Why Is the Delta Virus So Dangerous? How Does Coronavirus Infect Cells?

Like a wolf in sheep's clothing, many viruses hide from the human immune system using glycans that coat their external proteins. Computational biophysical chemist Rommie Amaro of the University of California, San Diego, and colleagues used structural and genetic data to produce the most detailed visualization yet of this covering. Using a supercomputer, they constructed the image atom by atom.

Within an hour of posting their simulation on Twitter on March 22, 2020, a researcher commented: What is the bare, uncoated loop protruding from the top of the protein?

Amaro had no answer. But 10 minutes after that comment, structural biologist Jason McLellan of the University of Texas at Austin responded: the uncoated loop binds to human cell receptors and is a receptor-binding domain (RBD), one of the spike's three sections.

Image source: Structural image from Lorenzo Casalino, Univ. California, San Diego; Graphic: Nik Spencer/Nature

In Amaro's simulation, the RBD rises above the glycan cloud, and two glycans hold it in place like a bicycle kickstand. When Amaro introduced mutations into the glycans in the computer model, the RBD collapsed. McLellan and his team devised a way to test the same experiment in the laboratory and, by June 2020, reported that mutating the two glycans reduced the spike protein's ability to bind a human cell receptor. McLellan says this was a role no one previously knew about in coronaviruses. Although researchers cannot yet reduce the two sugars, Amaro suggests that achieving this could reduce the virus's infectiousness.

Since the beginning of the Covid-19 pandemic, scientists have been developing a detailed understanding of how SARS-CoV-2 infects cells. Beyond understanding infection, they want to find better ways to stop it with improved treatments and vaccines and learn why new variants such as Delta are more contagious.

How Does Coronavirus Infect Cells?

What has emerged from 19 months of work, supported by many years of coronavirus research, is a detailed account of how SARS-CoV-2 invades human cells. Scientists have uncovered important adaptations that help the virus attach to human cells with surprising force and then hide inside. As SARS-CoV-2 leaves cells, it performs another important process to prepare its particles to infect more human cells. These are some of the tools that allowed the virus to spread so rapidly and claim millions of lives. Virologist Wendy Barclay of Imperial College London says, “This is why it is so difficult to control.”

Image source: Hui (Ann) Liu, Univ. Utah; Graphic: Nik Spencer/Nature

Spiky and Ready to Infect

Each SARS-CoV-2 virion (virus particle) has an outer surface studded with 24 – 40 randomly arranged spike proteins, key to fusion with human cells. In other viruses, such as influenza, fusion proteins are relatively rigid. However, according to work published in August 2020 by Martin Beck and colleagues at the Mac Planck Institute of Biophysics in Frankfurt, Germany, SARS-CoV-2 spikes are incredibly flexible and attached at three points.

This lets the spikes rotate, sway and turn, making it easier to scan the cell surface and for multiple spikes to bind a human cell. Beck says similar experimental data are unavailable for other coronaviruses, but it is reasonable to assume that ‘the characteristic is shared because spike protein sequences are highly evolutionarily conserved’.

Image source: Cryo-electron tomography images of SARS-CoV-2 (Scale bar: 30 nanometres.) Credit: B. Turoňová et al./Science

Early in the pandemic, researchers confirmed that the RBDs of SARS-CoV-2 spike proteins bind a familiar protein called the ACE2 receptor found in the membranes of most human throat and lung cells. This receptor also serves as the attachment point for SARS-CoV, the virus causing severe acute respiratory syndrome (SARS). However, compared with SARS-CoV, the new coronavirus (SARS-CoV-2) binds the ACE2 receptor 2-4 times more strongly, because several changes in the RBD stabilize the virus's binding sites.

The Problem of Variants

SARS-CoV-2 variants of concern tend to have mutations in the spike protein's S1 subunit, which houses the RBDs and is responsible for binding the ACE2 receptor. A second spike subunit, S2, triggers viral fusion with the host cell.

The Alpha variant, for example, contains 10 changes in the spike protein sequence, making the RBDs more likely to remain in the ‘up’ position. Structural biologist Priyamvada Acharya, who studies spike mutations at the Duke Human Vaccine Institute in North Carolina, says, “It helps the virus advance by making it easier to enter cells.”

The Delta variant, now spreading worldwide, has many mutations in its S1 subunit, including three in the RBD. These mutations appear to enhance Delta's ability for its RBD to bind more strongly to ACE2 and evade the immune system.

Restricted Entry

When the virus's spikes bind ACE2, other proteins on the host cell surface initiate a process that fuses the viral and cellular membranes. (Figure-4: Viral entry)

Source: Janet Iwasa, Univ. Utah; Graphic: Nik Spencer/Nature

The virus causing SARS, SARS-CoV, uses one of two host protease enzymes to enter cells: TMPRSS2 (tempress two) or cathepsin. TMPRSS2 provides a faster entry route, but SARS-CoV instead usually enters through an endosome—a lipid-surrounded bubble—using cathepsin L. Only when virions enter cells by this route can antiviral proteins catch them.

SARS-CoV-2 differs from SARS-CoV because it efficiently uses TMPRSS2, an enzyme abundant on the outside of respiratory cells. First, TMPRSS2 cuts a site in the spike's S2 subunit. The cut region exposes a series of hydrophobic amino acids that rapidly embed themselves in the nearest membrane—the host cell's. The extended spike then folds back on itself like a zipper, forcing the viral and cellular membranes to fuse.

Below is an animation of SARS-CoV-2 fusing with cells. Credit: Janet Iwasa, University of Utah

The virus then transfers its genome directly into the cell. According to work published in April by Barclay and colleagues at Imperial College, invading this way lets SARS-CoV-2 spread faster than SARS-CoV and avoid being caught in endosomes.

Why the Malaria Drug Plaquenil Did Not Work

The virus's rapid entry using TMPRSS2 explains why the malaria drug chloroquine, despite early promising laboratory results, did not work in clinical trials as a Covid-19 treatment. The laboratory experiments were found to have used cells relying solely on cathepsins for endosomal entry. Barclay explains, “When the virus infects and replicates in the human respiratory tract, it does not use endosomes. That is why chloroquine, an endosomal-disrupting drug, is ineffective in real life.”

The finding also points to proteases as a promising therapeutic option for preventing a virus from using TMPRSS2, cathepsin L or other proteases to enter host cells. The TMPRSS2 inhibitor ‘camostat mesylate’, approved to treat pancreatitis in Japan, blocked viral entry into lung cells but did not improve patient outcomes in early clinical trials.

Stefan Pöhlmann, head of the Infection Biology Unit at the German Primate Center in Göttingen, who pioneered research on ACE2 binding and the TMPRRS2 pathway, says, “In my view, we should have protease inhibitors as broad antivirals to combat new disease outbreaks and prevent future pandemics from the outset.”

Deadly Competition

The next steps of infection are even murkier. Chemist Janet Iwasa of the University of Utah, who made an annotated animation of the viral life cycle, says, “There are many more black boxes after cell entry. There is more uncertainty and there are competing hypotheses.”

Coronaviruses commandeer cellular machinery in many ways. Virologist Noam Stern-Ginossar and her team focused on three mechanisms by which SARS-CoV-2 suppresses the host mRNA environment in its favor. Stern-Ginossar says, “None is unique to this virus, but the combination, speed and scale of the effects make it unique.”

• First, the virus eliminates competition: when it seeks to enter a human cell, Nsp1, the first protein translated, commandeers host proteins to systematically break down all cellular mRNAs lacking a viral tag. But when Stern-Ginossar's team placed the same tag at the end of a host mRNA, the mRNA was not broken down.
• Second, infection reduces overall protein translation in the cell by 70%. Studies by two research teams again identify Nsp1 as the main culprit. This time it physically blocks the ribosomes' entry channel, preventing mRNA from entering. Stern-Ginossar says the small remaining translation capacity is reserved for viral RNAs.
• Finally, the virus switches off the cell's alarm system. This happens in several ways, but Stern-Ginossar's team identified a clear mechanism for SARS-CoV-2: the virus prevents cellular mRNA from leaving the nucleus, including instructions for proteins intended to alert the immune system to infection. A second team confirmed this finding and again pointed to Nsp1: the protein appears to constrict exit channels in the nucleus so that nothing can escape. Because gene transcripts cannot leave the nucleus, infected cells do not secrete most interferons —signaling proteins that alert the immune system to a virus's presence. SARS-CoV-2 is particularly effective at switching off this alarm: compared with other respiratory viruses, including SARS-CoV and respiratory syncytial virus (RSV), SARS-CoV-2 infection produces substantially lower interferon levels. In June 2021, researchers reported Alpha-variant mutations that suppress interferon production even more efficiently.

Stern-Ginossar says, “It is clear that SARS-CoV-2 is a very fast virus with a unique ability to prevent our immune system from recognizing infection and fighting it in the early stages.” By the time the immune system realizes there is a virus, so much virus is present that immune-response proteins sometimes enter the bloodstream faster than normal, which can be harmful. Early in the pandemic, people with very severe Covid-19 were understood to be harmed not only by the virus but also by an overactive immune response to SARS-CoV-2. Some proven treatments work by reducing this response.

Remodeling Station

After taking over host translation, the virus begins making changes, extensively reshaping the cell's interior and exterior to meet its needs.

First, some newly made viral spike proteins move to the cell surface and protrude from the host cell membrane. There, they activate a host calcium ion channel that creates a fatty covering on the cell's exterior—the same covering found in cells that naturally fuse, such as muscle cells. The infected cell then fuses with neighboring ACE2-expressing cells, forming large individual respiratory cells containing up to 20 nuclei.

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

On April 18, molecular biologist Mauro Giacca of King's College London and his team published a new finding: these fused structures, called syncytia, are induced by viral infections such as HIV and herpes simplex, but not by the SARS virus. Giacca hypothesizes that forming syncytia lets infected cells thrive for longer and shed increasing numbers of virions. ‘This is not a hit-and-run virus; it persists,’ he says. Qiang Sun and colleagues at the Chinese Academy of Medical Sciences in Beijing found that some Covid-19-infected cells form syncytia with lymphocytes, the body's own immune cells. This is an immune-evasion mechanism known in tumor cells, not viruses. Infected cells are proposed to evade immune detection by latching onto nearby immune scouts and fusing with them.

Proteins involved in constructing DMVs may be good drug targets given their apparent role in viral replication. For example, the host protein TMEM41B is needed to mobilize cholesterol and other lipids to expand ER membranes so that all the virus parts fit inside. Coronavirus researcher Vineet Menachery of the University of Texas Medical Branch in Galveston, who took part in the research, says, ‘Removing TMEM41B has a major effect on infection.’ The coronavirus transmembrane protein Nsp3 may also be a target: it forms a crown-like pore in DMV walls to release newly made viral RNA.

Most viruses with an outer covering, called an envelope, form it by assembling directly at the cell's edge and taking part of the cell's own plasma membrane as they leave. Newly made coronavirus proteins, however, take a different route.

For years, coronaviruses were thought to travel out of the cell through the Golgi complex, an organelle working like a postal service, packaging molecules in membranes and sending them to other parts of the cell. There, the virus forms a lipid envelope from the Golgi membrane. Virologist and cell biologist Carolyn Machamer, who has studied coronaviruses for 30 years at Johns Hopkins University in Maryland, says newly formed virions are then transported inside Golgi vesicles to the cell surface, where they are released.

However, in December, cell biologist at the United States National Heart, Lung, and Blood Institute in Bethesda, Maryland, Nihal Altan-Bonnet and colleagues reported detecting coronaviruses leaving cells through lysosomes—cellular waste bins filled with enzymes that break down cell parts. Altan-Bonnet says blocking the Golgi-based secretory pathway did not affect the amount of infectious virus released. The team's evidence suggests viral proteins form an envelope by budding into the ER, then take over lysosomes to leave the cell. Researchers are testing inhibitors that block lysosomal exit as potential antiviral candidates.

Leaving a cell through the Golgi or lysosomes is slow and inefficient compared with budding through a plasma membrane, so scientists do not know why SARS-CoV-2 does it. Machamer suspects the lipid composition of an envelope derived from the Golgi or a lysosome is somehow more beneficial for the virus than that of the plasma membrane. ‘If we understood this part a little better, there would be major opportunities for new antiviral therapeutics,’ she says.

The End

As the virus leaves the cell, another event makes it infectious: a rapid cut at a five-amino-acid site prepares it to attack its next target.

Where other coronaviruses have a single arginine amino acid at the junction of the spike's S1 and S2 subunits, SARS-CoV-2 has a five-amino-acid sequence: proline, arginine, arginine, alanine and arginine. ‘We focused on the site because it was unusual, and yes, it turned out to be necessary for invasion of lung cells,’ Pöhlmann said. In May 2020, he and colleagues reported that a host cell protein called furin recognizes and cuts this sequence, and that cleavage is ‘necessary’ for efficient entry into human lung cells.

This is not the first time researchers have identified a furin cleavage site in a virus; Barclay says highly pathogenic avian influenza viruses also have one. When a colleague sent Barclay a SARS-CoV-2 strain that had spontaneously lost its furin cleavage site in culture, her team found that infected ferrets shed fewer viral particles than those infected with the pandemic strain and did not transmit infection to nearby animals. As Barclay's team reported its findings in a September 2020 preprint, a Dutch study found that coronavirus with an intact furin cleavage site entered human airway cells faster than virus lacking it.

Furin is suspected to cut the site during virion assembly or just before release. Virologist Tom Gallagher of Loyola University Chicago says the timing may explain why the virus exits through the Golgi or lysosomes. ‘Once assembled, the virus enters an organelle where it can bathe in the presence of the furin protease.’

Gallagher says furin cleavage breaks the bond between the S1 and S2 subunits, loosening virion spike proteins so that they respond to a second cut by TMPRSS2 during cell entry, exposing the hydrophobic region that rapidly embeds in a host cell membrane. If furin has not cut the spikes beforehand—which is not always the case—they bypass TMPRSS2 and enter through the slower endosomal route, if available.

Two coronavirus variants, Alpha and Delta, have altered furin cleavage sites. In Alpha, the first proline amino acid changes to histidine (P681H); in Delta, it changes to arginine (P681R). Barclay says both changes make the sequence less acidic, and the more basic the amino acid sequence, the more efficiently furin recognizes and cuts it. ‘We can assume this made the virus even more effective at transmission.’

More furin cleavage means more spike proteins prepared to enter human cells. Menachery says fewer than 10% of SARS-CoV spike proteins are ready, although his team has not yet published its work counting ready spike proteins. In SARS-CoV-2, the proportion rises to 50%; in Alpha, it exceeds 50%. The team found that more than 75% of spikes in the highly contagious Delta variant were ready to infect a human cell.

Known Unknowns

The scientific community is still only scratching the surface of its understanding of SARS-CoV-2. MacLellan lists important unknowns: how many ACE2 receptors are needed to bind each spike protein, exactly when the S2 site is cleaved by TMPRSS2, and how many spikes are needed for virus–cell membrane fusion—and these concern cell entry alone. In April 2020, a University of California, San Francisco team identified at least 332 interactions between SARS-CoV-2 and human proteins.

Keeping up with the rapidly mutating virus is not easy. Experts agree that most mutations so far concern how effectively the virus spreads rather than how much harm it causes its host. A study this month reported that Delta replicates faster and at higher levels in people's lungs and throats than earlier versions of the virus.

However, Stern-Ginossar says it is not yet clear how Delta's mutations strengthen the variant in this way. ‘This is something many laboratories are trying to understand.’

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