Coronaviruses (round particles) with spike proteins (cyan) infect a host cell carrying sporadic ACE-2 receptors (pink). After membranes fuse and release viral components (purple). (violet) are released.
Newswise — In Europe, the pandemic initiated in 2020 by the SARS-CoV-2 virus is presently mostly managed. However, the exact mechanism behind this virus's high transmissibility remains unknown. A group of scientists, led by Dr. Simone Backes, Dr. Gerti Beliu, and Prof. Dr. Markus Sauer from Julius Maximilians University of Würzburg (JMU), has recently demonstrated in a publication in "Angewandte Chemie" that certain prior presumptions necessitate reevaluation.
For instance, the virus does not concurrently attach to multiple surface proteins and multiple cell receptors during the infection process. This hypothesis was previously proposed to explain the virus's enhanced infectivity. Additionally, the binding of a virus to a single receptor does not trigger the subsequent attachment of additional receptors to the virus. The research team from Würzburg has now presented compelling evidence that a solitary virus binds to a lone receptor, thereby enabling a remarkably efficient infection.
What could only be speculated about
The SARS-CoV-2 virus bears an average of 20 - 40 spike proteins on its outer surface. Utilizing these proteins, it adheres to ACE2 receptors present in the membrane of its target cells, such as those found in the human nose and throat. When these receptors are obstructed by antibodies, the cell becomes impervious to infection. "This indicates that the attachment of the virus to the ACE2 receptor plays a pivotal role in the infection process," elucidates Sauer.
Visualizing the ACE2 receptors and their interaction with the viral spike proteins through microscopy has been challenging thus far. Consequently, a significant portion of the understanding has relied on speculation, such as the possibility of viruses binding to multiple receptors using multiple spikes to facilitate cell entry.
Another consideration was that the ACE2 receptors may exist in pairs or groups of three within the membrane, allowing them to bind more effectively with the trimeric spike proteins. Alternatively, it was speculated that receptor grouping occurs only after binding to a spike protein. Both hypotheses heavily rely on the density of ACE2 receptors within the membrane.
Super-resolution microscopy made it clear
The researchers from Würzburg aimed to unravel this enigma by employing a method to make the receptors visible and countable. They accomplished this by labeling antibodies with dyes and utilizing different cell lines commonly employed as model systems for SARS-CoV infection. The technique employed was dSTORM (direct stochastic optical reconstruction microscopy), a super-resolution microscopy method known for its single-molecule sensitivity, which was developed by Markus Sauer's research group.
The findings revealed that Vero cells, commonly employed as a model for SARS-CoV-2 infection, possess only one to two ACE2 receptors per square micrometer of cell membrane. This quantity is relatively low compared to other membrane receptors, which typically range from 30 to 80 receptors per square micrometer, as highlighted by Sauer.
Backes explains that the average distance between adjacent ACE2 receptors is approximately 500 nanometers, which is significantly larger than the size of a virus particle measuring only 100 nanometers. Consequently, the notion that a virus particle with multiple spike proteins can simultaneously bind to multiple receptors appears highly improbable, according to her.
ACE2 receptors are always single
The question of whether the receptors are present as pairs or groups of three in the membrane has been addressed. Beliu, a group leader at the Rudolf Virchow Centre, states that the receptors are found individually in the membrane and remain so even after a viral spike protein has bound to them. It is noteworthy that a single spike attaching to a single receptor is sufficient for an infection to occur.
The JMU research team's findings have allowed them to refute several initial hypotheses regarding the interaction between viral particles and multiple ACE2 receptors. Additionally, their research demonstrated that host cells with higher ACE2 expression are more susceptible to infection, as anticipated. Nevertheless, the efficiency of infection is also influenced by factors such as the lipid composition of the membrane and other related elements.
What is next?
The JMU team aims to acquire comprehensive knowledge about the cell entry mechanism of coronaviruses to enhance their understanding of the infection process. This research endeavor could potentially lead to improved preventive measures and the development of more effective drugs against COVID-19. The Würzburg researchers plan to further investigate the entry mechanism using high-resolution light sheet microscopy, enabling detailed analysis and insights into the process.
Funding
The research described in the article received funding from several sources, including the European Research Council, the German Research Foundation (Deutsche Forschungsgemeinschaft), and the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung). These funding bodies provided support for the study conducted by the Würzburg researchers.
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