title: Biochemical and structural characterization of the interface mediating interaction between the influenza A virus non-structural protein-1 and a monoclonal antibody.
abstract: We have previously shown that a non-structural protein 1 (NS1)-binding monoclonal antibody, termed as 2H6, can significantly reduce influenza A virus (IAV) replication when expressed intracellularly. In this study, we further showed that 2H6 binds stronger to the NS1 of H5N1 than A/Puerto Rico/8/1934(H1N1) because of an amino acid difference at residue 48. A crystal structure of 2H6 fragment antigen-binding (Fab) has also been solved and docked onto the NS1 structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. In one of the models, the predicted molecular contacts between residues in NS1 and 2H6-Fab correlate well with biochemical results. Taken together, residues N48 and T49 in H5N1 NS1 act cooperatively to maintain a strong interaction with mAb 2H6 by forming hydrogen bonds with residues found in the heavy chain of the antibody. Interestingly, the pandemic H1N1-2009 and the majority of seasonal H3N2 circulating in humans since 1968 has N48 in NS1, suggesting that mAb 2H6 could bind to most of the currently circulating seasonal influenza A virus strains. Consistent with the involvement of residue T49, which is well-conserved, in RNA binding, mAb 2H6 was also found to inhibit the interaction between NS1 and double-stranded RNA.
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore.
authors.forename: Jianping
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore.
authors.forename: Chee-Keng
authors.initials: CK
authors.lastname: Mok
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore.
authors.forename: Vincent Tak Kwong
authors.initials: VT
authors.lastname: Chow
authors.affiliation: Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore.|National University of Singapore (Suzhou) Research Institute, Suzhou Industrial Park, Jiangsu 215123, China.
authors.forename: Y Adam
authors.initials: YA
authors.lastname: Yuan
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore.|Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.
authors.forename: Yee-Joo
authors.initials: YJ
authors.lastname: Tan
mesh_terms: D000072283:A549 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D000956:Antigens, Viral / Q000378:metabolism; D001665:Binding Sites; D001717:Birds / Q000821:virology; D018360:Crystallography, X-Ray; D004285:Dogs; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D053124:Influenza A Virus, H5N1 Subtype / Q000378:metabolism; D009980:Influenza A virus / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D012330:RNA, Double-Stranded / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D007140:Immunoglobulin Fab Fragments; D012330:RNA, Double-Stranded; D017361:Viral Nonstructural Proteins