Work

Structural insights into a broadly reactive nanobody that binds pathogenic and non-pathogenic lagoviruses

Year: 2026

Type: article

Abstract: nanobody, X-ray crystallography, calicivirus P athogenic genogroup I rabbit hemorrhagic disease virus (RHDV) is genetically related to non-pathogenic GII hare calicivirus (HaCV) in the Lagovirus genus (1).Lagovirus capsid protruding (P) domains, analogous to human noroviruses, engage histo-blood group antigens (HBGAs) as co-factors (2-10).In this study, we generated nanobody libraries against HaCV and RHDV-K5 (a biocontrol agent released in Australia in 2017) (11, 12) virus-like particles (VLPs) to identify nanobodies with broad reactivity.Two HaCV nanobodies (NB7 and NB9) and two K5 nanobodies (NB3 and NB6) were expressed and evaluated for cross-reactivity with HaCV, K5, RHDV-Ast89, and RHDV-Czech-351 (a biocontrol agent released in Australia in 1996) (11, 12) VLPs using a direct ELISA (Fig. 1) (13).NB7 bound HaCV (0.05 µg/mL), cross-reacted with K5 (3.13 µg/mL), and showed weak cross-reactivity to both RHDV Ast89 and Czech-351 (25-50 µg/mL).NB9 bound HaCV (<0.02 µg/mL) and showed no cross-reactivity to the three RHDVs.NB3 bound K5 (0.05 µg/mL), cross-reacted with both HaCV (0.05 µg/mL) and Ast89 (0.2 µg/mL), but not Czech-351.NB6 bound K5 (<0.02 µg/mL), cross-reacted with both Ast89 and Czech-351 (<0.02 µg/mL), and had no cross-reactivity to HaCV.Crystallization trials with the corresponding HaCV, Ast89, K5, and Czech-351 P domains yielded crystals only for the HaCV P domain and the NB7 complex (HaCV-NB7) (Table 1).Structural analysis showed that NB7 recognized a conformational epitope at the bottom of the HaCV P domain, where it interacted with residues from both monomers at the dimeric interface (Fig. 2A and B).Interestingly, the overall NB7 binding site on the HaCV P domain was similar to several broadly reactive diagnostic and therapeutic norovirus nanobodies (14-19).Affinity measurements found that NB7 bound strongly to the HaCV P domain (KD = 8.8 nM) (Fig. 2C).An amino acid sequence alignment revealed that the residues involved in NB7 binding are highly conserved among RHDVs (Fig. 2D).To obtain further structural insight into the NB7 conformational binding epitope, we determined the Czech-351-apo structure (Fig. 2E and Table 1) and, together with the Ast89-apo structure (PDB ID: 8CYL), performed superimpositions with the HaCV-NB7 and HaCV-apo structures.Most of the corresponding Czech-351 (Fig. 2F) and Ast89 (Fig. 2G) P domain loops and residues predicted to interact with NB7 aligned closely with the HaCV-apo structure, indicating the conformational NB7 epitope was preserved.However, structural comparison of HaCV-apo, HaCV-HBGA, and HaCV-NB7 revealed that NB7 binding induced extensive P domain loop rearrangements (Fig. 3A), including residues that bound NB7 and residues that engaged the HBGA (Fig. 3B) (20).Although certain P domain loop rearrangements in the HaCV-NB7 complex may be influenced by crystal lattice contacts, our extensive structural studies with norovirus P domain nanobody complexes show that nanobody binding did not result in substantial conformational changes in the P domain loops (14-19).

Source: Journal of Virology

DOI: 10.1128/jvi.01990-25