Deciphering the individual role of integrins in reovirus infection using Atomic Force Microscopy (AFM)

Details

Supervisors

Alsteens, David ; Melanie Köhler

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries

Abstract

Viruses are first order pathogens able to infect and cause diseases to a wide variety of living organisms, including humans. As they cannot replicate by themselves, they are obliged to infect host cells and hijack their cellular machineries to complete their life cycle. Thus, the understanding of the virus infection cycle at the molecular level and its dynamics is of crucial importance to be able to fight against this infection and the associated diseases. The particular case of reoviruses (studied in this thesis) appears to be different and not very important for the virology community at first glance, as they are not recognized as common human pathogens (diseases are mostly cause in the very young). However, the study of the infection cycle of this member of the Reoviridae family remains of particular interest, as it has been shown in the past years, that reoviruses are involved in triggering celiac disease and on the other hand in a very positive sense, can be used to specifically lyse cancer cells. Known from the literature, the whole infection process is largely defined by the very first interactions between the virus and its host cellular surface. Those interactions determine the mechanisms of virus attachment, uptake and finally their penetration into the cytosol. This thesis focuses on the role of β1 integrins in reovirus infection. It has been shown that integrins are involved in the virus internalization by activating (among others) the clathrin-mediated endocytosis pathway but a clear proof for actual binding/interaction between reoviruses and integrins is still missing. FD curve-based AFM is used to understand more about the dynamics of the molecular processes and quantitative parameters of this interaction. This technique provides an unprecedented time and spatial resolution, as well as allows to work under physiological conditions and at the single molecule/virus level. Consequently, it is the ultimate technique to carry on such a study. The interaction between reovirus (linked to the AFM tip) and β1 integrins is probed both on living cells and model surfaces. Extracting kinetic and thermodynamic parameters for different conditions/virus strains enables deciphering the interaction down to the single molecule level. The investigations allowed proving for the first time, that integrins function as a specific binding receptor for reovirus before its internalization into the endocytic pathway. On top of that, the divalent cation behaviour of integrin activation, described by the theory, was confirmed, manganese acting as the highest activator, calcium as the lowest and magnesium triggering an intermediate state of affinity. Moreover, it was shown that the interaction is more stable in presence of manganese (lower koff corresponding to a higher bond lifetime and higher multivalence (up to 4 parallel bonds on cells, maximum 3 in the presence of magnesium) and forces). It was then demonstrated that the change of binding probability associated to the presence of a divalent cation was due to a change of affinity for binding rather than a conformational change influencing the reovirus binding. Finally, the investigations did not allow to highlight an influence of sialic acid in a potential glycan-mediated enhancement of reovirus - integrin binding, as it was previously shown for JAM-A.