Structural Basis of Nanobodies Targeting the Prototype Norovirus

Abstract

Human norovirus infections are a major disease burden. In this study, we analyzed three new norovirus-specific Nanobodies that interacted with the prototype human norovirus (i.e., genogroup I genotype 1 [GI.1]). We showed that the Nanobodies bound on the side (Nano-7 and Nano-62) and top (Nano-94) of the capsid-protruding (P) domain using X-ray crystallography. Nano-7 and Nano-62 bound at a similar region on the P domain, but the orientations of these two Nanobodies clashed with the shell (S) domain and neighboring P domains on intact particles. This finding suggested that the P domains on the particles should shift in order for Nano-7 and Nano-62 to bind to intact particles. Interestingly, both Nano-7 and Nano-94 were capable of blocking norovirus virus-like particles (VLPs) from binding to histo-blood group antigens (HBGAs), which are important cofactors for norovirus infection. Previously, we showed that the GI.1 HBGA pocket could be blocked with the soluble human milk oligosaccharide 2-fucosyllactose (2′FL). In the current study, we showed that a combined treatment of Nano-7 or Nano-94 with 2′FL enhanced the blocking potential with an additive (Nano-7) or synergistic (Nano-94) effect. We also found that GII Nanobodies with 2′FL also enhanced inhibition. The Nanobody inhibition likely occurred by different mechanisms, including particle aggregation or particle disassembly, whereas 2′FL blocked the HBGA binding site. Overall, these new data showed that the positive effect of the addition of 2′FL was not limited to a single mode of action of Nanobodies or to a single norovirus genogroup. IMPORTANCE The discovery of vulnerable regions on norovirus particles is instrumental in the development of effective inhibitors, particularly for GI noroviruses that are genetically diverse. Analysis of these GI.1-specific Nanobodies has shown that similar to GII norovirus particles, the GI particles have vulnerable regions. The only known cofactor region, the HBGA binding pocket, represents the main target for inhibition. With a combination treatment, i.e., the addition of Nano-7 or Nano-94 with 2′FL, the effect of inhibition was increased. Therefore, combination drug treatments might offer a better approach to combat norovirus infections, especially since the GI genotypes are highly diverse and are continually changing the capsid landscape, and few conserved epitopes have so far been identified.

The Dynamic Life of Virus Capsids

“…The base of the P domain is only exposed to the antibody if there is extreme flexibility in the tether region between the shell and the P domain. This result has been further substantiated by more recent results in genotypes GI.1 [63] and GII.4 [64], where various epitopes are clearly buried in the compressed particle and only exposed if the P domain is allowed to lift off the shell.…”

“…In either case, if any of these normally buried sites are immunodominant, then the immune response might focus on producing antibodies that bind poorly or not at all to the contracted capsid. This hypothesis is supported by the findings that the immune response to MNV included antibodies to the buried shell domain [69] and from studies on human genotypes GII.10 [62], Gi.1 [63], and GII.4 [64]. Essentially, the highly flexible nature of the P domain could be a "moving target" for the immune response.…”

“…This hypothesis is supported by the findings that the immune response to MNV included antibodies to the buried shell domain [69] and from studies on human genotypes GII. 10 [62], Gi.1 [63], and GII.4 [64]. Essentially, the highly flexible nature of the P domain could be a "moving target" for the immune response.…”

Bile Salts Alter the Mouse Norovirus Capsid Conformation: Possible Implications for Cell Attachment and Immune Evasion

“…The base of the P domain could only be exposed to the antibody if there is extreme flexibility in the tether region between the shell and the P domain, suggesting that the extreme flexibility of the P domain of the cryo-EM structures is biologically relevant. This was further substantiated by studies on genotypes GI.1 (15) and GII.4 (16), where various epitopes are only exposed if the P domain is allowed to lift off the shell. Studies on MNV demonstrated that a number of the antibodies raised against MNV recognized the buried shell domain and were nonneutralizing (17).…”

“…Both are likely possible due to the extreme flexibility of the linker. In the case of human noroviruses, a number of antibodies have been shown to bind to the buried P1 domain (14,15) and, similarly, this appears to only be possible through the flexibility of the linker region. From the structures presented here, it appears that an additional function for this linker flexibility may be to optimize receptor binding valency on the membrane surface, signaled by metabolites found in the organ where infection occurs, while burying potential epitopes only found in the expanded form.…”