Direct Blockade of the Norovirus Histo-Blood Group Antigen Binding Pocket by Nanobodies.

Noroviruses are the leading cause of outbreaks of acute gastroenteritis. These viruses usually interact with histo-blood group antigens (HBGAs), which are considered essential cofactors for norovirus infection. This study structurally characterizes nanobodies developed against the clinically important GII.4 and GII.17 noroviruses with a focus on the identification of novel nanobodies that efficiently block the HBGA binding site. Using X-ray crystallography, we have characterized nine different nanobodies that bound to the top, side, or bottom of the P domain. The eight nanobodies that bound to the top or side of the P domain were mainly genotype specific, while one nanobody that bound to the bottom cross-reacted against several genotypes and showed HBGA blocking potential. The four nanobodies that bound to the top of the P domain also inhibited HBGA binding, and structural analysis revealed that these nanobodies interacted with several GII.4 and GII.17 P domain residues that commonly engaged HBGAs. Moreover, these nanobody complementarity-determining regions (CDRs) extended completely into the cofactor pockets and would likely impede HBGA engagement. The atomic level information for these nanobodies and their corresponding binding sites provide a valuable template for the discovery of additional "designer" nanobodies. These next-generation nanobodies would be designed to target other important genotypes and variants, while maintaining cofactor interference. Finally, our results clearly demonstrate for the first time that nanobodies directly targeting the HBGA binding site can function as potent norovirus inhibitors. IMPORTANCE Human noroviruses are highly contagious and a major problem in closed institutions, such as schools, hospitals, and cruise ships. Reducing norovirus infections is challenging on multiple levels and includes the frequent emergence of antigenic variants, which complicates designing effective, broadly reactive capsid therapeutics. We successfully developed and characterized four norovirus nanobodies that bound at the HBGA pockets. Compared with previously developed norovirus nanobodies that inhibited HBGA through disrupted particle stability, these four novel nanobodies directly inhibited HBGA engagement and interacted with HBGA binding residues. Importantly, these new nanobodies specifically target two genotypes that have caused the majority of outbreaks worldwide and consequently would have an enormous benefit if they could be further developed as norovirus therapeutics. To date, we have structurally characterized 16 different GII nanobody complexes, a number of which block HBGA binding. These structural data could be used to design multivalent nanobody constructs with improved inhibition properties.

Structural Basis for Rabbit Hemorrhagic Disease Virus Antibody Specificity.

Rabbit hemorrhagic disease virus (RHDV) typically causes a fatal disease in rabbits. In Australia, RHDV was imported to control the feral rabbit population, while it poses a severe threat to native rabbits in other countries. RHDV variants are genetically diverse and serological studies using antibodies isolated from infected rabbits or raised against RHDV virus-like particles (VLPs) have found RHDV variants antigenically distinct. In this study, we determined the X-ray crystal structure of an RHDV GI.2 (N11 strain) protruding (P) domain in complex with a diagnostic monoclonal antibody (2D9) Fab. We showed that 2D9 interacted with conserved and variable residues on top of the P domain with nanomolar affinity. To better illustrate 2D9 specificity, we determined the X-ray crystal structure of an RHDV GI.1b (Ast89 strain) that was a 2D9 non-binder. Structural analysis indicated that amino acid substitutions on the GI.1b P domain likely restricted 2D9 binding. Interestingly, a model of the GI.2 P domain-Fab complex superimposed onto a cryo-EM structure of an RHDV VLP revealed that 2D9 Fab molecules clashed with neighboring Fabs and indicated that there was a reduced antibody binding occupancy. Moreover, the RHDV GI.2 histo-blood group antigen (HBGA) co-factor binding site appeared obstructed when 2D9 was modeled on the VLP and suggested that 2D9 might also function by blocking HBGA attachment. Overall, this new data provides the first structural basis of RHDV antibody specificity and explains how amino acid variation at the binding site likely restricts 2D9 cross-reactivity. IMPORTANCE Isolated RHDV antibodies have been used for decades to distinguish between antigenic variants, monitor temporal capsid evolution, and examine neutralizing capacities. In this study, we provided the structural basis for an RHDV GI.2 specific diagnostic antibody (2D9) binding and reveal that a small number of amino acid substitutions at the binding site could differentiate between RHDV GI.2 and GI.1b. This novel structural information provides a framework for understanding how RHDV displays a specific antigenic epitope and engages an antibody at the atomic level. Importantly, part of the 2D9 binding region was earlier reported to contain a neutralizing epitope and our structural modeling as well as recent human norovirus antibody-mediated neutralization studies, suggest that the 2D9 antibody has the potential to block HBGA attachment. These new findings should aid in characterizing antigenic variants and advance the development of novel monoclonal antibodies for diagnostics and therapeutics.

Nanobody-Mediated Neutralization Reveals an Achilles Heel for Norovirus.

Human norovirus frequently causes outbreaks of acute gastroenteritis. Although discovered more than five decades ago, antiviral development has, until recently, been hampered by the lack of a reliable human norovirus cell culture system. Nevertheless, a lot of pathogenesis studies were accomplished using murine norovirus (MNV), which can be grown routinely in cell culture. In this study, we analyzed a sizeable library of nanobodies that were raised against the murine norovirus virion with the main purpose of developing nanobody-based inhibitors. We discovered two types of neutralizing nanobodies and analyzed the inhibition mechanisms using X-ray crystallography, cryo-electron microscopy (cryo-EM), and cell culture techniques. The first type bound on the top region of the protruding (P) domain. Interestingly, this nanobody binding region closely overlapped the MNV receptor-binding site and collectively shared numerous P domain-binding residues. In addition, we showed that these nanobodies competed with the soluble receptor, and this action blocked virion attachment to cultured cells. The second type bound at a dimeric interface on the lower side of the P dimer. We discovered that these nanobodies disrupted a structural change in the capsid associated with binding cofactors (i.e., metal cations/bile acid). Indeed, we found that capsids underwent major conformational changes following addition of Mg2+ or Ca2+ Ultimately, these nanobodies directly obstructed a structural modification reserved for a postreceptor attachment stage. Altogether, our new data show that nanobody-based inhibition could occur by blocking functional and structural capsid properties.IMPORTANCE This research discovered and analyzed two different types of MNV-neutralizing nanobodies. The top-binding nanobodies sterically inhibited the receptor-binding site, whereas the dimeric-binding nanobodies interfered with a structural modification associated with cofactor binding. Moreover, we found that the capsid contained a number of vulnerable regions that were essential for viral replication. In fact, the capsid appeared to be organized in a state of flux, which could be important for cofactor/receptor-binding functions. Blocking these capsid-binding events with nanobodies directly inhibited essential capsid functions. Moreover, a number of MNV-specific nanobody binding epitopes were comparable to human norovirus-specific nanobody inhibitors. Therefore, this additional structural and inhibition information could be further exploited in the development of human norovirus antivirals.

Structural Basis of Nanobodies Targeting the Prototype Norovirus.

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.

Natural extracts, honey, and propolis as human norovirus inhibitors.

Norovirus is the most important cause of acute gastroenteritis, yet there are still no antivirals, vaccines, or treatments available. Several studies have shown that norovirus-specific monoclonal antibodies, Nanobodies, and natural extracts might function as inhibitors. Therefore, the objective of this study was to determine the antiviral potential of additional natural extracts, honeys, and propolis samples. Norovirus GII.4 and GII.10 virus-like particles (VLPs) were treated with different natural samples and analyzed for their ability to block VLP binding to histo-blood group antigens (HBGAs), which are important norovirus co-factors. Of the 21 natural samples screened, date syrup and one propolis sample showed promising blocking potential. Dynamic light scattering indicated that VLPs treated with the date syrup and propolis caused particle aggregation, which was confirmed using electron microscopy. Several honey samples also showed weaker HBGA blocking potential. Taken together, our results found that natural samples might function as norovirus inhibitors.

Structural heterogeneity of a human norovirus vaccine candidate.

Human norovirus virus-like particles (VLPs) are assumed to be morphologically and antigenically similar to virion particles. The norovirus virion is assembled from 180 copies of the capsid protein (VP1) and exhibits T = 3 icosahedral symmetry. In this study, we showed that the vaccine candidate GII.4c VP1 formed T = 1 and T = 3 VLPs, but mainly assembled into T = 4 icosahedral particles that were composed of 240 VP1 copies. In contrast, another clinically important genotype, GII.17, almost exclusively folded into T = 3 VLPs. Interestingly, the GII.4c T = 1 particles had higher binding capacities to norovirus-specific Nanobodies than to GII.4c T = 3 and T = 4 particles. Our data indicated that the occluded Nanobody-binding epitopes on the T = 1 particles were more accessible compared to the larger T = 3 and T = 4 particles. Overall, this new data revealed that GII.4c VLPs had a preference for forming the T = 4 icosahedral symmetry and future studies with varied sized norovirus VLPs should take caution when examining antigenicity.

Heterologous expression of human norovirus GII.4 VP1 leads to assembly of T=4 virus-like particles.

Human noroviruses are a leading cause of acute gastroenteritis, yet there are still no vaccines or antivirals available. Expression of the norovirus capsid protein (VP1) in insect cells typically results in the formation of virus-like particles (VLPs) that are morphologically and antigenically comparable to native virions. Indeed, several different norovirus VLP candidates are currently used in clinical trials. So far, structural analysis of norovirus VLPs showed that the capsid has a T = 3 icosahedral symmetry and is composed of 180 copies of VP1 that are folded into three quasi-equivalent subunits (A, B, and C). In this study, the VLP structures of two norovirus GII.4 genetic variants that were identified in 1974 and 2012 were determined using cryo-EM. Surprisingly, we found that greater than 95% of these GII.4 VLPs were larger than virions and 3D reconstruction showed that these VLPs exhibited T = 4 icosahedral symmetry. We also discovered that the T = 4 VLPs presented several novel structural features. The T = 4 particles assembled from 240 copies of VP1 that adopted four quasi-equivalent conformations (A, B, C, and D) and formed two distinct dimers, A/B and C/D. The protruding domains were elevated ∼21 Šoff the capsid shell, which was ∼7 Šmore than in the previously studied GII.10 T = 3 VLPs. A small cavity and flap-like structure at the icosahedral two-fold axis disrupted the contiguous T = 4 shell. Overall, our findings indicated that GII.4 VP1 sequences assemble into T = 4 VLPs and these larger particles might have important consequences for VLP-based vaccine development.

Structure of the hexagonal surface layer on Caulobacter crescentus cells.

Many prokaryotic cells are encapsulated by a surface layer (S-layer) consisting of repeating units of S-layer proteins. S-layer proteins are a diverse class of molecules found in Gram-positive and Gram-negative bacteria and most archaea1-5. S-layers protect cells from the outside, provide mechanical stability and also play roles in pathogenicity. In situ structural information about this highly abundant class of proteins is scarce, so atomic details of how S-layers are arranged on the surface of cells have remained elusive. Here, using purified Caulobacter crescentus' sole S-layer protein RsaA, we obtained a 2.7 Å X-ray structure that shows the hexameric S-layer lattice. We also solved a 7.4 Šstructure of the S-layer through electron cryotomography and sub-tomogram averaging of cell stalks. The X-ray structure was docked unambiguously into the electron cryotomography map, resulting in a pseudo-atomic-level description of the in vivo S-layer, which agrees completely with the atomic X-ray lattice model. The cellular S-layer atomic structure shows that the S-layer is porous, with a largest gap dimension of 27 Å, and is stabilized by multiple Ca2+ ions bound near the interfaces. This study spans different spatial scales from atoms to cells by combining X-ray crystallography with electron cryotomography and sub-nanometre-resolution sub-tomogram averaging.