Virus World

SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry.

Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.

Published in Cell (July 7, 2023):

https://doi.org/10.1016/j.cell.2023.06.005

The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into human cells is an essential step for virus transmission and development of COVID 19. Although the lung epithelial cells are its initial target, SARS-CoV-2 also can infect endothelial cells. Endothelial cells are the major constituents of the vascular system and cardiovascular complication is a hallmark of severe COVID-19. Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for SARS-CoV-2. However, the possible involvement of other cellular components in the viral entry is not fully understood.  A team of researchers from the Boston University School of Medicine (BUSM) has identified extracellular vimentin as an attachment factor that facilitates SARS-CoV-2 entry into human cells. Vimentin is a structural protein that is widely expressed in the cells of mesenchymal origin such as endothelial cells and a potential novel target against SARS-CoV-2, which could block the infection of the SARS-CoV-2. "Severe endothelial injury, vascular thrombosis, and obstruction of alveolar capillaries (tiny air sacs scattered throughout the lungs) are common features of severe COVID-19.

Identification of vimentin as a host attachment factor for SARS-CoV-2 can provide new insight into the mechanism of SARS-CoV-2 infection of the vascular system and can lead to the development of novel treatment strategies," said corresponding author Nader Rahimi, Ph.D., associate professor of pathology & laboratory medicine at BUSM. The researchers used liquid chromatography–tandem mass spectrometry (LC-MS/MS) and identified vimentin as a protein that binds to the SARS-CoV-2 spike (S) protein and facilitates SARS-CoV-2 infection. They also found that depletion of vimentin significantly reduces SARS-CoV-2 infection of human endothelial cells. In contrast, over-expression of vimentin with ACE2 significantly increased the infection rate. "More importantly, we saw that the CR3022 antibody inhibited the binding of vimentin with CoV-2-S-protein, and neutralized SARS-CoV-2 entry into human cells," explained Rahimi. 

Findings Published in P.N.A.S. (Jan. 25, 2022):

https://doi.org/10.1073/pnas.2113874119 

In Europe, the pandemic triggered in 2020 by the SARS-CoV-2 coronavirus is now largely under control. But why this virus is able to spread so efficiently remains unclear. A team of researchers led by Dr. Simone Backes, Dr. Gerti Beliu and Prof. Dr. Markus Sauer of the Julius Maximilians University of Würzburg (JMU) has now shown in a publication in "Angewandte Chemie" that some previous assumptions need to be reconsidered. For example, the virus does not bind with several surface proteins simultaneously to several receptors of the cell to be infected. This assumption has previously been an attempt to explain how viruses increase their infectivity. Binding to a single receptor also does not lead to the subsequent docking of further receptors to the virus. The Würzburg research group has now provided evidence that a single virus binds to a single receptor, opening the door for a highly efficient infection.

What could only be speculated about

SARS-CoV-2 carries an average of 20 - 40 spike proteins on its surface. With these, it binds to ACE2 receptors in the membrane of its target cells, for example in the nose and throat of humans. When these receptors are blocked with antibodies, the cell can no longer be infected. Making the ACE2 receptors and their interaction with the viral spike proteins visible microscopically has not been possible so far. Therefore, much was left to speculation - such as whether the viruses bind to multiple receptors with multiple spikes to facilitate entry into the cell. It was also considered that the receptors are present in the membrane in pairs or groups of three rather, so that they can bind more efficiently to the trimeric spike proteins. Or that they are only combined into such groups after binding to a spike protein. Both depend strongly on the density of the ACE2 receptors in the membrane.

Super-resolution microscopy made it clear

The Würzburg researchers wanted to elucidate this mystery: They labeled antibodies with dyes to make the receptors visible and countable. To do this, they used various cell lines that are used as model systems for SARS-CoV infection, and the single-molecule sensitive super-resolution microscopy method dSTORM, developed in Markus Sauer's research group. It turned out that Vero cells, for example, which are often used as a model for SARS-CoV-2 infection, only have one to two ACE2 receptors per square micrometre of cell membrane. This is very few: "In other membrane receptors, this number is often between 30 and 80," Sauer added. "The average distance between neighbouring ACE2 receptors is about 500 nanometres. It is thus much larger than a virus particle, which measures only 100 nanometres," says Backes. The idea that a virus particle with multiple spike proteins can bind to multiple receptors simultaneously is therefore very unlikely, she adds.

ACE2 receptors are always single

The following open question: Are the receptors also present as pairs or groups of three in the membrane? "No. They only occur there singly. And it stays that way even when a viral spike protein has bound to them," says Beliu, group leader at the Rudolf Virchow Center. For an infection, it is sufficient if a single spike binds to a single receptor. With these results, the JMU team was able to disprove many of the original hypotheses about the interaction of viral particles with multiple ACE2 receptors. It also showed that host cells with higher ACE2 expression are more easily to infect, as expected. However, the lipid composition of the membrane and other factors also influence infection efficiency.

What is next?

The JMU team wants to gather as much detailed knowledge as possible about the cell entry mechanism of coronaviruses in order to better understand the infection process. This could ultimately contribute to better prevention and the development of better drugs against COVID-19. Next, the Würzburg researchers want to analyse the entry mechanism with high-resolution light sheet microscopy.

Published March 2023:

https://doi.org/10.1002/anie.202300821 

In a major breakthrough an international team of scientists, led by the University of Bristol, has potentially identified what makes SARS-CoV-2 highly infectious and able to spread rapidly in human cells. The findings, published in Science today [20 October] describe how the virus's ability to infect human cells can be reduced by inhibitors that block a newly discovered interaction between virus and host, demonstrating a potential anti-viral treatment.  Unlike other coronavirus, which cause common colds and mild respiratory symptoms, SARS-CoV-2, the causative agent of COVID-19, is highly infective and transmissive. Until now, major questions have remained unanswered as to why SARS-CoV-2 readily infects organs outside of the respiratory system, such as the brain and heart. To infect humans, SARS-CoV-2 must first attach to the surface of human cells that line the respiratory or intestinal tracts. Once attached, the virus invades the cell then replicates multiple copies of itself. The replicated viruses are then released leading to the transmission of SARS-CoV-2.  The virus's process of attachment to and invasion of human cells is performed by a viral protein, called the 'Spike' protein. Understanding the process by which the 'Spike' protein recognises human cells is central to the development of antiviral therapies and vaccines to treat COVID-19.

In this breakthrough study, the research groups in Bristol's Faculty of Life Sciences, Professor Peter Cullen from the School of Biochemistry; Dr. Yohei Yamauchi, Associate Professor and virologist from the School of Cellular and Molecular Medicine, and Dr. Boris Simonetti, a senior researcher in the Cullen lab, used multiple approaches to discover that SARS-CoV-2 recognises a protein called neuropilin-1 on the surface of human cells to facilitate viral infection. Yohei, Boris and Pete explained: "In looking at the sequence of the SARS-CoV-2 Spike protein we were struck by the presence of a small sequence of amino acids that appeared to mimic a protein sequence found in human proteins which interact with neuropilin-1. This led us to propose a simple hypothesis: could the Spike protein of SARS-CoV-2 associate with neuropilin-1 to aid viral infection of human cells? Excitingly, in applying a range of structural and biochemical approaches we have been able to establish that the Spike protein of SARS-CoV-2 does indeed bind to neuropilin-1. "Once we had established that the Spike protein bound to neuropilin-1 we were able to show that the interaction serves to enhance SARS-CoV-2 invasion of human cells grown in cell culture. Importantly, by using monoclonal antibodies—lab-created proteins that resemble naturally occurring antibodies—or a selective drug that blocks the interaction we have been able to reduce SARS-CoV-2's ability to infect human cells. This serves to highlight the potential therapeutic value of our discovery in the fight against COVID-19."

Intriguingly, scientists at the Technical University of Munich, Germany and the University of Helsinki, Finland, have independently found that neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity. Together the Bristol researchers concluded: "To defeat COVID-19 we will be relying on an effective vaccine and an arsenal of anti-viral therapeutics. Our discovery of the binding of the SARS-CoV-2 Spike to neuropilin-1 and its importance for viral infectivity provides a previously unrecognised avenue for anti-viral therapies to curb the current COVID-19 pandemic."

Origina studies published in Science (Oct. 20, 2020):

https://doi.org/10.1126/science.abd3072

https://doi.org/10.1126/science.abd2985