Periodic Reporting for period 1 - CoVentry (Multiscale study of the interactions between corona viruses of various pathogenicity and cell membrane components in the early stages of virus entry)
Reporting period: 2022-05-23 to 2024-05-22
Understanding how viruses attach to and enter h ost cells is critical in the study of infectious diseases. This attachment process is the first step in viral infection and plays a key role in determining how effectively a virus can spread. Viruses bind to a membrane molecule which is needed for viral entry, referred as viral receptor. In the case of SARS-CoV-2, this interaction is mediated by the viral protein spike. However, the cellular membrane is a complex environment comprised of hundreds of molecular species, which the virus has to navigate to reach the receptor. To do so, viruses also establish weak and transient bonds to several additional molecules. Heparan sulfate (HS), a complex sugar molecule found on the surface of human cells, is known to be exploited as an attachment factor by various viruses. SARS-CoV-2 spike has been shown to interact with HS and the strength of this interaction to vary among the variants that emerged during the pandemic.
Traditionally, studies of molecular interactions are performed using isolated molecular species. Researchers often purify the spike protein and the viral receptor and measure the strength of the bond formed between them. However, this method does not accurately represent the complex environment a virus encounters when approaching a cell. In reality, the viral particle simultaneously interacts with several membrane components, whose relevance and interplay cannot be fully appreciated when studied separately. This creates a significant gap between the biophysical characteristics determined in these studies and the actual biological question.
The CoVentry project calls for a paradigm shift in studying biophysical interactions during viral entry. By focusing on the interaction between the spike protein and the complete host cell membrane, the project aimed to understand the role of various membrane components in regulating viral attachment and entry and their potential effects on virus tropism (the ability of a virus to infect particular cell types).
Traditionally, studies of molecular interactions are performed using isolated molecular species. Researchers often purify the spike protein and the viral receptor and measure the strength of the bond formed between them. However, this method does not accurately represent the complex environment a virus encounters when approaching a cell. In reality, the viral particle simultaneously interacts with several membrane components, whose relevance and interplay cannot be fully appreciated when studied separately. This creates a significant gap between the biophysical characteristics determined in these studies and the actual biological question.
The CoVentry project calls for a paradigm shift in studying biophysical interactions during viral entry. By focusing on the interaction between the spike protein and the complete host cell membrane, the project aimed to understand the role of various membrane components in regulating viral attachment and entry and their potential effects on virus tropism (the ability of a virus to infect particular cell types).
Studying the entire membrane significantly increases the system's complexity and complicates data interpretation. Therefore, the project required developing and optimizing stable and reliable in-vitro systems that closely mimic the biological environment in a controlled setting. Spike proteins were immobilized on liposomes, which are small artificial lipid vesicles resembling the viral envelope that surrounds SARS-CoV-2 particles. This system allowed for high purity, precise control over their composition, and easy comparison between variants. The binding of these liposomes was studied on a supported lipid bilayer, a lipid membrane formed above a glass substrate, produced from the cell membrane. These "native" bilayers maintain the cell membrane composition but are stable over time, unlike living cells which react to external stimuli. They are also compatible with highly sensitive biophysical and microscopy techniques needed for measurements at the single particle and molecule level. Throughout the project, we employed total internal reflection microscopy, a technique that focuses on imaging the sample surface with extremely high sensitivity, to study the binding of single particles, and atomic-force microscopy (AFM) to analyse single molecular bonds.
The study included the original SARS-CoV-2 strain isolated in Wuhan and three widespread variants: Alpha, Delta, and Omicron (BA.1). The first result is a clear increase in virus binding to pulmonary cells for Omicron compared to earlier variants. Analysis of the binding kinetics—how fast the virus attaches to and leaves the cell membrane—revealed that the increase is due to faster binding. This suggested the increased use by Omicron of a common molecule on the cell surface, offering many attaching points, and thus fast binding.
HS was identified as a major factor in the increased interaction with the virus. Initially, HS had a weak interaction with early variants of the virus and removing HS from the cell surface increased virus binding for all variants except Omicron. This is likely because HS's long sugar chain hides SARS-CoV-2's main receptor, ACE2. However, Omicron has a 10-fold increase in affinity for HS, making it an important binding factor. Single molecule studies using AFM confirmed this increased affinity for Omicron and revealed subtle differences in the Alpha and Delta variants. These differences may help the virus anchor to the surface via HS but also increase its mobility, improving its chances of engaging ACE2.
The study included the original SARS-CoV-2 strain isolated in Wuhan and three widespread variants: Alpha, Delta, and Omicron (BA.1). The first result is a clear increase in virus binding to pulmonary cells for Omicron compared to earlier variants. Analysis of the binding kinetics—how fast the virus attaches to and leaves the cell membrane—revealed that the increase is due to faster binding. This suggested the increased use by Omicron of a common molecule on the cell surface, offering many attaching points, and thus fast binding.
HS was identified as a major factor in the increased interaction with the virus. Initially, HS had a weak interaction with early variants of the virus and removing HS from the cell surface increased virus binding for all variants except Omicron. This is likely because HS's long sugar chain hides SARS-CoV-2's main receptor, ACE2. However, Omicron has a 10-fold increase in affinity for HS, making it an important binding factor. Single molecule studies using AFM confirmed this increased affinity for Omicron and revealed subtle differences in the Alpha and Delta variants. These differences may help the virus anchor to the surface via HS but also increase its mobility, improving its chances of engaging ACE2.
Our results reveal that SARS-CoV-2 binding to the cell membrane is strongly influenced by HS. These findings also allow us to speculate on how virus attachment affects the symptoms and severity of the resulting disease. HS is much more abundant in the airway than ACE2, and Omicron's high affinity for HS gives the virus more attachment points. This makes it more likely to infect cells in the upper airway, leading to milder symptoms but higher transmission, as observed in Omicron infections. In contrast, the weak but dynamic interaction of Alpha and Delta with HS increases the likelihood of infection in the lungs and more efficient targeting of the viral receptor, leading to more severe symptoms.
The use of complex membrane models instead of isolated receptors, as proposed in the project, extends beyond SARS-CoV-2 research and opens new avenues in the biophysics field. This approach allows for a more comprehensive understanding of viral behaviour in a realistic context, providing a more reliable basis for developing therapies. Insights gained from this study could be applied to other viruses, improving how we study and combat a wide range of viral infections and ultimately greatly benefitting public health.
In conclusion, the CoVentry project has advanced our scientific understanding of SARS-CoV-2 and its variants, demonstrating the critical and shifting role of HS in viral binding. It has also pioneered new tools for studying viral interactions at the crossroads of biology and biophysics. This new knowledge opens new scientific questions to explore, such as how the virus moves on the cell surface and which parts of the spike protein and HS are responsible for the interaction, which we are currently pursuing. Additionally, it paves the way for improved antiviral strategies and highlights the importance of using sophisticated models to study virus-host interactions.
The use of complex membrane models instead of isolated receptors, as proposed in the project, extends beyond SARS-CoV-2 research and opens new avenues in the biophysics field. This approach allows for a more comprehensive understanding of viral behaviour in a realistic context, providing a more reliable basis for developing therapies. Insights gained from this study could be applied to other viruses, improving how we study and combat a wide range of viral infections and ultimately greatly benefitting public health.
In conclusion, the CoVentry project has advanced our scientific understanding of SARS-CoV-2 and its variants, demonstrating the critical and shifting role of HS in viral binding. It has also pioneered new tools for studying viral interactions at the crossroads of biology and biophysics. This new knowledge opens new scientific questions to explore, such as how the virus moves on the cell surface and which parts of the spike protein and HS are responsible for the interaction, which we are currently pursuing. Additionally, it paves the way for improved antiviral strategies and highlights the importance of using sophisticated models to study virus-host interactions.