMPRSS2, an enzyme foundin high amounts on the outside of respiratory
cells. First, TMPRSS2 cuts a site on the spike’s
S2 subunit8. That cut exposes a run of hydrophobic amino acids that rapidly buries itself inthe closest membrane — that of the host cell.
Next, the extended spike folds back onto itself,
like a zipper, forcing the viral and cell membranes to fuse (see ‘Viral entry up close’).
The virus then ejects its genome directly
into the cell. By invading in this springloaded manner, SARS-CoV-2 infects fasterthan SARS-CoV and avoids being trapped inendosomes, according to work published in
April by Barclay and her colleagues at Imperial
College London9.
The virus’s speedy entry using TMPRSS2
explains why the malaria drug chloroquine
didn’t work in clinical trials as a COVID-19 treatment, despite early promising studies in thelab10. Those turned out to have used cells that
rely exclusively on cathepsins for endosomal
entry. “When the virus transmits and replicatesin the human airway, it doesn’t use endosomes,
so chloroquine, which is an endosomal disrupting drug, is not effective in real life,” says Barclay.

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Nature | Vol 595 | 29 July 2021 | 641

Feature

LIFE CYCLE OF THE PANDEMIC CORONAVIRUS
A simplified account of how SARS-CoV-2 enters and exits cells.

Nucleocapsid
Spike proteinRNA

M protein

TMPRSS2
ACE2
Stage 2: Inside the cell
Viral RNA is translated into
non-structural proteins (NSPs) thatquickly suppress the translation ofhost messenger RNAs in favour of
those belonging to the virus.
Endoplasmic
reticulum (ER)TMPRSS2 cuts
the spike protein

Deadly competition

Viral proteins(NSPs)

NSPs

Viral RNA

Ribosome

The spikes unravel and pullthe membrane of the virus
and host cell together

ER remodelling

DMVs

Stage 3: Remodelling the cell
The virus transforms the cell’s ER —an internal membrane network —into bubble-like structures called
double-membrane vesicles (DMVs).
These might provide a safe havenfor more viral RNA to be replicatedand translated.

Golgi
apparatus

Furin cutStage 5: The last slice
A host enzyme named furin
makes a crucial cut at a siteof five amino acids on the
spike protein. This prepares
the virus to strike anothercell. Variants have a higher
proportion of snipped spike
proteins, helping them to
infect cells more efficiently.

Stage 4: Exit
Once the newly made molecules
assemble into a complete virus
particle, this leaves the cell throughan organelle called the Golgi
apparatus, or perhaps throughlysosomes, which are cellular
rubbish bins.

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The next steps of infection are murkier. “There
are a lot more black boxes once you are inside
the cell,” says chemist Janet Iwasa at the University of Utah in Salt Lake City, who is developing
an annotated animation of the viral life cycle.
“There’s more uncertainty, and competing
hypotheses.”
After the virus shoots its RNA genome into
the cell, ribosomes in the cytoplasm translate
two sections of viral RNA into long strings ofamino acids, which are then snipped into
16 proteins, including many involved in RNA
synthesis. Later, more RNAs are generated
that code for a total of 26 known viral proteins,
including structural ones used to make newvirus particles, such as the spike, and otheraccessory proteins. In this way, the virus
begins churning out copies of its own messenger RNA. But it needs the cell’s machineryto translate those mRNAs into proteins.
Coronaviruses take over that machinery inmany ways. Virologist Noam Stern-Ginossar
and her team at the Weizmann Institute of Science in Rehovot, Israel, zoomed in on threemechanisms by which SARS-CoV-2 suppresses
the translation of host mRNA in favour of its
own. None are exclusive to this virus, but the
combination, speed and magnitude of theeffects seem unique, says Stern-Ginossar.
First, the virus eliminates the competition:
viral protein Nsp1, one of the first proteinstranslated when the virus arrives, recruits hostproteins to systematically chop up all cellular mRNAs that don’t have a viral tag. When
Stern-Ginossar’s team put that same tag on
the end of a host mRNA, the mRNA was not
chopped up12.
Second, infection reduces overall proteintranslation in the cell by 70%. Nsp1 is again themain culprit, this time physically blocking theentry channel of ribosomes so mRNA can’t get
inside, according to work from two researchteams 13,14. The little translation capacitythat remains is dedicated to viral RNAs, says
Stern-Ginossar.
Finally, the virus shuts down the cell’s alarm

SOURCE: HUI (ANN) LIU, UNIV. UTAH (HTTPS://ANIMATIONLAB.UTAH.EDU/COVA);
GRAPHIC: NIK SPENCER/NATURE

Stage 1 : Viral entry
The virus’s spike protein binds to a
receptor on the host cell called ACE2.
Then, the host molecule TMPRSS2
cleaves the spike protein, exposing
parts that fuse the viral membranewith that of the host.

The discovery also points to proteaseinhibitors as a promising therapeutic option toprevent a virus from using TMPRSS2, cathepsin L or other proteases to enter host cells.
One TMPRSS2 inhibitor, camostat mesylate,which is approved in Japan to treat pancreatitis, blocked viral entry into lung cells8, but
the drug did not improve patients’ outcomesin an initial clinical trial11.
“From my perspective, we should have suchprotease inhibitors as broad antivirals available to fight new disease outbreaks and prevent
future pandemics at the very beginning,” says
Stefan Pöhlmann, director of the Infection
Biology Unit at the German Primate Center
in Göttingen, who has led research on ACE2
binding and the TMPRSS2 pathway.

SOURCE: JANET IWASA, UNIV. UTAH (HTTPS://ANIMATIONLAB.UTAH.EDU/
COVA); GRAPHIC: NIK SPENCER/NATURE

system. This happens in numerous ways, but
Stern-Ginossar’s team identified one clear
mechanism for SARS-CoV-2: the virus prevents
cellular mRNA from getting out of the nucleus,
including instructions for proteins meant toalert the immune system to infection. A second team confirmed this finding, and again
pointed to Nsp1: the protein seems to jam up
exit channels in the nucleus so nothing canescape15.
Because gene transcripts can’t get out of thenucleus, the infected cells don’t release many
interferons — these are signalling proteins thatalert the immune system to the presence of avirus. SARS-Cov-2 is particularly efficient at
shutting down this alarm system: comparedwith other respiratory viruses, including
SARS-CoV and respiratory syncytial virus,
SARS-CoV-2 infection induces significantly
lower levels of interferons16. And this June,
researchers reported mutations in the Alphavariant that seem to enable it to subdue interferon production even more efficiently17.
“It’s clear that SARS-CoV-2 is a very fastvirus that has a unique ability to prevent
our immune system from recognizing andcombating infection in the first stages,” says
Stern-Ginossar. By the time the immune system does realize there is a virus, there is somuch of it that immune-response proteinssometimes flood the bloodstream at a fasterrate than normal — which can cause damage.
Doctors saw early in the pandemic that somepeople with COVID-19 who become very ill are
harmed by an overactive immune response to
SARS-CoV-2, as well as by the virus itself. Some
proven treatments work by dampening down
this immune response.

Renovation station
Once the virus has taken over host translation, it starts a home makeover, extensively
remodelling the interior and exterior of thecell to its needs.
First, some of the newly made viral spike
proteins travel to the surface of the cell andpoke out of the host-cell membrane. There,
they activate a host calcium-ion channel,
which expels a fatty coating onto the outsideof the cell — the same coating found on cells
that naturally fuse together, such as muscle
cells. At this point, the infected cell fuses toneighbouring cells expressing ACE2, developing into massive individual respiratory cells
filled with up to 20 nuclei.
These fused structures, called syncytia,
are induced by viral infections such as HIV
and herpes simplex virus, but not by the
SARS virus, says molecular biologist Mauro
Giacca at King’s College London, who led theteam that published the finding in April18. He
hypothesizes that forming syncytia allows
infected cells to thrive for long periods of time,
churning out more and more virions. “This is
not a hit-and-run virus,” he says. “It persists.”

VIRAL ENTRY UP CLOSE

Virus and host-cell membranes fuse after the TMPRSS2 enzyme cuts a SARS-CoV-2 spike protein.
This exposes hydrophobic amino acids in the spike that rapidly embed themselves into thenearest membrane — that of the host cell.
Virus

Spike
protein

TMPRSS2

Cut

ACE2

Hydrophobic
amino acids
attach to nearest
membrane

Host cell

A second team, led by researcher Qiang Sun at
the Chinese Academy of Medical Sciences in
Beijing, found that some COVID-19-infected
cells even form syncytia with lymphocytes —one of the body’s own immune cells19. This isa known mechanism of immune evasion bytumour cells, but not by viruses. It suggeststhat infected cells avoid immune detection
by simply grabbing on to and merging withnearby immune scouts.
On the inside of the cell, even more change
is occurring. Like other coronaviruses,
SARS-CoV-2 transforms the long, thin endoplasmic reticulum (ER), a network of flat
membranes involved in protein synthesis andtransport, into double-membrane spheres,“There are a lot more
black boxes once you
are inside the cell.”
as if the ER were blowing bubbles. These double-membrane vesicles (DMVs) might provide
a safe place for viral RNA to be replicated andtranslated, shielding it from innate immune
sensors in the cell, but that hypothesis is still
being investigated.
Proteins involved in making DMVs could begood drug targets, because they seem to benecessary for viral replication. For instance,a host protein, TMEM41B, is needed to mobilize cholesterol and other lipids to expand
the ER membranes so that all the virus parts
will fit inside20. “When you take TMEM41B
out, it has a major impact on infection,” says
Vineet Menachery, a coronavirus researcher
at the University of Texas Medical Branch in
Galveston, who was involved in the research.
The coronavirus transmembrane protein Nsp3
could also be a target: it creates a crown-likepore in the walls of the DMVs to shuttle out

newly made viral RNA21.
Most viruses that have an outer wrapping,known as an envelope, form this feature byassembly directly at the edge of the cell,
co-opting some of the cell’s own plasma
membrane on their way out. But newly madecoronavirus proteins take a different path.
For years, evidence has suggested that
coronaviruses are transported out of the cellthrough the Golgi complex, an organelle thatworks like a post office, packaging molecules
in membranes and sending them off to otherparts of the cell. There, the virus forms a lipid
envelope from the Golgi complex’s membrane;
newly formed virions are then carried inside
Golgi vesicles to the cell surface, where they
are spat out of the cell, says virologist and cellbiologist Carolyn Machamer at Johns Hopkins
University in Baltimore, Maryland, who hasstudied coronaviruses for 30 years.
But in December, cell biologist Nihal
Altan-Bonnet at the US National Heart, Lung,
and Blood Institute in Bethesda, Maryland,
and her colleagues reported that they had
detected coronaviruses leaving the cellthrough lysosomes — cellular rubbish bins
full of enzymes that break down cell parts22.
Blocking the Golgi-based secretory pathway
didn’t seem to affect the amount of infectious
virus being released, says Altan-Bonnet. Her
team’s evidence22 suggests that viral proteins
form an envelope by budding into the ER, thentake over lysosomes to get out of the cell. The
researchers are currently testing inhibitorsthat block the lysosomal exit process as potential antiviral candidates.
Leaving a cell through either the Golgi orlysosomes is slow and inefficient comparedwith budding out of a plasma membrane, so
scientists don’t know why SARS-CoV-2 does it.
Machamer suspects that the lipid compositionof a Golgi- or lysosome-derived envelope issomehow more beneficial to the virus than one

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FeatureMore furin cuts mean more spike proteinsprimed to enter human cells. In SARS-CoV, lessthan 10% of spike proteins are primed, says
Menachery, whose lab group has been quantifying the primed spike proteins but is yet to
publish this work. In SARS-CoV-2, that percentage rises to 50%. In the Alpha variant, it’s morethan 50%. In the highly transmissible Delta variant, the group has found, greater than 75% of
spikes are primed to infect a human cell.

Fused cell structures (syncytia) seen in cells expressing the SARS-CoV-2 spike protein(green). Nuclei are in blue and the cell skeleton is in red.

from the plasma membrane. “If we understood
this part a little bit better, there would be great
opportunities for novel antiviral therapeutics,”
she says.

Last slice
On the way out of the cell, one more event
makes this virus into an infectious juggernaut:a quick snip at a site of five amino acids prepares the virus to strike its next target.
Where other coronaviruses have a single
arginine amino acid at the junction of the S1and S2 subunits of the spike, SARS-CoV-2 has aline of five amino acids: proline, arginine, arginine, alanine and arginine. “Because the site
was unusual, we focused on it, and it turned
out that, yes, the site is essential for invasion oflung cells,” says Pöhlmann. In May 2020, he andhis colleagues reported that a host-cell protein
called furin recognizes and clips that string ofamino acids — and the cut is “essential” for thevirus to enter human lung cells efficiently23.
It’s not the first time that researchers haveidentified a furin cleavage site on a virus;
highly pathogenic avian influenza viruses also
have it, says Barclay. When a colleague sent
Barclay a strain of SARS-CoV-2 in culture that
had spontaneously lost the furin cleavage site,
her team found that ferrets infected with thisstrain shed viral particles in lower amounts
than did those infected with the pandemicstrain, and did not transmit the infection to
nearby animals9. At the same time as Barclay’s
team reported its results in a September 2020
preprint, a study in the Netherlands also foundthat coronavirus with an intact furin cleavage
site enters human airway cells faster than do“We would hypothesize that
this is the virus getting evenbetter at transmitting.”
in a host-cell membrane, says Gallagher. If
spikes are not pre-clipped by furin —and they
aren’t always — they bypass TMPRSS2, andenter through the slower endosomal pathway, if at all.
Two coronavirus variants, Alpha and Delta,
have altered furin cleavage sites. In the Alphavariant, the initial proline amino acid ischanged to a histidine (P681H) ; in the Delta
variant, it is changed to an arginine (P681R).
Both changes make the sequence less acidic,
and the more basic the string of amino acids,
the more effectively furin recognizes andcuts it, says Barclay. “We would hypothesizethat this is the virus getting even better attransmitting.”

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those without it24.
Furin is suspected to cut the site at somepoint during virion assembly, or just before
release. The timing might explain why the virus
exits through the Golgi or lysosomes, says Tom
Gallagher, a virologist at Loyola University
Chicago in Illinois. “The virus, once assembled,
moves into an organelle where it can be bathedin the presence of the furin protease.”
By snipping the bond between the S1 and S2
subunits, the furin cut loosens up virion spike
proteins so that during cell entry they respondto a second cut by TMPRSS2, which exposes
the hydrophobic area that rapidly buries itself

The scientific community is still scratching thesurface of its understanding of SARS-CoV-2.
Key unknowns include the number of ACE2
receptors needed to bind to each spike protein; when exactly the S2 site is cleaved by
TMPRSS2; and the number of spikes neededfor virus–cell membrane fusion, says McLellan— and that’s just for entry. In April 2020, a teamat the University of California, San Francisco,identified at least 332 interactions between
SARS-CoV-2 and human proteins25.
It is not easy to keep pace with the quickly
mutating virus. Most mutations so far are associated with how effectively the virus spreads,
not with how much the virus damages the host,experts agree. This month, a study reported
that the Delta variant grew more rapidly and at
higher levels inside people’s lungs and throats
than did earlier versions of the virus26.
But it is not yet certain how Delta’s mutations have supercharged the variant in thisway, says Stern-Ginossar. “This is something
many labs are trying to figure out.”
Megan Scudellari is a science journalist in
Boston, Massachusetts.

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MAURO GIACCA

Known unknowns