Fig 1.
Nanobodies cross-reactivites and inhibition of VLP attachment to PGM.
Nanobody cross-reactivities were analyzed using a panel of GII and GI noroviruses in direct ELISA. P domains, 15 μg/ml, (GII.1, GII.2, GII.4, GII.10, GII.12, GII.17) or VLPs, 4 μg/ml, (GI.1 and GI.11) were detected with a panel of serially diluted Nanobodies (A) Nano-85, (B) Nano-4 (C) Nano-26 (D) Nano-42. Nano-85 exhibited the broadest reactivity range and detected all GII noroviruses at 0.4 μg/ml or less and cross-reacted strongly with GI.11 VLPs (<0.8 μg/ml). Nano-26 recognized GII.1, GII.2, GII.4, GII.10, GII.12, and GII.17 P domains. Nano-4 bound GII.10, GII.17, GII.12, and GII.1 P domains. Nano-42 could only detect GII.10 and GII.4 2012, whereas Nano-27, Nano-32, Nano-14 did not cross-react with any examined P domains. All experiments were performed in triplicate (error bars are shown) and the cutoff was set at an OD490 of 0.15 (dashed line). (E, F) PGM blocking assay was used as a surrogate neutralization assay. GII.10 VLPs were pretreated with serially diluted Nanobodies and added on PGM coated plates. (E) Inhibition of GII.10 VLPs binding to PGM. Nano-14 and Nano-32 inhibited 50% (IC50) of the binding at 1.7 to 2.6 μg/ml, respectively. For Nano-26, the IC50 value was 6.6 μg/ml. Nano-85 showed only weak blocking potential. (F) Inhibition of GII.4 VLPs binding to PGM. Nano-85 and Nano-26 blocked the binding with IC50 values of 2.2 μg/ml and 2.5 μg/ml, respectively. Binding is expressed as a percentage of the untreated VLP binding (100%). 50% inhibition is shown as a dashed line. All experiments were performed in triplicate (error bars are shown).
Table 1.
Thermodynamic properties of Nanobody binding to the P domain.
Fig 2.
Variety of Nanobody binding sites on the GII.10 domain.
The X-ray crystal structures of the P domain-Nanobody complexes were superimposed onto each other. Nano-85 and Nano-25 complex structures were previously published in [31]. GII.10 P domain is colored light gray (chain A) and dark gray (chain B), Nano-14 (red), Nano-42 (dark purple), Nano-32 (yellow), Nano-27 (blue), Nano-26 (cyan), Nano-4 (pink), Nano-85 (orange), and Nano-25 (dark green). HBGA binding sites are marked black and newly identified additional fucose binding sites are marked blue. One Nanobody bound on the top of the P domain (Nano-14), two Nanobodies bound on the side (Nano-32 and Nano-25) and five Nanobodies bound on the bottom (Nano-85, Nano-4, Nano-26, Nano-42 and Nano-27). Nano-32, Nano-26 and Nano-14 were involved in a dimeric interaction with the P domain, whereas the binding of Nano-4, Nano-25, Nano-27, and Nano-85 was monomeric. Nanobody binding footprints are marked on the bottom and top of the P domain.
Table 2.
Data collection and refinement statistics for P domain Nanobody complex structures.
Table 3.
Summary of Nanobodies analyzed in this study.
Fig 3.
Nano-14 in complex with the GII.10 domain.
(A) The X-ray crystal structure of the GII.10 domain Nano-14 complex was determined to 1.7Å resolution. The molecular replacement in C121 space group indicated one P domain dimer and one Nanobody per asymmetric unit. P domain chain A (light blue), chain B (salmon), Nano-14 (red). The Nano-14 bound to the top of the P1 subdomain in the canyon between two monomers. (B) The Nano-14 binding site overlapped with the binding pocket of HBGAs (as an example B-trisaccharide is shown in green sticks) (C) A close-up view of GII.10 P domain and Nano-14 interacting residues. The P domain hydrogen bond interactions included side-chain and main chain interactions from both monomers. R299, W381, K449, D403, and E333 from chain A and Glu384 from chain B formed direct hydrogen bonds with Nano-14: D53, D1, F102, T103, T104, M106, and W109. P domain E382 and R299 were involved in electrostatic interactions with Nano-14 residues D1 and H32. Hydrophobic interactions involved P domain chain A: W381, H298, R299, V361, A363 and Nano-14: F102, I100, V101, M106, and H32. Two additional interactions were observed with P domain chain B residues: direct hydrogen bond with Q384 and Pi-sulfur interaction with H358.
Fig 4.
Nanobody binding epitopes on the GII P domain sequence alignment.
Eleven different GII genotype P domain sequences were aligned using ClustalX. The GII.10 capsid sequence was used as the consensus sequence, other sequences include GII.1 (U07611), GII.2 (HCU75682), GII.3 (DQ093066), Saga-2006 GII.4 (AB447457), NSW-2012 GII.4 (JX459908), GII.5 (BD011877), GII.6 (BD093064), GII.7 (BD011881), GII.8 (AB039780) GII.10, and GII.12 (AB044366). For clarity only GII.10 residues are shown. The binding epitope of a broadly reactive monoclonal antibody 5B18 MAb (light blue) is shown for the reference. The GII.10 residues interacting with Nano-4 (pink), Nano-14 (red), Nano-25 (dark green), Nano-27 (blue), Nano-32 (yellow), Nano-42 (deep violet), and Nano-85 (orange) are colored accordingly. The asterisks mark conserved amino acids. P domain residues interacting with HBGAs are boxed.
Fig 5.
Nano-32 in complex with the GII.10 domain.
The X-ray crystal structure of the GII.10 domain Nano-32 complex was determined to 2.1Å resolution. The asymmetric unit cell contained one P domain dimer and two Nanobodies in space group P41212. The interface with the surface area of 650 Å2 was considered biologically relevant. (A) The complex was colored according to Fig 3 with Nano-32 (yellow). (B) The Nano-32 bound to the side of the P1 subdomain in the cleft between two monomers. (C) A close-up view of GII.10 P domain and Nano-32 interacting residues. The P domain hydrogen bond interactions included side chain and main chain interactions from both monomers. Direct hydrogen bonds were formed with chain A: R287, N344, W343, D316 and chain B: R492 and W519 with Nano-32: D1, N5, S25, L45, S101, and D123. Electrostatic interactions formed between P domain chain A: R287, E236, chain B: D247 and Nano-32: D1 and K120. Hydrophobic interactions involved chain A: P314 and chain B: P518, V248 and Nano-32: V2, L45, Y95, and A119.
Fig 6.
Nano-4 GII.17 domain and Nano-42 GII.10 P domain complex structures.
(A, B) The X-ray crystal structure of the GII.17 domain Nano-4 complex was determined to 1.7Å resolution. Unit cell contained one P domain and one Nanobody. Only one relevant interface with a surface area of 532 Å2 was found. GII.17 P domain is colored violet (Chain A) and light green (Chain B), Nano-4 is shown in hot pink. A close up view shows the formation of the extended network of hydrogen bonds between P domain residues: T483, E486, D516, N520, Y523, S524 and Nano-4: R99, R100, Y102, T106, G112, and Y113. Two hydrophobic interactions were formed between P domain: Y523, A526 and Nano-4: Y113, A109. Five electrostatic interactions (P domain: R482, E486, D516) contributed to binding to Nano-4: D104, R100. (C, D) The structure of GII.10 P domain and Nano-42 was solved to 2.0Å resolution with the unit cell containing one P domain dimer and two Nanobodies. The Nano-42 binding site had an interface surface area of 621 Å2. GII.10 P domain is colored as in Fig 3 and Nano-42 is colored deep purple. Five direct hydrogen bonds involved P domain residues: D526, W528, N530, T534 and Nano-42 residues: T31, Y100, S101, and S56, one electostatic interaction formed between P domain F532 and Nano-42 K96. Two hydrophobic interactions were formed between P domain residues V529, A536 and Nano-42 residues Y100, V54. Ethylene glycol molecule is shown in green sticks and participates in six direct hydrogen bonds with P domain and Nanobody.
Fig 7.
Nano-26 Nano-85 GII.10 P domain double complex structure.
GII.10 P domain Nano-26 Nano-85 crystal diffracted to 2.3Å in a C121 space group. Unit cell contained a P domain dimer with two Nano-85 and two Nano-26 molecules. (A) GII.10 P domain is colored as in Fig 3 with Nano-26 (cyan) and Nano-85 (orange). The Nano-85 and Nano-26 binding site in a double complex were identical to binding sites in individual complexes. (B) Nano-26 binds to the cleft between two P domain monomers at the bottom of the P domain dimer. (C) Close up view on the interactions between P domain residues and a Nano-26. Seven direct hydrogen bonds formed between P domain chain B: D269, L272, E274, E471, E472, T276 and Nano-26: V2, R26, R99, and Y104. P domain chain A: I231, P488 and chain B: E271, D316, Y470, and P475 were involved in hydrophobic interactions and two electrostatic interactions with Nano-26: V2, I28, F30, M31, K75, and A102.
Fig 8.
Structure of the GII.10 P domain Nano-27 complex.
The X-ray crystal structure of GII.10 P domain Nano-27 complex was solved to 2.9Å resolution. GII.10 P domain is colored according to Fig 3 and Nano-27 (blue). (A) Nano-27 bound to the lower part of P domain monomer. (B) Nano-27 forms an extensive network of hydrogen bonds and hydrophobic interactions with P domain residues. Six P domain residues: R484, G491, R492, T493, E496, and T534 were involved in ten direct hydrogen bonds and two electrostatic interactions with Nano-27: D54, E64, R98, Y52, S56, T57, and E64. Three P domain residues: R484, A536, and P537 were involved in four hydrophobic interactions with Nano-27: W58, Y52, and L47.
Fig 9.
Nanobody treatment of GII.10 norovirus VLPs causes capsid deformation.
GII.10 VLPs were treated with each Nanobody for 1 h at room temperature and applied on EM grids for negative staining. Nano-14 treated VLPs preserved the initial morphology, whereas Nano-26, Nano-85 and Nano-42 binding caused changes in particle integrity. Nano-85 treated VLPs were largely broken with a few small-size particles. Nano-4, Nano-27 and Nano-42 treated VLPs tended to shift to the smaller form, whereas Nano-32 treated VLPs formed large aggregates. Negative stain EM images were obtained at 50,000 magnification. The scale bar represents ~50 nm.
Fig 10.
Nanobody treatment leads to changes in norovirus capsid morphology.
(A) DLS profiles of Nanobody treated GII.10 VLPs. (B) Average diameters of treated VLPs. Nano-26, Nano-32 and Nano-85 binding caused the formation of large molecular weight aggregates. All experiments were performed in triplicates. (C) Concentrated stool suspension was treated with Nano-14, Nano-26, Nano-85, and 250 mM citrate buffer and subsequently with 50 U of RNAse. Genome copies were quantified with RT-qPCR. Nano-26, Nano-85, and citrate caused a significant decrease in genome copy levels compared to Nano-14. (D) 10% stool suspension was treated with Nanobodies or GHCl and diluted twice with PBS to decrease viral lysis efficiency. Genome copies levels were measured as before and indicated additional lysis in samples pre-treated with Nano-85, Nano-26 and GHCl. Statistical analysis was performed using one-way ANOVA test. Significant differences (P≤0.05) between the treated samples and a negative control (Nano-14 treatment) are marked with stars.
Fig 11.
Broadly reactive Nanobodies affect the capsid of prevalent norovirus strains.
VLPs of two prevalent norovirus strains GII.4 and GII.17 were incubated with Nano-26 and Nano-85 for 1 h at room temperature. (A) Samples were then applied on EM grids and stained with 1% uranyl acetate. Both Nanobodies degraded GII.4 VLPs. Nano-26 exposure of GII.17 VLPs caused the appearance of VLPs with smaller diameters, whereas Nano-85 seemed to be ineffective, although VLPs appeared to be partially deformed. EM images were obtained at 50,000 magnification, the scale bar represents ~100 nm. (B) DLS profiles of GII.4 2012 and GII.17 VLPs treated with Nano-26 and Nano-85 confirmed the EM observations.
Fig 12.
Nanobody binding in context of the whole particle.
Nanobodies GII.10 P domain complex structures were superimposed with A/B dimer (A) or C/C dimer (B) of the GII.10 VLP cryo-EM structure. A view from 5-fold axes (A) or 3-fold axis is presented (B). Two Nanobodies bound to the relatively more exposed sites, Nano-26 (cyan) and Nano-32 (yellow). Nano-4 (hot pink), Nano-14 (red), Nano-25 (dark green), Nano-27 (blue), Nano-42 (dark purple), and Nano-85 (orange) bound to occluded epitopes on the bottom of the P domain and were poorly visible in context of the whole particle.