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Characterization of human norovirus binding to gut-associated bacterial ligands.

ABSTRACT: OBJECTIVE:Research suggests human norovirus binding to histo-blood group antigen (HBGA)-like molecules on enteric bacteria may enhance viral pathogenesis; however, the properties of these bacterial ligands are not well known. Previous work identified, but did not characterize, seven norovirus-binding bacteria. To further examine this bacteria-virus binding interaction, enteric bacteria were analyzed via Western blot with anti-HBGA antibodies and lectins targeting HBGA-associated sugar components. Virus overlay assays using capsids from six different human norovirus strains further identified responsible ligands and strain dependent binding properties. RESULTS:Each bacterial species possessed varying degrees of HBGA-like activity, and lectin binding further elucidated potential sugar residues involved (N-acetyl-galactosamine, ?-D-galactose or ?-L-fucose). Both GI and GII norovirus capsids bound specific bacterial ligand sizes, and generally corresponded to anti-HBGA Western blot patterns. A 35-kDa band reacted with all HBGA antibodies, bound all six of the noroviruses tested, and had a high affinity for the lectins. Collectively, this work characterizes the varying carbohydrate residues potentially responsible for norovirus-bacteria interactions and provides a basis for future ligand identification.

SUBMITTER: Almand EA

PROVIDER: S-EPMC6755701 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Characterization of human norovirus binding to gut-associated bacterial ligands.

Almand Erin A EA Moore Matthew D MD Jaykus Lee-Ann LA

BMC research notes 20190923 1

<h4>Objective</h4>Research suggests human norovirus binding to histo-blood group antigen (HBGA)-like molecules on enteric bacteria may enhance viral pathogenesis; however, the properties of these bacterial ligands are not well known. Previous work identified, but did not characterize, seven norovirus-binding bacteria. To further examine this bacteria-virus binding interaction, enteric bacteria were analyzed via Western blot with anti-HBGA antibodies and lectins targeting HBGA-associated sugar co ...[more]

PMID: 31547886

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Project description:Human noroviruses (HuNoVs) are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs) expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP) to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2) and the protruding domain (P domain) encoding gene (3' terminal fragment of ORF2) of HuNoVs GI.1 and GII.4 were fused with 5' terminal fragment of INP encoding gene (inaQn). The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an oral vaccine for HuNoVs.

Project description:Emergent strains of human norovirus seed pandemic waves of disease. These new strains have altered ligand binding and antigenicity characteristics. Study of viral variants isolated from immunosuppressed patients with long-term norovirus infection indicates that initial virus in vivo evolution occurs at the same antigenic sites as in pandemic strains. Here, cellular ligand binding and antigenicity of two cocirculating strains isolated from a patient with long-term norovirus infection were characterized. The isolated GII.4 viruses differed from previous strains and from each other at known blockade antibody epitopes. One strain had a unique sequence in epitope D, including loss of an insertion at residue 394, corresponding to a decreased relative affinity for carbohydrate ligands. Replacement of 394 with alanine or restoration of the contemporary strain epitope D consensus sequence STT improved ligand binding relative affinity. However, monoclonal antibody blockade of binding potency was only gained for the consensus sequence, not by the alanine insertion. In-depth study of unique changes in epitope D indicated that ligand binding, but not antibody blockade of ligand binding, is maintained despite sequence diversity, allowing escape from blockade antibodies without loss of capacity for binding cellular ligands.IMPORTANCE Human norovirus causes ?20% of all acute gastroenteritis and ?200,000 deaths per year, primarily in young children. Most epidemic and all pandemic waves of disease over the past 30?years have been caused by type GII.4 human norovirus strains. The capsid sequence of GII.4 strains is changing over time, resulting in viruses with altered ligand and antibody binding characteristics. The carbohydrate binding pocket of these strains does not vary over time. Here, utilizing unique viral sequences, we study how residues in GII.4 epitope D balance the dual roles of variable antibody binding site and cellular ligand binding stabilization domain, demonstrating that amino acid changes in epitope D can result in loss of antibody binding without ablating ligand binding. This flexibility in epitope D likely contributes to GII.4 strain persistence by both allowing escape from antibody-mediated herd immunity and maintenance of cellular ligand binding and infectivity.

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Characterization of human norovirus binding to gut-associated bacterial ligands.

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Characterization of human norovirus binding to gut-associated bacterial ligands.

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