Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo

Erika Crouch,Nikolaos Nikolaidis,Francis X Mccormack,Barbara Mcdonald,Kimberly Allen,Michael J Rynkiewicz,Tanya M Cafarella,Mitchell White,Kara Lewnard,Nancy Leymarie,Joseph Zaia,Barbara A Seaton,Kevan L Hartshorn

doi:10.1074/jbc.m111.300673

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.

Highlights

surfactant protein D (SP-D) plays important roles in the defense against influenza A
Our studies have shown that combinatorial mutagenesis of the residues that flank the calcium ion at the lectin site of human SP-D can greatly enhance interactions with specific strains of influenza A virus (IAV) both in vitro and in vivo
Previous studies have shown that exogenous native human SP-D can rescue SP-D null mice challenged with specific strains of IAV [6]; this is the first study demonstrating antiviral effects of the more therapeutically practical trimeric NCRDs

Summary

Background

SP-D plays important roles in the defense against influenza A. Viral interactions require calcium and are inhibited by monosaccharide ligands of SP-D, directly implicating the lectin activity of the SP-D carbohydrate recognition domain (CRD) [11] This is consistent with crystallographic studies of recombinant trimeric neck plus carbohydrate recognition domains (NCRDs) that have demonstrated binding of the equatorial hydroxyl groups of mannoses to calcium at the lectin site (14 –16). We employed a novel surface plasmon resonance assay, viral binding and neutralization assays, mass spectrometry of HA glycans, and a new in vivo model of IAV infection to examine interactions between a recently developed combinatorial SP-D mutant and influenza A virus. The findings demonstrate the potential for collectin NCRD-based antiviral interventions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION