Structural Basis for Broad Detection of Genogroup II Noroviruses by a Monoclonal Antibody That Binds to a Site Occluded in the Viral Particle

Hansman, Grant S.; Taylor, David W.; McLellan, Jason S.; Smith, Thomas J.; Georgiev, Ivelin S.; Tame, J.R.H.; Park, Sam Yong; Yamazaki, Makoto; Gondaira, Fumio; Miki, Motohiro; Katayama, Kazuhiko; Murata, Kazuyoshi; Kwong, Peter D.

doi:10.1128/jvi.06868-11

Cited by 81 publications

(126 citation statements)

References 74 publications

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“…These cryptic epitopes are often very conserved and therefore provide cross-serotypic neutralization. We previously identified a broadly reactive norovirus mAb [53] and Nano-85 that bound to a conserved region that was occluded in the context of native-size particles [31]. Here, we identified four novel Nanobodies (Nano-4, Nano-26, Nano-42, and Nano-27) that bound to the similar internal and poorly accessible epitopes as Nano-85.…”

Section: Discussionmentioning

confidence: 99%

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Koromyslova

Hansman

2018

Biophysical Journal

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Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses. PLOS Author summaryWe determined the binding sites of six novel human norovirus specific Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) using X-ray crystallography. The unique Nanobody recognition epitopes were correlated with their potential neutralizing capacities. We showed that one Nanobody (Nano-26) bound numerous genogroup II genotypes and interacted with highly conserved capsid residues. Four Nanobodies (Nano-4, Nano-26, Nano-27, and Nano-42) bound to occluded regions on the intact particles and impaired normal capsid morphology and particle integrity. One Nanobody (Nano-14) bound contiguous to the HBGA pocket and interacted with several residues involved in binding HBGAs. We found that the Nanobodies delivered multiple inhibition mechanisms, which included steric obstruction, allosteric interference, and disruption of the capsid stability. Our data suggested that the HBGA pocket might not be an ideal target for drug development, since the surrounding regio...

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Section: Discussionmentioning

confidence: 99%

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Koromyslova

Hansman

2018

Biophysical Journal

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“…The binding sites of both Nano-85 and Nano-25 were located on the lower region of the P1 subdomain. In order to better understand the nanobody binding in the context of the intact particles, the X-ray crystal structures of the GII.10 P domain Nano-25/Nano-85 complexes were manually positioned in the cryo-EM structure of the GII.10 VLP as previously described (11). The P dimers of the nanobody complexes unambiguously fit into the cryo-EM P dimer density map.…”

Section: Figmentioning

confidence: 99%

“…The S domain forms a scaffold surrounding the RNA, whereas the P domains extend off the S domain and likely contain the main determinants for strain diversity. There are two other high-resolution cryo-electron microscopy (cryo-EM) structures of norovirus particles, GV.1 murine norovirus and GII.10 human norovirus (10,11). One major structural distinction among these particles is the position of the P domain on the S domain.…”

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Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

Koromyslova

Hansman

2015

J Virol

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Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses, and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different nanobodies. One nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly comprised highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues also were recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain nanobody complex structures into a cryoelectron microscopy particle structure revealed that both nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ϳ10 to 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. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability. IMPORTANCEWe characterized two different nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich enzyme-linked immunosorbent assay. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 not only will work as a diagnostic reagent in norovirus detection systems but also could function as a broadly reactive GII norovirus antiviral.

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“…Several cross-reactive MAbs have been described for noroviruses, and epitopes for most of them have been mapped within the most conserved regions of the norovirus capsid protein (C-terminal P or S domain). As suggested previously, cross-reactive MAbs could potentially be used in diagnostic assays (3,24,36,46,51); however, Oliver et al (45) described a cross-reactive MAb that recognized a linear epitope in the S domain of the VLPs from bovine noroviruses that did not detect native virions in fecal samples from experimental animals. They proposed that because the S domain forms the innermost domain of the capsid, the epitope is not accessible in native virions.…”

Section: Discussionmentioning

confidence: 99%

Multiple Antigenic Sites Are Involved in Blocking the Interaction of GII.4 Norovirus Capsid with ABH Histo-Blood Group Antigens

Parra

Abente

Sandoval-Jaime

et al. 2012

J Virol

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Structural Basis for Broad Detection of Genogroup II Noroviruses by a Monoclonal Antibody That Binds to a Site Occluded in the Viral Particle

Cited by 81 publications

References 74 publications

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

Multiple Antigenic Sites Are Involved in Blocking the Interaction of GII.4 Norovirus Capsid with ABH Histo-Blood Group Antigens

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