Barbed and ready

It 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 with human cells2. For other types of virus, such as 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 2020 by 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 them to 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 are highly evolutionarily conserved, it is fair to assume the trait is shared, says Beck.

Cryo-electron tomography images of SARS-CoV-2 virions. (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 attach to a familiar protein called the ACE2 receptor, which adorns the outside of most human throat and lung cells. This receptor is also the docking point for SARS-CoV, the virus that causes severe acute respiratory syndrome (SARS). But compared with SARS-CoV, SARS-CoV-2 binds to ACE2 an estimated 2–4 times more strongly4, because several changes in the RBD stabilize its virus-binding hotspots5.

Worrying variants of SARS-CoV-2 tend to have 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 the host cell’s membrane.)

The Alpha variant, for example, includes ten changes in the spike-protein sequence, which result in RBDs being more likely to stay in 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 mutations in the S1 subunit, including three in the RBD that seem to improve the RBD’s ability to bind to ACE2 and evade the immune system7.

Restricted entry

Once the viral spikes bind to ACE2, other proteins on the host cell’s surface initiate a process that leads to the merging of viral and cell membranes (see ‘Viral entry up close’).

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

The virus that causes SARS, SARS-CoV, uses either of two host protease enzymes to break in: TMPRSS2 (pronounced ‘tempress two’) or cathepsin L. TMPRSS2 is the faster route in, but SARS-CoV often enters instead through an 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 found in 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 in the 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.

An animation of the way SARS-CoV-2 fuses with cells.Credit: Janet Iwasa, University of Utah

The virus then ejects its genome directly into the cell. By invading in this spring-loaded manner, SARS-CoV-2 infects faster than SARS-CoV and avoids being trapped in endosomes, 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 the lab10. Those turned out to have used cells that rely exclusively on cathepsins for endosomal entry. “When the virus transmits and replicates in the human airway, it doesn’t use endosomes, so chloroquine, which is an endosomal disrupting drug, is not effective in real life,” says Barclay.

The discovery also points to protease inhibitors as a promising therapeutic option to prevent 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’ outcomes in an initial clinical trial11.

“From my perspective, we should have such protease 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: Structural image from Lorenzo Casalino, Univ. California, San Diego (Ref. 1); Graphic: Nik Spencer/Nature

In Amaro’s simulation, when the RBD lifted up above the glycan cloud, two glycans swooped in to lock it into place, like a kickstand on 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, the collaborators had reported that mutating the two glycans reduced the ability of the spike protein to bind to a human cell receptor1 — a role that no one has previously recognized in coronaviruses, McLellan says. It’s possible that snipping out those two sugars could reduce the 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, is a 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 and then 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 the tools that have enabled the virus to spread so quickly and claim millions of lives. “That’s why it’s so difficult to control,” says Wendy Barclay, a virologist at Imperial College London.

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

• NEWS FEATURE
• 28 July 2021

How the coronavirus infects cells — and why Delta is so dangerous

Scientists are unpicking the life cycle of SARS-CoV-2 and how the virus uses tricks to evade detection.

• Megan Scudellari

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A computer simulation of the structure of the coronavirus SARS-CoV-2.Credit: Janet Iwasa, University of Utah

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’s almost unrecognizable,” says Amaro, a computational biophysical chemist at the University of California, San Diego.

Many viruses have glycans covering their outer proteins, camouflaging them from the human immune system like a wolf in sheep’s clothing. But last year, Amaro’s laboratory group and collaborators created the most detailed visualization yet of this coat, based on 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, uncoated loop 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: the uncoated loop was a receptor binding domain (RBD), one of three sections of the spike that bind to receptors on human cells (see ‘A hidden spike’).

Better late than never! A Promisory Abstract from Nature..

1. nature  
2. letters  
3. article

• Letter
• Published: 11 July 2018

Reprogramming human T cell function and specificity with         non-viral genome targeting

• Theodore L. Roth, 
• Cristina Puig-Saus, 
• […]
• Alexander Marson 

Nature volume 559, pages 405–409 (2018)Cite this article

• 71k Accesses

• 292 Citations

• 589 Altmetric

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Abstract

Decades of work have aimed to genetically reprogram T cells for therapeutic  purposes1,2using recombinant viral vectors, which do not target transgenes to                     specific genomic sites3,4.                                                                                                                           

The need for viral vectors has slowed down research and clinical use as their                                         manufacturing and testing is lengthy and expensive.                                                                

Genome editing brought the promise of specific and efficient insertion of large            transgenes into target cells using homology-directed repair5,6.  

 Here we developed a CRISPR–Cas9 genome-targeting system that does not                        require viral vectors, allowing rapid and efficient insertion of large DNA                      sequences (greater than one kilobase) at specific sites in the genomes of                                                    primary human T cells, while preserving cell viability and function.                                             This permits individual or multiplexed modification of endogenous genes.                               

First, we applied this strategy to correct a pathogenic IL2RA mutation in cells                                          from patients with monogenic autoimmune disease, and demonstrate improved                                    signalling function. Second, we replaced the endogenous T cell receptor                                (TCR) locus with a new TCR that redirected T cells to a cancer antigen.                                        The resulting TCR-engineered T cells specifically recognized tumour antigens                                   and mounted productive anti-tumour cell responses in vitro and in vivo.                                  

Together, these studies provide preclinical evidence that non-viral genome                        targeting can enable rapid and flexible experimental manipulation and                               therapeutic engineering of primary human immune cells.

 

I WAS WRONG!

 I was wrong! I though the Chinese government was finally doing an exemplary public service  when they released science based Covid-19 information to the United Nations and to the World. 

 Today, November 5, I learned that I was wrong! It was a brave young Chinese journalist who released the information to the world and she is now paying the price. Today, Zhang Zhan is dying in a Chinese prison because she released the Covid-19 information to the United Nations. The Chinese government accused her of provoking trouble and disturbing the public order.

 As a journalist myself who discovered years ago that journalistic objectivity does not exist, my blood is boiling! I am angry for believing the Chinese government was anything but a callous group of old farts without humanitarian ethics. I am angry at myself for crediting a cruel Communist dictatorship with the decency to inform the world of a deadly pandemic creating virus. A government punishing the one journalist in China with the self sacrificing courage to speak up.

 The Chinese government would have allowed millions of people to die if it were not for the courage of Zhang Zhan. Today, I ask for her immediate release and for her hospitalization, preferably in some other country. She is an international heroine!

 Today, I am disturbing the world order by accusing the Chinese leaders of cruelty, stupidity and ridiculous politics. Zhang Zhan is being punished for telling the truth and saving millions of lives. If the Chinese leaders want to save face, I recommend they move their collective butts quickly to save Zhang Zhan or I will call for an international boycott of Chinese products and BEJING CAN GO BACK TO THE STONE AGE! 

How is this for disturbing the public order?

Signed: Nelson Joseph Raglione

International Journalist.