Specific Sites of N-Linked Glycosylation on the Hemagglutinin of H1N1 Subtype Influenza A Virus Determine Sensitivity to Inhibitors of the Innate Immune System and Virulence in Mice

Abstract

Oligosaccharides on the hemagglutinin (HA) and neuraminidase of influenza A virus (IAV) are a target for recognition by lectins of the innate immune system, including soluble surfactant protein-D and the macrophage mannose receptor on airway macrophages. Glycans attached to the head of H1 subtype of IAV differ markedly in number and location. A reverse genetic approach was used to define the importance of particular N-glycosylation sites on H1 in determining sensitivity to innate immune defenses and virulence in mice. The HA of A/PR/8/34 (PR8, H1N1) and A/Brazil/11/78 (Brazil, H1N1) express zero and four glycosylation sites on the head of HA, respectively. Site-directed mutagenesis was used to add (PR8) or delete (Brazil) glycosylation sites, and IAV expressing wild-type or mutant HA were generated on a PR8 backbone. Addition or removal of particular glycans modulated sensitivity to mouse lung fluids but was not a major factor determining susceptibility of airway macrophages to infection. PR8 is a mouse-adapted virus, and mutations in multiple IAV genes have been shown to contribute to virulence, yet addition of glycosylation to PR8 HA was sufficient to attenuate disease. In contrast, removal of glycans from Brazil HA resulted in severe disease and death. These studies provide insight regarding the mechanisms by which IAV can induce disease in mice. Moreover, reduced glycosylation of HA is likely to be an important factor associated with adaptation of human IAV to growth in mouse lung.

Addition of Glycosylation to Influenza A Virus Hemagglutinin Modulates Antibody-Mediated Recognition of H1N1 2009 Pandemic Viruses

“…Alternatively, high-mannose glycans at Asn 136 might be orientated in a manner that is prohibitive to effective recognition by MBL and SP-D. For seasonal H1N1, loss of a high-mannose glycan from Asn 104 has been associated with resistance to conglutinin, a bovine-specific collectin (58). Addition of Asn 104 and Asn 144 together, but not either site alone, to PR8 HA was associated with enhanced sensitivity to MBL and SP-D (20), suggesting that for seasonal H1 IAV both sites express glycans recognized by collectins but that multivalent interactions increase binding avidity and therefore anti-IAV activity.…”

“…Site-directed mutagenesis was used to add or remove N-linked glycosylation sites from the Auck/09 HA (20 …”

“…SP-D and MBL bind to HA and NA (15,18,19), with the majority of binding to HA. Sitedirected mutagenesis and reverse genetics (RG) approaches have demonstrated the importance of particular N-glycans on the head of seasonal H1 (20) and H3 IAV (21,22) as ligands for MBL and SP-D.…”

A Single Amino Acid Substitution in the Hemagglutinin of H3N2 Subtype Influenza A Viruses Is Associated with Resistance to the Long Pentraxin PTX3 and Enhanced Virulence in Mice

“…All research complied with the University of Melbourne's Animal Experimentation Ethics guidelines and policies. At various times postinfection, mice were culled, and samples including blood, bronchoalveolar lavage fluid (BALF), thymus, and lungs were collected as described (28,30). Plaque assay was used to determine titers of infectious virus in clarified homogenates prepared from the lungs of IAV-infected mice.…”

“…Subconfluent monolayers of mouse LA-4 airway epithelial cells or MDCK cells in eight-well chamber slides (Lab-Tek; Nunc) were incubated with increasing dilutions of IAV for 1 h at 37˚C, washed three times, and incubated in serum-free media, as described (28). At 7-8 h postinfection, cells were fixed in 80% (v/v) acetone and stained with anti-NP mAb MP3.1092.IC7, followed by FITC-conjugated rabbit anti-mouse Ig (Dako).…”