Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly (1)

We investigated occluded epitopes and capsid flexibility using a panel of Nanobodies raised against GII.10 norovirus virus-like particles (VLPs). We characterized two Nanobodies, Nano-25 and Nano-85, which bind to the human norovirus capsid. We showed that Nano-85 was broadly reactive to GII noroviruses and was also able to detect norovirus virions from clinical samples. Moreover, Nano-85 had weak binding to mouse norovirus and human genogroup I (GI) norovirus. Nano-25 was found to be strain specific for GII.10. X-ray crystal structures of the human norovirus protruding (P) domain in complex with Nano-85 and Nano-25 revealed that the binding sites were located at a lower region on the P domain. The Nano-85 binding site mainly comprised of amino acids highly conserved among the genetically distinct human GII noroviruses. Superposition of the P domain Nanobody complex structures onto a cryo-EM norovirus particle structure revealed that both Nanobodies bound at occluded sites on the particles. The flexible hinge region, which contains ~10-12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the Nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble in vitro. This suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Our data indicated that Nano-85 has the potential to function as a diagnostic reagent for the detection of human norovirus in clinical specimens and also as a possible antiviral.

Rabbit hemorrhagic disease virus binding to histo-blood group antigens (2)

We analyzed a rabbit hemorrhagic disease virus (RHDV, isolate N11) using X-ray crystallography. This was the first report showing the histo-blood group antigen (HBGA) binding site on a calicivirus other than human norovirus. The HBGA binding pocket was significantly different from previously predicted binding sites and located at a dimeric interface on the side of the P domain. Residues from both monomeric subunits were involved in HBGA binding through a network of direct hydrogen bonds and water-mediated interactions. Amino acids directly interacting with the ABH-fucose of the HBGA were highly conserved among different RHDV strains, suggesting that the HBGA binding pocket might have been preserved over time. Interestingly, several HBGA binding characteristics between RHDV and human GII noroviruses were similar, which indicates a possible convergent evolution of HBGA binding interactions and discloses the possibility to use RHDV as a surrogate model for human norovirus.

Human norovirus’ fondness of histo-blood group antigens (3)

We investigated P domains from epidemic GII.4 human noroviruses from 2004, 2006, and 2012 and co-crystallized them with a panel of HBGAs. Using X-ray crystallography, we demonstrated, for the first time, that the interaction between HBGAs and the norovirus P domain was dynamic and involved P domain loop movements and alternative HBGA conformations. As an example, a P domain loop was found in different positions in various structures to allow Lewis HBGA binding. Moreover, we observed alternative HBGA conformations and HBGA rotations. Interestingly, many of the GII.4 P domain-HBGA interactions, we identified, were negative in earlier ELISA-based studies. GII.4 noroviruses have dominated outbreaks over the past decade and our results presented a comprehensive picture of the evolution of HBGA recognition in GII.4 human noroviruses. The complexity of GII.4 norovirus-HBGA binding mechanisms could explain their worldwide dominance in the human population over the past decade.

Structural analysis of a feline norovirus protruding domain (4)

We determined the first structure of a feline norovirus P domain using X-ray crystallography. The unbound GIV.2 P domain structure was reminiscent of human norovirus P domains, except for a novel P2 subdomain α-helix and an extended P1 subdomain interface loop. The feline norovirus P domain was unable to bind to HBGAs, since these newly identified structural features likely hindered the binding. These results indicated that feline norovirus might have evolved to bind ligands other than HBGAs.

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