title: Mutagenesis of surfactant protein D informed by evolution and x-ray crystallography enhances defenses against influenza A virus in vivo.
abstract: The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. crouch@path.wustl.edu
authors.forename: Erika
authors.initials: E
authors.lastname: Crouch
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Nikolaidis
authors.forename: Francis X
authors.initials: FX
authors.lastname: McCormack
authors.forename: Barbara
authors.initials: B
authors.lastname: McDonald
authors.forename: Kimberly
authors.initials: K
authors.lastname: Allen
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Rynkiewicz
authors.forename: Tanya M
authors.initials: TM
authors.lastname: Cafarella
authors.forename: Mitchell
authors.initials: M
authors.lastname: White
authors.forename: Kara
authors.initials: K
authors.lastname: Lewnard
authors.forename: Nancy
authors.initials: N
authors.lastname: Leymarie
authors.forename: Joseph
authors.initials: J
authors.lastname: Zaia
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Seaton
authors.forename: Kevan L
authors.initials: KL
authors.lastname: Hartshorn
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology; D018360:Crystallography, X-Ray; D060467:Disease Resistance / Q000235:genetics*; D019143:Evolution, Molecular; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009976:Orthomyxoviridae Infections / Q000821:virology; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D037663:Pulmonary Surfactant-Associated Protein D / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D000998:Antiviral Agents; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D037663:Pulmonary Surfactant-Associated Protein D