Probing and elucidating the dynamics of virus-membrane interaction via plasma membrane mimics

Sammanfattning: Virus infection is initiated by the attachment of a virion to a susceptible cell’s plasma membrane, in a highly dynamic and well-orchestrated process that encompasses various steps and engages numerous viral and cellular factors. These dynamic steps may include initial non-specific binding to ubiquitous cell-membrane ligands, diffusion across the membrane to a suitable entry site and virus engagement with various receptors and co-receptors on the cell surface. Molecules and processes involved may vary across virus species, but it is likely that in all cases the dynamics of virus-membrane interactions need to be carefully fine-tuned to optimize the entry process. Nevertheless, the investigation and characterization of the involved biomolecular interactions are often oversimplified to isolated virus-receptor pairs engagement, ignoring the complexity of the membrane and the dynamic behaviors of the interaction. This doctoral thesis aims to shed light on this critical sphere of virus-membrane interactions, focusing on how viruses dynamically engage with plasma membrane molecules to successfully infect the cell. To facilitate this research, we utilized an innovative approach of using plasma membrane mimics to explore how viruses dynamically interact with the plasma membrane across several chosen contexts.Central to this project was the development and optimization of cell membrane mimics consisting of supported lipid bilayers (SLBs) reflecting the compositional complexity of the membrane. In the first paper, we developed a method to quantify the membrane material in a lipid vesicle sample in terms of total membrane surface area. This method is vital for the full utilization of our membrane mimics, which in many cases require the mixing of different vesicles in specific ratios.Subsequently, cell membrane mimics of complex composition were used to investigate the molecular mechanisms modulating virus-membrane interactions in several chosen contexts. These investigations relied primarily on total internal reflection fluorescent microscopy to characterize the attachment and detachment behavior of individual virus particles from the membrane. Firstly, studies focused on norovirus, a pathogen that infects cells in the gastrointestinal tract. The virus is known to bind to histo-blood group antigens (HGBA), specific glycans found on intestinal epithelial cells membranes. However, susceptibility to norovirus infection varies between individuals, and the difference is correlated to the specific glycan expression of the intestinal cells. This suggests that the differences in susceptibility might be related to differences in virus-membrane interaction dynamics. Using membranes constructed from lipids extracted from human intestinal enteroids (HIE) derived from susceptible and non-susceptible individuals, it was determined that norovirus associates similarly to both susceptible and non-susceptible membranes but dissociates slower from susceptible membranes. Using native supported lipid bilayers (nSLBs), bilayers derived from plasma membrane extracts of the HIEs, we then investigated the contribution of different carbohydrate moieties to interaction kinetics. This investigation revealed that virus binding to fucose residues on HBGAs is only part of the interaction, and that the virus also binds to sialic acid to a similar degree. It was also found that binding occurs primarily to membrane glycoproteins, and not membrane glycolipids.nSLBs were further found to be highly useful complements to virological investigations in a number of contexts. First, we studied the effect of isoform 4 of apolipoprotein E (ApoE4) on herpes simplex virus 1 (HSV-1) binding kinetics to plasma membrane SLBs. ApoE4 is a lipid binding protein that has been found, in conjunction with HSV-1, to be a risk factor for Alzheimer's disease. We showed that membrane-bound ApoE4 does not affect HSV-1 binding kinetics, but that viruses coated with ApoE4 demonstrate faster dissociation from susceptible membranes than non-coated viruses, indicating that the protein facilitates the release of new virions from the infected cell. This provides a mechanistic understanding of the overall pro-viral effect of ApoE, observed in infection experiments.Second, nSLBs from respiratory epithelial cells were used to quantify binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the plasma membrane. SARS-CoV-2 has caused one of the largest pandemic in modern history and the virus has shown the ability to rapidly mutate, causing periodic surges of new cases, with newer strains spreading more easily. These mutations have often been linked to the viral spike glycoprotein, responsible for viral attachment and entry. Investigations using spike-decorated liposomes as virus-mimetics, revealed that virus-membrane interaction dynamics vary for different variants of concerns. Specifically, the late Omicron variant, a highly transmissible variant, shows significantly increased affinity to susceptible membranes. This increased affinity was primarily due to increased association to the membrane. Experiments also showed that membrane-bound heparan sulfate has an inhibiting effect on virus binding to ACE2, for earlier variants.In summary, we have successfully implemented membrane mimics with different levels of complexity to investigate virus-membrane interactions. This thesis demonstrates their potential in virology research in several contexts, including measuring the avidity of viruses to membranes, evaluating the relative contributions of different attachment factors to kinetics, and the influence of viral and cellular factors on binding.