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Abstract

Norovirus is one of the most common agents associated with acute gastroenteritis. Despite its discovery 50 years ago, as of now there is no cure or treatment, and knowledge about the infection cycle of norovirus is still limited. Using cryo-electron microscopy, we determined the structure of different clinically relevant GII.4 strain virus like particles (VLPs), and an engineered version of GII.4 that is in use as vaccine candidate. Unexpectedly we found that these empty particles were composed of 240 copies of VP1 instead of the typical 180. We could show that this conformation is only adopted by GII.4 strain VLPs, and that norovirus virions most likely assemble into T=3 icosahedral particles. Conversely, VP1 of GII.17, another strain that has become clinically relevant in the last years, assembled into the characteristic T=3 icosahedral capsid, as determined by cryo-electron microscopy. Moreover, we characterized a panel of GII.4 specific Nanobodies and determined their binding sites and influence on particle integrity using cryo-electron microscopy. Several Nanobodies had a strong inhibition potential in surrogate assays against GII.4 VLPs, but cross-reactivity to other strains was low. Structures of Nanobody/VLP complexes revealed that the Nanobodies bound to a similar site on the lower part of the P domain. Moreover, the structures showed that the P domains were flexibly rotated to distinct positions, but generally particle conformation remained intact. Likewise, we also worked on three GI.1 specific Nanobodies and found two Nanobodies with great inhibition potential. Importantly, we found that combination of Nanobody with the human milk oligosaccharide 2’FL was able to synergistically improve attachment inhibition of VLPs to histo-blood group antigens, suggesting the use of combinational treatment against norovirus infection. In another study, we found that murine norovirus, which is commonly used as a model system for human norovirus, is undergoing severe structural rearrangements after incubation with ions, such as MgCl2 and CaCl2. The normally extended P domains are collapsing onto the shell, accompanied by a rotational movement. Most likely, this movement is important to facilitate infection. Importantly, we also characterized a Nanobody that was binding to the lower part of the P domain, effectively inhibiting MNV infection by blocking this movement. Taken together, these results broaden the understanding on general norovirus structure and how noroviruses are interacting with antivirals and cofactors. Our basic research broadens the understanding of norovirus internal differences and capsid flexibilities, and can ultimately aid in the development of a more effective treatment for norovirus infections.