Probing molecular interactions between Norovirus and histo-blood group antigens using atomic force microscopy

Details

Supervisors

David Alsteens

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Norovirus is an important pathogen that causes acute gastroenteritis worldwide, with an estimated 685 million cases of illness annually, including 200 million cases in children under the age of five, and contributes to approximately 50,000 child deaths each year. It is a highly infectious and resistant pathogen, with a rapid transmission rate. A thorough understanding of its viral lifecycle, especially its binding process to host cells, is essential for the development of infection prevention strategies. While research has shown that noroviruses can bind to histo-blood group antigens (HBGAs) upon entry into host cells, their role as specific receptors or attachment factors requires further investigation. The expression of different HBGAs on our cells is determined by the expression of specific enzymes encoded in our genes, resulting in different HBGAs profiles through divergent biosynthetic pathways. This study aims to identify different binding patterns between the virus and HBGAs to elucidate genetic susceptibility to norovirus infection. The predominant human norovirus genotype GII.4 is studied in the form of virus-like particles (VLPs) due to the lack of suitable systems to grow and replicate the virus. The different HBGAs studied in this work are Lewis A, Lewis B, antigen B and antigen H. To investigate norovirus interactions at the molecular level, an advanced technique known as atomic force microscopy (AFM), based on single-molecule force spectroscopy (SMFS), was used. This technique allows the calculation of kinetic and thermodynamic parameters, offering detailed insights into unbinding forces, bond lifetimes and the energy barriers associated with these interactions. This study started with in vitro experiments on model surfaces grafted with different HBGAs and probed with a functionalized AFM tip bearing the virus. Initially, screening experiments confirmed effective norovirus binding to most HBGAs, except for Lewis A that did not show significant binding and was therefore excluded from further analysis. After, contact time experiments enabled the determination of the association rate constant (kon), while dynamic force spectroscopy (DFS) experiments provided data on the dissociation rate constant (koff). These experiments allowed the determination of the thermodynamic dissociation constant (KD), an essential indicator of the affinity between binding partners. Finally, in vivo studies were conducted on living Caco-2 cells at varying maturation stages using a confocal microscope coupled to AFM to measure binding affinities under physiological conditions and explore the potential impact of the degree of cellular differentiation on binding interactions. The results of this study revealed that Lewis B and antigen B had low KD values, indicating high binding affinities with norovirus. This observation implies that individuals expressing these antigens on their cell surface may be more susceptible to norovirus infection. Conversely, antigen H demonstrated a significantly higher KD value, suggesting a lower affinity for the virus, which could confer greater resistance to norovirus infection in individuals expressing this antigen. Live cell experiments demonstrated that 14-day old cells yielded higher binding frequencies compared to fresh cells, suggesting a potential influence of cellular differentiation on these interactions or the involvement of additional receptors in norovirus binding.